Principles of Plant breeding and biotechnology.pptx

Lecture note

1. 1 Dr. Zekeria Yusuf (PhD) Plant Breeding and Biotechnology (BIOG 541 & 522)

2. Introduction Plant breeding is a science based on principles of genetics and cytogenetics . It aims at improving the genetic makeup of the crop plants. Improved varieties are developed through plant breeding. Its objectives are to improve yield, quality, disease-resistance, drought and frost-tolerance and important characteristics of the crops. Plant breeding has been crucial in increasing production of crops to meet the ever increasing demand for food. Some well known achievements are development of semi-dwarf wheat and rice varieties, noblization of canes (sugarcanes), and production of hybrid and composite varieties of maize….. 2 Dr. Zekeria Yusuf (PhD)

3. Introduction…. Crop improvement means combining desirable characteristics in one plant and then multiplying it. The job of a plant breeder is to select plants with desired characters, cross them and then identify the offspring that combine the attributes of both parents. Then multiply the progeny to supply to farmers, growers or planters. The modern age of plant breeding began in the early part of the 20thC , after Mendel’s work was rediscovered. Today plant breeding is a specialized technology based on genetics. It is now clearly understood that within a given environment, crop improvement has to be achieved through superior heredity. 3 Dr. Zekeria Yusuf (PhD)

4. Introduction…. Plant breeding is the art, science and technology of changing the heredity of plants for human welfare. Nature of Plant Breeding: 1. Art •In earlier days man depends on his skills and judgement in selecting better plants. He knew nothing about the inheritance of characters, role of environment in producing them and the basis of variation in various plant characters. His method of selection was designed without the understanding of principles of inheritance. •Therefore during primitive time plant breeding was largely an art and very less science was involved in that. Even today success of selection depends upon ability of the person involved in the selection. 4 Dr. Zekeria Yusuf (PhD)

5. Introduction…. 2. Science •Plant breeding is considered as the current phase of crop evolution. As the knowledge of genetics and other related science progresses plant breeding become less art and more science. •Especially, discovery of Mendel̕s work in 1900 added a lot to the knowledge of science. •Selection of desirable plant even today is an art it depends on the skill of a person. But alone skill is not enough, modern plant breeding is a combined effort of art and understanding and use of genetic principles. 3. Technology Product of all plant breeding activities, whether dependent on the art or science, is improved variety, hybrids, synthetics and composites. This product is utilized by farmers for commercial cultivation. Therefore, plant breeding can be rightly viewed as a technology since it generates a useful product. 5 Dr. Zekeria Yusuf (PhD)

6. Role of Plant Breeding: Human beings are dependent on the plants for: Food :- Breeding of field crops provides us food either directly (food grains) or indirectly (meat and milk). 2. Shelter :- In addition to food by produce of agriculture farms are used in making shelter by farmers of rural areas. 3. Clothing :- Breeding for fibre crops like cotton provides clothes for the human population. 4. Fuels :- Crops like Euphorbia and Jatropha are used for Biofuel production. Breeding of such crops tackles the problems of energy production for rapidly increasing human population. Now a days, Maize is also used as an important source of Ethanol production. 5. Drugs :- Breeding of medicinal plants plays an important role in production of many important drugs. These drugs are used for treatment of various human and animal diseases. 6. Entertainment:- Flowers play an essential role in peoples celebrations and everyday lives like weddings, Christmas etc. most of the medicinal plants are seasonal in nature. Shifting the seasonal timing of reproduction is a major goal of plant breeding efforts to produce novel varieties that are better adapted to local environments and changing climatic conditions. 6 Dr. Zekeria Yusuf (PhD)

7. Plant breeding is the process by which humans change certain aspects of plants over time in order to introduce desired characteristics Plant Breeding Concept Increase crop productivity

8. Plant Domestication Domestication: The process by which people try to control the reproductive rates of animals and plants. Without knowledge on the transmission of traits from parents to their offspring. Plant Breeding : The application of genetic analysis to development of plant lines better suited for human purposes. Plant Breeding and Selection Methods to meet the food, feed, fuel, and fiber needs of the world Genetic Engineering to increase the effectiveness and efficiency of plant breeding. 8 Dr. Zekeria Yusuf (PhD)

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11. Domestication Plant Breeding activities began at least 10.000 years ago in the Fertile Crescent with plant domestication Challenges: transition from nomadic to a sedentary  lifestyle Increase plant yield Increase number of edible plants ( reduce toxicity )

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13. Geography of crop domestication Vavilov’s eight centers of origin where crops were first tamed. Turns out that centers of diversity do not coincide with Vavilov’s centers of origin. Areas with lots of wild relatives and primitive versions of modern crops can be invaluable sources of genes for plant breeders and geneticists. 13 Dr. Zekeria Yusuf (PhD)

14. Concept first devised by Vavilov in 1919 Archaeological evidence suggests that hunter-gatherers independently began cultivating food plants in 24 regions,….” (Purugannan and Fuller, 2009) Centres of Plant Domestication 14 Dr. Zekeria Yusuf (PhD)

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18. What is a domestication syndrome? A domestication syndrome describes the properties that distinguish a certain crop from it’s wild progenitor. Typically such characteristics are: larger fruits or grains more robust plants more determinate growth / increased apical dominance loss of natural seed disperal fewer fruits or grains decrease in bitter substances in edible structures changes in photoperiod sensitivity synchronized flowering 18 Dr. Zekeria Yusuf (PhD)

19. Tomato - Fewer and Larger Fruits 19 Dr. Zekeria Yusuf (PhD)

20. Sunflowers - reduced branching, larger seeds, increased seed set per head ‏ 20 Dr. Zekeria Yusuf (PhD)

21. Wheat - reduced seed shattering, increased seed size 21 Dr. Zekeria Yusuf (PhD)

22. Squash – larger, fleshier fruits 22 Dr. Zekeria Yusuf (PhD)

23. Corn – reduced fruitcase, softer glume, more kernels per cob, no dispersal, reduced branching, apical dominance 23 Dr. Zekeria Yusuf (PhD)

24. Lettuce – leaf size/shape, fewer secondary compounds 24 Dr. Zekeria Yusuf (PhD)

25. Rice – no shattering, larger grains 25 Dr. Zekeria Yusuf (PhD)

26. Crop consequences of domestication: More ‘yield’ of desirable part. Non-shattering - seed are easier to harvest. Big seeds - domesticated bean seed are 5-8 times as large as their wild relatives. Improved quality - remove or lower toxic substances. Increased protein, oil, sugar concentration, which means improved flavor, storage ability. 26 Dr. Zekeria Yusuf (PhD)

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29. Methods for identifying domestication genes Biparental QTL mapping Association Mapping Using Unrelated Individuals QTL Mapping Using Advanced Intercross Populations Genomic scans Genome Resequencing and Screening for Selection 29 Dr. Zekeria Yusuf (PhD)

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31. Classical Examples… Teosinte branched 1 ( tb1 ) QTL of maize controls the difference in apical dominance in maize and teosinte tb1 , it acts as transcriptional regulators, a class of genes involved in the transcriptional regulation of cell cycle Teosinte glume architecture1 ( tga1 ) was identified as a QTL controlling the formation of the casing that surrounds the kernels of the maize ancestor, teosinte tga1 is a member of the squamosa -promoter binding protein ( SBP ) family of transcriptional regulators Fruitweight2.2 ( fw2.2 ) was identified as a large effect QTL controlling 30% of the difference in fruit mass between wild and cultivated tomato fw2.2 acts as a negative regulator of cell division in the fruit, perhaps via some role in cell-to cell communication Q is a major gene involved in wheat domestication that affects a suite of traits, including The tendency of the spike (ear) to shatter, The tenacity of the chaff surrounding the grain, & The spike is elongated as in wild wheat or compact like the cultivated forms 31 Dr. Zekeria Yusuf (PhD)

32. shattering4 ( sh4 ) is a major QTL controlling whether the seed fall off the plant (shatter) as in wild rice or adhere to the plant as in cultivated rice sh4 encodes a gene with homology to Myb3 transcription factors. A single amino acid change in the predicted DNA binding domain converts plants from shattering to non-shattering Rc is a domestication-related gene required for red pericarp in rice Two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14- bp deletion within exon 6 - originated in japonica cultivar and spread into indica cultivars. 32 Dr. Zekeria Yusuf (PhD)

33. Super-domestication The processes that lead to a domesticate with dramatically increased yield that could not be selected in natural environments without new technologies. The array of genome manipulations enable barriers to gene exchange to be overcome and have lead to super-domesticates with – dramatically increased yields, – resistances to biotic and abiotic stresses, and with – new characters for the market place. Hybrid rice can be considered a super-domesticate Conversion of a crop from C3 to C4 photosynthesis would certainly be a super-domesticate. Plantbreeders+GenomicScientists ⇨ Superdomestication 33 Dr. Zekeria Yusuf (PhD)

34. Domestication ‘ Domestication is the process by which humans actively interfere with and direct crop evolution. ’ It involves a genetic bottleneck: Often only few genes are actively selected and account for large shifts in phenotype. Crops exhibit various levels of domestication. 34 Dr. Zekeria Yusuf (PhD)

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38. selective sweep A selective sweep is the reduction or elimination of variation among the nucleotides in neighboring DNA of a mutation as the result of recent and strong positive natural selection A strong selective sweep results in a region of the genome where the positively selected haplotype (the mutated allele and its neighbours ) is essentially the only one that exists in the population, resulting in a large reduction of the total genetic variation in that chromosome region. 38 Dr. Zekeria Yusuf (PhD)

39. selective sweep 39 Dr. Zekeria Yusuf (PhD)

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42. Domestication is a process The distinction ‘domesticated’ or ‘not domesticated’ is an over-simplification Some crops have moved further along this process further than others. We can recognize different levels of domestication How can we decide which level? 42 Dr. Zekeria Yusuf (PhD)

43. Different domestication traits were selected for progressively Distinction between selection under domestication vs. crop diversification  more targeted, ‘conscious’ selection during diversification ‘Slow’ rate of evolution of different domestication traits despite faster rates suggested by models Artificial selection can be “similar across different taxa , geographical origins and time periods” 43 Dr. Zekeria Yusuf (PhD)

44. Parallel evolution for “sticky glutinous varieties” in rice and foxtail millets, all through selection at the waxy locus Most QTL studies suggest that many domestication traits are controlled by a few genes of large effect – not though in sunflower Population genomic studies in maize suggest 2 – 4% of genes show evidence of artificial selection 44 Dr. Zekeria Yusuf (PhD)

45. Domestication of Maize 45 Dr. Zekeria Yusuf (PhD)

46. The evolution of non-shattering in the archaeological record 46 Dr. Zekeria Yusuf (PhD)

47. The genetic basis of the evolution of non-shattering Non-shattering is often regarded as the hallmark of domestication in most seed crops because it renders a plant species primarily dependent on humans for survival and propagation: rice gene sh4 (similar to the genes encoding MYB -like transcription factors in maize) rice quantitative trait locus ( QTL ) qSH1 , which encodes a homeobox -containing protein the wheat gene Q, which is similar to genes of the AP2 family in other plants In sunflower likely controlled by multiple genes 47 Dr. Zekeria Yusuf (PhD)

48. Domestication genes in plants Maize and rice domestication seem to suggest few loci of large effect are important Sunflower domestication seems to suggest many loci of small to intermediate effect are important 9 domestication genes in plants so far, as well as 26 other loci known to underlie crop diversity Of the 9 domestication loci, 8 encode transcriptional activators. More than half of crop diversification genes encode enzymes.  Domestication seems to be associated with changes in transcriptional regulatory networks, whereas crop diversification involves a larger proportion of enzyme-encoding loci (lots of them loss-of-function alleles). 48 Dr. Zekeria Yusuf (PhD)

49. The role of polyploidy in domestication 49 Dr. Zekeria Yusuf (PhD)

50. Towards resolving the genetic basis of domestication in the Compositae Artificial selection through domestication but HOW ? 50 Dr. Zekeria Yusuf (PhD)

51. Some fundamental questions in domestication genetics Which genes show strong signs of selection in different crops? Can we see common patterns in taxa that have been domesticated for similar purposes? Can we see dissimilar categories of genes under selection in different crop types despite their close phylogenetic relationship (e.g. sunflower and jerusalem artichoke)? 51 Dr. Zekeria Yusuf (PhD)

52. Bioinformatics pipeline: Methodological ‘bottom-up’ approach 1.) Input: EST libraries of crop, progenitor and outgroup 2.) Genes that are orthologous in all taxa are identified 3.) These genes are scanned for signs of strong positive selection 4.) Such genes are compared to all known proteins in Arabidopsis 5.) Functional characteristics of best fits in Arabidopsis genomic database ( TAIR ) are annotated 52 Dr. Zekeria Yusuf (PhD)

53. Some preliminary results Preliminary results from candidate domestication gene search in Compositae crops: Several stress response genes are under selection in leaf and oil seed crops Other interesting candidate domestication genes:  safflower: fatty acid metabolism  sunflower: nitrate assimilation  Jerusalem artichoke: lateral root formation 53 Dr. Zekeria Yusuf (PhD)

54. What to do with candidate genes? Confirm their role underlying traits - functional analysis (introgression/transgenes) ‏ - expression - population genetic work confirm associations with fitness association mapping with traits of interest 54 Dr. Zekeria Yusuf (PhD)

55. What to do with candidate genes? Applications: breeding / improvement conservation of genetic diversity identification of taxon boundaries understanding adaptation/domestication comparative analysis – other taxa 55 Dr. Zekeria Yusuf (PhD)

56. Crop improvement Phenotype – based selection Slow, ineficient but can be effective Using genetics to inform breeding Marker-assisted selection Marker-assisted introgression Transformation Efficient (if you have the gene) but controversial 56 Dr. Zekeria Yusuf (PhD)

57. Transgenics controversy Advantages: Targeted to specific gene Any gene can be changed / introduced from any species Fast and efficient Disadvantages: Safety issues Regulations / legal issues Requires expertise and technology 57 Dr. Zekeria Yusuf (PhD)

58. Transgenics controversy Advantages: Huge improvements in phenotype of interest possible Yield improvements Health / nutrition benefits Reduce herbicide / pesticide / fertilizer use New products – pharmaceuticals, chemicals, etc. Disadvantages: Little regulation for health/environmental safety Loss of genetic diversity Reliance on big seed companies 58 Dr. Zekeria Yusuf (PhD)

59. Types of crops: Grain crops - wheat, rice, corn, sorghum, barley, oats. Oil crops - olive, linseed, sesame, sunflower, soybean, coconut, palm, corn, peanut, canola. Fiber crops - cotton, flax, hemp, jute, kenaf , sisal. Forage crops - alfalfa, clovers, other legumes, many grasses, including tall fescue. Spice / drug crops - tobacco, black pepper, cinnamon. Fruit crops, vegetable crops, ornamentals, forest trees , etc. 59 Dr. Zekeria Yusuf (PhD)

60. 9000 BC First evidence of plant domestication in the hills above the Tigris river 1694 Camerarius first to demonstrate sex in (monoecious) plants and suggested crossing as a method to obtain new plant types 1714 Mather observed natural crossing in maize 1761-1766 Kohlreuter demonstrated that hybrid offspring received traits from both parents and were intermediate in most traits, first scientific hybrid in tobacco 1866 Mendel: Experiments in plant hybridization 1900 Mendel’s laws of heredity rediscovered 1944 Avery, MacLeod, McCarty discovered DNA is hereditary material 1953 Watson, Crick, Wilkins proposed a model for DNA structure 1970 Borlaug received Nobel Prize for the Green Revolution Berg, Cohen, and Boyer introduced the recombinant DNA technology 1994 ‘FlavrSavr’ tomato developed as first GMO 1995 Bt-corn developed Selected milestones in plant breeding 60 Dr. Zekeria Yusuf (PhD)

61. Landmarks in Plant B reeding 1694 1866 1953 Camerarius crossing as a method to obtain new plant types Mendel Empirical evidence on heredity Watson, Crick, W ilkins & Rosalind Franklin model for DNA structure 1923 Wallace First commercial hybrid corn

62. “ The Green Revolution ” (1960) Norman Borlaug Challenge: improve wheat and maize to meet the production needs of developing countries High yielding semi-dwarf , lodging resistant wheat varieties

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64. Future Challenges Challenge : Increase of human population by 60-80%, requiring to nearly double the global food production Multidisciplinary Field Biometry / Statistics Pathology

65. Challenges before Plant Breeder : Increasing population: at present, the world population stand at 6.3 billion and will reach at 10-12 billion during the next 50-70 years. The main problem from breeding respect is that the population is growing faster than increases in food productivity, to reduce the use of harmful agrochemicals and to produce nutritious and healthful food is greater today. 2. Squeezing arable land : Day-by-day the total arable land for agriculture is decreasing due to urbanization and industrial development. Breeders have to tackle this problem by releasing improved varieties of major crops which gives better production per unit area. 65 Dr. Zekeria Yusuf (PhD)

66. Challenges before Plant Breeder… 3. Erratic rainfall : esp in tropics rainfall is erratic, unpredictable and unevenly distributed. Over 80% of the annual rainfall is received in the four rainy months of June to September. Therefore, varieties which can tolerate dry spells and perform better at low water availability are needed to be develop by Indian Breeders. 4. Mechanization:- The variety developed by plant breeders should give response to application of fertilizers, manures, irrigation and should be suitable for mechanical cultivation. 66 Dr. Zekeria Yusuf (PhD)

67. Research Institutes, Universities, Governmental Services, Private Companies, Non-Governmetal Organizations, Breeders, Farmers… ….are working hard to breed plants for a better agriculture with less environmental impacts Take-Home M essage

68. Scientific disciplines and technologies of plant breeding Genetics Botany Plant physiology Agronomy Pathology and entomology Statistics Biochemistry 68 Dr. Zekeria Yusuf (PhD)

69. Importance of Plant breeding • Plant breeding allowed civilization to form and its continual success is critical to maintaining our way of life • Problem: Feeding 9 billion (+) people with the same (or fewer) inputs Same or less acreage Same or less fertilizer, pesticides, water Adapting to climate and environmental change 69 Dr. Zekeria Yusuf (PhD)

70. Goals of Plant breeding •Plant breeding aims to improve the characteristics of plants so that they become more desirable agronomically and economically. The specific objectives may vary greatly depending on the crop under consideration. Increase the frequency of favorable alleles within a line ( favoring additive effects ) • Increase the frequency of favorable genotypes within a line ( with dominance and interaction effects ) • Better adapt crops to specific environments – Region-specific cultivars ( high location G x E ) – Stability across years within a region ( low year to-year G x E ) Food (yield and nutritional value), feed, fibre , pharmaceuticals(antibodies),landscape, industrial need ( eg . Crops are being produced in regions to which they are not native). 70 Dr. Zekeria Yusuf (PhD)

71. Objectives of Plant Breeding • Development of pure (i.e. highly inbred) lines with high per se performance, • Development of pure lines with high hybrid performance (either with each other or with a testcross), • Less emphasis on developing outbred (random-mating) populations with improved performance • Development of lines with high regional G x E, low year G x E. Note: Details among plant species vary because of origin, mode of reproduction, ploidy levels, and traits of greater importance and adjustments were made to adapt to specific situations. 71 Dr. Zekeria Yusuf (PhD)

72. Objectives of Plant Breeding... The prime objective of plant breeding is to develop superior plants over the existing ones in relation to their economic use. The objectives of plant breeding differ from crop to crop. A brief account of some important objectives are: Higher productivity/yield- Increased yield has been the ultimate aim of most plant breeders. This can be achieved by developing more efficient genotypes having greater physiological efficiency. 2. Improved quality- Improved quality of agricultural products has contributed a lot to the human well-being. Quality characters vary from one crop to another crop. For example, Grain size, colour , milling, and baking qualities in wheat ( Triticum aestivum ). 3. Disease and Insect Resistance- Resistance varieties offer the cheapest and most convenient method of disease and insect management. In some cases, they offer only feasible means of control. eg . Rust in Wheat. 72 Dr. Zekeria Yusuf (PhD)

73. Objectives of Plant Breeding... 4. Varieties for new seasons- The varieties for new seasons have been developed by adjusting the growth cycle of the variety to suit better to the available growing season. 5. Modification of agronomic characteristics- modification of agronomic characteristics such as plant height, tillering , branching, erect or trailing habit etc. is often desirable. For example, dwarfness in cereals is generally associated with lodging resistance and fertilizer responsiveness. 6. Change in maturity duration- it permits new crop rotations and often extends the crop area. Development of wheat varieties suitable for late planting has permitted rice-wheat rotation. This objective is more desirable especially in those areas where multiple cropping system has been followed. 73 Dr. Zekeria Yusuf (PhD)

74. Objectives of Plant Breeding... 7. Photo and thermo insensitivity- Development of photo and thermo insensitive wheat and photo insensitive rice varieties has permitted their cultivation in new areas. 8. Synchronous maturity- Synchronous maturity is highly desirable in crops where several pickings are necessary. Eg . Mungbean , pigeon pea, cotton etc. 9. Non-shattering characteristics- It would be of great value in crops like mung , castor, soybean etc. where shattering is a major problem in case of many commercial varieties. 10. Determinate growth- Development of varieties with determinate growth is desirable in crops like mung , pigeon pea, cotton, etc. 74 Dr. Zekeria Yusuf (PhD)

75. Objectives of Plant Breeding... 11. Dormancy- Dormancy plays both beneficial and harmful role according to the need of grower. For example, if we want next crop immediate after harvesting of previous crop, in such case dormancy is not required. But if we want to store the seed for its future purpose, a period of dormancy is essential. 12. Elimination of toxic substances: some crops have toxic substances which must be eliminated to make them safe for consumption. For example, • Khesari ( Lathyrus odoratus ) seeds have a neurotoxin, β- N- oxalyl - α-β- diaminopropionic acid ( BOAA ) that causes paralysis in humans. •Similarly, elimination of Erusic acid from Brassica oil and Gossypol from seed cotton is necessary to make them fit for consumption. 75 Dr. Zekeria Yusuf (PhD)

76. Objectives of Plant Breeding... 13. Moisture Stress and Salt Tolerance: development of varieties for a rainfed area and saline soils would help to increase crop production. 14. Wider Adaptability : it helps in stabilizing the crop production over region and seasons. 15. Useful for Mechanical Cultivation : the variety developed should give response to application of fertilizers, manures and irrigation, suitable for mechanical cultivation etc. 76 Dr. Zekeria Yusuf (PhD)

77. Animal and tree breeding • Similar goals, but since mostly outcrossing , the goal is to create high-performing populations, not inbred lines • Generally speaking, inbreeding is bad in animals and many trees • Focus on finding those parents with the best transmitting abilities (highest breeding values) • Less of a G x E focus with animals, less of a focus on line and hybrid breeding 77 Dr. Zekeria Yusuf (PhD)

78. Special features exploited by plant breeders • Selfing allows for the capture of specific genotypes, and hence the capture of interactions between alleles and loci (dominance and epistasis ) – Homozygous for selfed lines – Heterozygous for crossed lines • Often high reproductive output (relative to animal breeding) • Seeds allow for multigeneration progeny testing, wherein individuals are chosen on the performance of their progeny, or of their sibs – Allows for better control over G x E by testing over multiple sites/years 78 Dr. Zekeria Yusuf (PhD)

79. Historical plant breeding • Early origins – Creation of new lines through species crosses (allopolyploids) – Visual selection – Early domestication (selection for specific traits for ease of harvesting) • Biometrical school – Using crosses to predict average performance under inbreeding or crossing or response to selection – Better management of G x E 79 Dr. Zekeria Yusuf (PhD)

80. Modern Breeding Tools Increase of breeding effectiveness and efficiency In vitro culture Genomic tools Genomic engineering

81. Classic/ traditional tools Emasculation Hybidization Wide crossing Selection Chromosome counting Chromosome doubling Male sterility Triploidy Linkage analysis Statistical tools Advanced tools Mutagenesis Tissue culture Haploidy In situ hybridization Molecular markers Marker-assisted selection DNA sequencing Plant genomic analysis Bioinformatics Microarray analysis Primer design Plant transformation 81 Dr. Zekeria Yusuf (PhD)

82. Plant Breeding Methods Conventional breeding Mutation or c rossing to introduce variability Selection based on morphological characteres Growth of selected seeds Challenge : reduce the time needed to complete a breeding program

83. Plant Breeding Methods Conventional Methods: 1.Plant introduction 2. Pureline selection 3. Mass selection 4. Pedigree method 5. Bulk method 6. Single Seed descent method 7. Back cross method 8. Hetrosis breeding Modern Methods 1. Mutation breeding 2. Polyploidy breeding 3. Transgenic breeding 4. Molecular breeding 83 Dr. Zekeria Yusuf (PhD)

84. Symbols for basic crosses F: The symbol F (for filial) denotes the progeny of a cross between two parents. Ⓧ : The symbol is the notation for selfing . S: The S notation is also used with numeric subscripts. In one usage S0 = F1 ; another system indicates S0 = F2 . 84 Dr. Zekeria Yusuf (PhD)

85. Modern tools Used in Plant Breeding • Molecular markers: – Initially low density for QTL mapping, introgression of major genes into elite germplasm – With high-density markers, association mapping and MAS/genomic selection • New statistical tools: – Mixed model methods – Bayesian approaches to handle high-dimensional data sets – New methods to deal with G x E • Other technologies: – Better standardization of field sites ( laser-tilled fields, GPS, better micro- and macro-environmental measurements ) – High throughput phenotypic scoring – DH (double haploid) lines 85 Dr. Zekeria Yusuf (PhD)

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87. Integrated Approaches • How do we best combine the rich history of quantitative genetics and classical plant breeding with the new tools from genomics and other advances? • Key: Quantitative genetics has all of the machinery needed to fully incorporate these new sources of information 87 Dr. Zekeria Yusuf (PhD)

88. Basic steps in plant breeding Objective Germplasm Selection Evaluation 88 Dr. Zekeria Yusuf (PhD)

89. Activities in plant breeding: 1. Creation of variation: variation means differences among individuals of a population or species for a specific character. Genetic variation is the source of raw material for selection. These are heritable and are transmitted from one generation to other. Such variation is useful in selection. Success of a breeding program usually depends on the desired genetic variation . It can be done in following ways i.e. domestication, germplasm collection, plant introduction, hybridization, polyploidy, mutation, somaclonal variation and genetic engineering. 89 Dr. Zekeria Yusuf (PhD)

90. Activities in plant breeding…. 2. Selection: During selection, the individual plant or group of plants having the desired characters are picked up from a population eliminating the undesirable ones. Those plants are selected which are looking promising for the character on thee basis of phenotype. The selected plants are then allowed to grow for setting their seeds. Seeds are selected and again a new crop is developed. This process is repeated again and again till the desired result is achieved. Selection acts on the genetic variation present in a population and produces a new population with improved characters. 90 Dr. Zekeria Yusuf (PhD)

91. Activities in plant breeding…. 3. Evaluation: The newly selected lines/strains/populations are tested for yield and other traits and their performance is compared with existing best varieties called Checks. If the new lines/strain/population shows superior performance to the checks, it is released and notified as a new variety. 4. Multiplication: This step concerns with large scale certified seed production of the released and notified variety. 5. Distribution: Certified seed is ultimately sold to the farmers who use it for commercial crop cultivation. 91 Dr. Zekeria Yusuf (PhD)

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93. Scope of plant breeding From times immemorial, the plant breeding has been helping the mankind. With knowledge of classical genetics, number of varieties have been evolved in different crop plants. Since the population is increasing at an alarming rate, there is need to strengthened the food production which is serious challenge to those scientists concerned with agriculture. Advances in molecular biology have sharpened the tools of the breeders, and brighten the prospects of confidence to serve the humanity. The application of biotechnology to field crop has already led to the field testing of genetically modified crop plants. Genetically engineered rice, maize, soybean, cotton, oilseeds rape, sugar beet and alfalfa cultivars are expected to be commercialized before the close of 20th century. Genes from varied organisms may be expected to boost the performance of crops especially with regard to their resistance to biotic and abiotic stresses. In addition, crop plants are likely to be cultivated for recovery of valuable compounds like pharmaceuticals produced by genes introduced into them through genetic engineering. It may be pointed out that in Europe hirudin , an anti-thrombin protein is already being produced from transgenic Brassica 93 Dr. Zekeria Yusuf (PhD)

94. What is Germplasm ? Germplasm broadly refer to the hereditary material (total content of gene)transmitted to the offspring through germ cell. It can also be described as a collection of genetic resources for an organism. • For plants, the germplasm may be stored as a seed ,stem, Callus, Whole plant in nurseries . • In case of animals- Genes, Body parts stored in gene bank/ cryobank . Germplasm provide the raw material (genes) which the breeder uses to develop commercial crop varieties. 94 Dr. Zekeria Yusuf (PhD)

95. Sources of germplasm for plant breeding Germplasm may be classified into five major types – advanced (elite) germplasm , improved germplasm (cultivars and varieties), landraces, wild or weedy relatives, and Genetic stocks. The major sources of variability for plant breeders may also be categorized into three broad groups – domesticated plants, undomesticated plants, and other species or genera. 95 Dr. Zekeria Yusuf (PhD)

96. 1. Undomesticated plants When desired genes are not found in domesticated cultivars, plant breeders may seek them from wild populations. When wild plants are used in crosses, they may introduce wild traits that have an advantage for survival in the wild (e.g., hard seed coat, shattering, indeterminacy) but are undesirable in modern cultivation. These undesirable traits have been selected against through the process of domestication. Wild germplasms have been used as donors of several important disease- and insect resistance genes and genes for adaptation to stressful environments. The cultivated tomato has benefited from such introgression by crossing with a variety of wild Licopersicon spp. Other species such as potato, sunflower, and rice have benefited from wide crosses. In horticulture, various wild relatives of cultivated plants may be used as rootstock in grafting (e.g., citrus, grape) to allow cultivation of the plant in various adverse soil and climatic conditions. 96 Dr. Zekeria Yusuf (PhD)

97. 2. Domesticated plants Domesticated plants are those plant materials that have been s ubjected to some form of human selection and are grown for food or other uses. There are various types of such material: 1. Commercial cultivars: There are two forms of this material – c urrent cultivars and retired or obsolete cultivars . These are products of formal plant breeding for specific objectives. It is expected that such genotypes would have superior gene combinations, be adapted to a growing area, and have a generally good performance. The obsolete cultivars were taken out of commercial production because they may have suffered a set back (e.g., susceptible to disease) or higher performing cultivars were developed to replace them. If desirable parents are found in commercial cultivars, the breeder has a head start on breeding since most of the gene combinations would already be desirable and adapted to the production environment. 97 Dr. Zekeria Yusuf (PhD)

98. 2. Breeding materials Ongoing or more established breeding programs maintain variability from previous projects. These intermediate breeding products are usually genetically narrow-based because they originate from a small number of genotypes or populations. For example, a breeder may release one genotype as a commercial cultivar after yield tests. Many of the genotypes that made it to the final stage or have unique traits will be retained as breeding materials to be considered in future projects. Similarly, genotypes with unique combinations may be retained. 98 Dr. Zekeria Yusuf (PhD)

99. 3. Landraces Landraces are farmer-developed and maintained cultivars. They are developed over very long periods of time and have coadapted gene complexes. They are adapted to the growing region and are often highly heterogeneous. Landraces are robust, having developed resistance to the environmental stresses in their areas of adaptation. They are adapted to unfavorable conditions and produce low but relatively stable performance. Landraces, hence, characterize subsistence agriculture. They may be used as starting material in mass selection or pure-line breeding projects. 99 Dr. Zekeria Yusuf (PhD)

100. 4. Plant introductions The plant breeder may import new, unadapted genotypes from outside the production region, usually from another country (called plant introductions). These new materials may be evaluated and adapted to new production regions as new cultivars, or used as parents for crossing in breeding projects. 100 Dr. Zekeria Yusuf (PhD)

101. Plant introductions… Plant introduction is the process of importing new plants or cultivars of well-established plants from the area of their adaptation to another area where their potential is evaluated for suitability for agricultural or horticultural use. First, the germplasm to be introduced is processed through a plant quarantine station at the entry port, to ensure that no pest and diseases are introduced along with the desired material. Once this is accomplished, the material is released to the researcher for evaluation in the field for adaptation. The fundamental process of plant introductions as a plant breeding approach is acclimatization. The inherent genetic variation in the introduced germplasm serves as the raw material for adaptation to the new environment, enabling the breeder to select superior performers to form the new cultivar. 101 Dr. Zekeria Yusuf (PhD)

102. Plant introductions… When the plant introduction is commercially usable as introduced without any modification, it is called a primary introduction. However, more often than not, the breeder makes selections from the variable population, or uses the plant introduction as a parent in crosses. The products of such efforts are called secondary introductions. Some plant introductions may not be useful as cultivars in the new environment. However, they may be useful in breeding programs for specific genes they carry. Many diseases, plant stature, compositional traits, and genes for environmental stresses have been introduced by plant breeders. As a plant breeding method, plant introductions have had a significant impact on world food and agriculture. 102 Dr. Zekeria Yusuf (PhD)

103. 5. Genetic stock Genetic stock: consists of products of specialized genetic manipulations by researchers (e.g., by using mutagenesis to generate various chromosomal and genomic mutants). 6. Other species and genera: Gene transfer via crossing requires that the parents be cross-compatible or cross-fertile. Crossing involving parents from within a species is usually successful and unproblematic. However, as the parents become more genetically divergent, crossing (wide crosses) is less successful, often requiring special techniques (e.g., embryo rescue) for intervening in the process in order to obtain a viable plant. Sometimes, related species may be crossed with little difficulty. 103 Dr. Zekeria Yusuf (PhD)

104. Germplasm enhancement There are occasions when breeders are compelled to look beyond the advanced germplasm pool to find desirable genes. T The desired genes may reside in unadapted gene pools. Breeders are frequently reluctant to use such materials because the desired genes are often associated with undesirable effects ( unadapted , unreproductive , yield reducing factors). Hence, these exotic materials often cannot be used directly in cultivar development. Instead, the materials are gradually introduced into the cultivar development program through crossing and selecting for intermediates with new traits , while maintaining a great amount of the adapted traits. To use wild germplasm , the unadapted material is put through a preliminary breeding program to transfer the desirable genes into adapted genetic backgrounds. The process of the initial introgression of a trait from an undomesticated source (wild) or agronomically inferior source, to a domesticated or adapted genotype is called prebreeding or germplasm enhancement. 104 Dr. Zekeria Yusuf (PhD)

105. The major uses of germplasm enhancement may be summarized as follows: 1. Preventions of genetic uniformity and the consequences of genetic vulnerability. 2. Potential crop yield augmentation. History teaches us that some of the dramatic yield increases in major world food crops, such as rice, wheat, and sorghum, were accomplished through introgression of unadapted genes (e.g., dwarf genes). 3. Introduction of new quality traits (e.g., starch, protein). 4. Introduction of disease- and insect-resistance genes. 5. Introduction of environment-resistance genes (e.g., drought resistance). Prebreeding can be expensive to conduct and time consuming as well. 105 Dr. Zekeria Yusuf (PhD)

106. Genetic vulnerability Genetic vulnerability is brought about largely by the manner in which breeders go about developing new and improved cultivars for modern society. Genetic vulnerability is a term used to indicate the genetic homogeneity and uniformity of a group of plants that predisposes it to susceptibility to a pest, pathogen, or environmental hazard of large-scale proportions . A case in point is the 1970 epidemic of southern leaf blight ( Helminthosporium maydis ) in the USA that devastated the corn industry. This genetic vulnerability in corn was attributed to uniformity in the genetic background in corn stemming from the widespread use of T-cytoplasm in corn hybrid seed production. Genetic uniformity per se is not necessarily the culprit in vulnerability of crops. In fact, both producers and consumers sometimes desire and seek uniformity in some agronomic traits. The key issue is commonality of genetic factors. Genetically dissimilar crops can share a trait that is simply inherited and that predisposes them to susceptibility to an adverse biotic or abiotic factor. A case in point is the chestnut blight ( Cryphonectria parasitica ) epidemic that occurred in the USA in which different species of the plant were affected. 106 Dr. Zekeria Yusuf (PhD)

107. Conservation of Plant Genetic Resources Why conserve plant genetic resources? There are several reasons why plant genetic resources should be conserved: 1. Plant germplasm is exploited for food, fiber, feed, fuel, and medicines by agriculture, industry, and forestry. 2. As a natural resource, germplasm is a depletable resource. 3. Without genetic diversity, plant breeding cannot be conducted. 4. Genetic diversity determines the boundaries of crop productivity and survival. 5. As previously indicated, variability is the life blood of plant breeding. As society evolves, its needs will keep changing. Similarly, new environmental challenges might arise (e.g., new diseases, abiotic stresses) for which new variability might be needed for plant improvement. 107 Dr. Zekeria Yusuf (PhD)

108. Conservation of Plant Genetic Resources… When a genotype is unable to respond fully to the cultural environment, as well as to resist unfavorable conditions thereof, crop productivity diminishes. The natural pools of plant genetic resources are under attack from the activities of modern society – urbanization, indiscriminate burning, and the clearing of virgin land for farming, to name a few. These and other activities erode genetic diversity in wild populations. Consequently, The actions of plant breeders also contribute to genetic erosion as previously indicated. High-yielding, narrow genetic-based cultivars are penetrating crop production systems all over the world, displacing the indigenous high-variability landrace cultivars. Genetic erosion Genetic erosion can be defined as the decline in genetic variation in cultivated or natural populations largely through the action of humans. Loss of genetic variation may be caused by natural factors, and by the actions of crop producers, plant breeders, curators of germplasm repositories, and others in society at large. 1. Natural factors: Genetic diversity can be lost through natural disasters such as large-scale floods, wild fires, and severe and prolonged drought. These events are beyond the control of humans. 108 Dr. Zekeria Yusuf (PhD)

109. 2. Action of farmers: Right from the beginnings of agriculture, farmers have engaged in activities that promote genetic erosion. These include clearing of virgin land in, especially, germplasm -rich tropical forests, and the choice of planting material (narrow genetic-based cultivars). Farmers, especially in developed economies, primarily grow improved seed, having replaced most or all landraces with these superior cultivars. Also, monoculture tends to narrow genetic diversity as large tracts of land are planted to uniform cultivars. Extending grazing lands into wild habitats by livestock farmers, destroys wild species and wild germplasm resources. 3. Action of breeders: Farmers plant what breeders develop. Some methods used for breeding (e.g., pure lines, single cross, multilines ) promote uniformity and a narrower genetic base. When breeders find superior germplasm , the tendency is to use it as much as possible in cultivar development. For example: in soybean, most of the modern cultivars in the USA can be traced back to about half a dozen parents. This practice causes severe reduction in genetic diversity. 109 Dr. Zekeria Yusuf (PhD)

110. Problems with germplasm conservation In spite of good efforts by curators of germplasm repositories to collect and conserve diversity, there are several ways in which diversity in their custody may be lost. The most obvious loss of diversity is attributed to human errors in the maintenance process (e.g., improper storage of materials leading to loss of variability). Also, when germplasm is planted in the field, natural selection pressure may eliminate some unadapted genotypes . Also, there could be spontaneous mutations that can alter the variability in natural populations. Hybridization as well as genetic drift incidences in small populations are also consequences of periodic multiplication of the germplasm holdings by curators. 110 Dr. Zekeria Yusuf (PhD)

111. Germplasm collection Planned collections ( germplasm explorations or expeditions) are conducted by experts to regions of plant origin. These trips are often multidisciplinary, comprising members with expertise in botany, ecology, pathology, population genetics, and plant breeding. Familiarity with the species of interest and the culture of the regions to be explored are advantageous. Most of the materials collected are seeds, even though whole plants and vegetative parts (e.g., bulbs, tubers, cuttings , etc.) and even pollen may be collected. Because only a small amount of material is collected, sampling for representativeness of the population’s natural variability is critical in the collection process, in order to obtain the maximum possible amount of genetic diversity. For some species whose seed is prone to rapid deterioration, or are bulky to transport, in vitro techniques may be available to extract small samples from the parent source. Collectors should bear in mind that the value of the germplasm may not be immediately discernible. Materials should not be avoided for lack of obvious agronomically desirable properties. It takes time to discover the full potential of germplasm . 111 Dr. Zekeria Yusuf (PhD)

112. Germplasm collection… Seed materials vary in viability characteristics. These have to be taken into account during germplasm collection, transportation, and maintenance in repositories. Based on viability, seed may be classified into two main groups – orthodox and recalcitrant seed: Orthodox seeds: These are seeds that can prolong their viability under reduced moisture content and low temperature in storage. Examples include cereals, pulses, and oil seed. Of these, some have superior (e.g., okra) while others have poor (e.g., soybean) viability under reduced moisture cold storage. 2. Recalcitrant seeds: Low temperature and decreased moisture content are intolerable to these seeds ( e.g.,coconut , coffee, cocoa). In vitro techniques might be beneficial to these species for long-term maintenance. 112 Dr. Zekeria Yusuf (PhD)

113. Germplasm collection… The conditions of storage differ depending on the mode of reproduction of the species: 1. Seed propagated species: these seeds are first dried to about 5% moisture content and then usually placed in hermetically sealed moisture-proof containers before storage. Vegetatively propagated species: these materials may be maintained as full plants for long periods of time in field gene banks, nature reserves, or botanical gardens. Alternatively, cuttings and other vegetative parts may be conserved for a short period of time under moderately low temperature and humidity. For long-term storage, in vitro technology is used. 113 Dr. Zekeria Yusuf (PhD)

114. Types of plant germplasm collections There are four types of plant genetic resources maintained by germplasm repositories – base collections, backup collections, active collections, and breeders’ or working collections. These categorizations are only approximate since one group can fulfill multiple functions. 1. Base collections: These collections are not intended for distribution to researchers, but are maintained in long-term storage systems. They are the most comprehensive collections of the genetic variability of species. Entries are maintained in the original form. Storage conditions are low humidity at subfreezing temperatures (−10 to − 18°C ) or cryogenic (−150 to − 196°C ), depending on the species. Materials may be stored for many decades under proper conditions. 2. Backup collections: The purpose of backup collections is to supplement the base selection. In case of a disaster at a center responsible for a base collection, a duplicate collection is available as insurance. In the USA, the National Seed Storage Laboratory at Fort Collins, Colorado, is a backup collection center for portions of the accessions of the Centro Internationale de Mejoramiento de Maiz y Trigo ( CIMMYT ) and the International Rice Research Institute ( IRRI ). 114 Dr. Zekeria Yusuf (PhD)

115. 3. Active collections: Base and backup collections of germplasm are designed for long-term unperturbed storage. Active collections usually comprise the same materials as in base collections, however, the materials in active collections are available for distribution to plant breeders or other patrons upon request. They are stored at 0°C and about 8% moisture content, and remain viable for about 10– 15years . To meet this obligation, curators of active collections at germplasm banks must increase the amount of germplasm available to fill requests expeditiously. Because the accessions are more frequently increased through field multiplication, the genetic integrity of the accession may be jeopardized. 4. Working or breeders’ collections: Breeders’ collections are primarily composed of elite germplasm that is adapted. They also include enhanced breeding stocks with unique alleles for introgression into these adapted materials. In these times of genetic engineering, breeders’ collections include products of rDNA research that can be used as parents in breeding programs. 115 Dr. Zekeria Yusuf (PhD)

116. Managing plant genetic resources The key activities of curators of germplasm banks include: Regeneration of accessions, Characterization, Evaluation, Monitoring seed viability and genetic integrity during storage, & Maintaining redundancy among collections. Germplasm banks receive new materials on a regular basis. These materials must be properly managed so as to encourage and facilitate their use by plant breeders and other researchers. 116 Dr. Zekeria Yusuf (PhD)

117. 1. Periodic Regeneration The regeneration of seed depends on the life cycle and breeding system of the species as well as cost of the activity. To keep costs to a minimum and to reduce loss of genetic integrity, it is best to keep regeneration and multiplication to a bare minimum. It is a good strategy to make the first multiplication extensive so that ample original seed is available for depositing in the base and duplicate or active collections. A major threat to genetic integrity of accessions during regeneration is contamination (from outcrossing or accidental migration),which can change the genetic structure. Other factors include differential survival of alleles or genotypes within the accession, and random drift. The isolation of accessions during regeneration is critical, especially in cross-pollinated species, to maintaining genetic integrity. This is achieved through proper spacing, caging, covering with bags, hand pollination, and other techniques. Regeneration of wild species is problematic because of high seed dormancy, seed shattering, high variability in flowering time, and low seed production. Some species have special environmental requirements (e.g., photoperiod, vernalization ) and hence it is best to rejuvenate plants under conditions similar to those in the places of their origin, to prevent selection effect, which can eliminate certain alleles. Dr. Zekeria Yusuf (PhD) 117

118. 2. Characterization: Curators of germplasm banks characterize their accessions, an activity that entails a systematic recording of selected traits of an accession. Traditionally, these data are limited to highly heritable morphological and agronomic traits. However, with the availability of molecular techniques, some germplasm banks have embarked upon molecular characterization of their holdings. For example, CIMMYT has used the simple sequence repeat ( SSR ) marker system for characterizing the maize germplasm in their holding. Passport data are included in germplasm characterization. These data include an accession number, scientific name, collection site (country, village), source (wild, market), geography of the location, and any disease and insect pests. To facilitate data entry and retrieval, characterization includes the use of descriptors. These are specific pieces of information on plant or geographic factors that pertain to the plant collection . The International Plant Genetic Resources Institute ( IPGRI ) has prescribed guidelines for the categories of these descriptors. Descriptors have been standardized for some species such as rice. 118 Dr. Zekeria Yusuf (PhD)

119. 3. Evaluation: Genetic diversity is not usable without proper evaluation. Preliminary evaluation consists of readily observable traits . Full evaluations are more involved and may include obtaining data on cytogenetics , evolution, physiology, and agronomy. More detailed evaluation is often done outside of the domain of the germplasm bank by various breeders and researchers using the specific plant traits such as disease resistance, productivity, and quality of product are important pieces of information for plant breeders. Without some basic information of the value of the accession, users will not be able to make proper requests and receive the most useful materials for their work. 119 Dr. Zekeria Yusuf (PhD)

120. 4. Monitoring seed viability and genetic integrity: During storage, vigor tests should be conducted at appropriate intervals to ensure that seed viability remains high. During these tests, abnormal seedlings may indicate the presence of mutations. 5. Exchange: The ultimate goal of germplasm collection, rejuvenation, characterization, and evaluation is to make available and facilitate the use of germplasm . There are various computer-based genetic-resource documentation systems worldwide, some of which are crop-specific. These systems allow breeders to rapidly search and request germplasm information. There are various laws regarding, especially, international exchange of germplasm . Apart from quarantine laws, various inspections and testing facilities are needed at the checkpoint of germplasm . 120 Dr. Zekeria Yusuf (PhD)

121. 6. Germplasm storage technologies: Once collected, germplasm is maintained in the most appropriate form by the gene bank with storage responsibilities for the materials. Plant germplasm may be stored in the form of pollen, seed, or plant tissue. Woody ornamental species may be maintained as living plants. Indoor maintenance is done under cold storage conditions, with temperatures ranging from −18 to − 196°C . i . Seed storage: Seeds are dried to the appropriate moisture content before being placing in seed envelopes. These envelopes are then arranged in trays that are placed on shelves in the storage room. The storage room is maintained at − 18°C , a temperature that will keep most seeds viable for up to 20 years or more. The curator of the laboratory and the staff periodically sample seeds of each accession to conduct a germination test. When germination falls below a certain predetermined level, the accession is regrown to obtain fresh seed. 121 Dr. Zekeria Yusuf (PhD)

122. ii. Field growing: Accessions are regrown to obtain fresh seed or to increase existing supplies (after filling orders by scientists and other clients). To keep the genetic purity, the accessions are grown in isolation, each plant covered with a cotton bag to keep foreign sources of pollen out and also to ensure self-pollination. iii. Cryopreservation: Cryopreservation or freeze-preservation is the storage of materials at extremely low temperatures of between −150 to − 196°C in liquid nitrogen. Plant cells, tissue, or other vegetative material may be stored this way for a long time without loosing regenerative capacity. Whereas seed may also be stored by this method, cryopreservation is reserved especially for vegetatively propagated species that need to be maintained as living plants. Shoot tip cultures are obtained from the material to be stored and protected by dipping in a cryoprotectant (e.g., a mixture of sugar and polyethylene glycol plus dimethylsulfoxide ). 122 Dr. Zekeria Yusuf (PhD)

123. iv. In vitro storage: Germplasm of vegetatively propagated crops is normally stored and distributed to users in vegetative forms such as tubers, corms, rhizomes, and cuttings. However, it is laborious and expensive to maintain plants in these forms. In vitro germplasm storage usually involves tissue culture . There are several types of tissue culture systems (suspension cells, callus, meristematic tissues). To use suspension cells and callus materials, there must be an established system of regeneration of full plants from these systems, something that is not available for all plant species yet. Consequently, meristem cultures are favored for in vitro storage because they are more stable. The tissue culture material may be stored using the method of slow growth (chemicals are applied to retard the culture temperature) or cryopreservation. v. Molecular conservation: The advent of biotechnology has made it possible for researchers to sequence DNA of organisms. These sequences can be searched for genes at the molecular level. Specific genes can be isolated by cloning and used in developing transgenic products. 123 Dr. Zekeria Yusuf (PhD)

124. What is germplasm conservation? Plant germplasm is the genetic source material used by the plant breeders to develop new cultivars. They may include : • Seeds Other plant propagules such as Leaf Stem Pollen Cultured cells Which can be grown into mature plant? Germplasm provide the raw material (genes) which the breeder used to develop commercial crop varieties. 124 Dr. Zekeria Yusuf (PhD)

125. Need for Conservation of plant Germplasm •• Storage of Economically important, endangered, rare species and make them available when needed. • The conventional methods of storage failed to prevent losses caused due to various reasons. • Human dependence on plant species for food and many different uses. E.g. Basic food crops, building materials, oils, lubricants, rubber & other latexes, resins, waxes, perfumes, dyes fibres and medicines. • Species extinction and many others are threatened and endangered – deforestation. • Great diversity of plants is needed to keep the various natural ecosystems functioning stably– interactions between species. • Aesthetic value of natural ecosystems and the diversity of plant species. 125 Dr. Zekeria Yusuf (PhD)

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128. In-situ Preservation Preservation of the germplasm in their natural habitat The conservation of domesticated and cultivated species in the farm or in the surroundings. However, there is a heavy loss or decline of species, populations and ecosystem composition, which can lead to a loss of biodiversity, due to habitat destruction and the transformations of these natural environments; therefore, in situ methods alone are insufficient for saving endangered species. 128 Dr. Zekeria Yusuf (PhD)

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130. Ex-situ preservation 1. To maintain the biological material outside their natural habitats. 2. Storage in seed banks, field gene collections, in vitro collections and botanical gardens 3. Ex situ conservation is a viable way for saving plants from extinction, and in some cases, it is the only possible strategy to conserve certain species 4. In vitro conservation is especially important for vegetatively propagated and for non-orthodox seed plant species 130 Dr. Zekeria Yusuf (PhD)

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133. Disadvantages of Ex-situ Conservation • Some plants do not produce fertile seeds. • Loss of seed viability • Seed destruction by pests, etc. • Poor germination rate. • This is only useful for seed propagating plants. • It’s a costly process. 133 Dr. Zekeria Yusuf (PhD)

134. In vitro method for germplasm conservation In vitro method employing shoots, meristems and embryos are ideally suited for the conservation of germplasm . The plant with recalcitrant seeds and genetically engineered can also be preserved by this in vitro approach. There are several advantages associated with in vitro germplasm conservation Large quantities of material can be preserved in small space The germplasm preserved can be maintained in an environment free from pathogens. It can be protected against the nature’s hazards From the germplasm stock large number of plants can be obtained whenever needed. 134 Dr. Zekeria Yusuf (PhD)

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136. CRYOPRESERVATION Cryopreservation (Greek, krayos -frost) literally mean in the frozen state. The principal involved in cryopreservation to bring the plant cells and tissue cultures to a zero metabolism or nondividing state by reducing the temperature in the presence of caryoprotectants . Cryopreservation broadly means the storage of germplam at very low temperature. Over solid carbon dioxide (at 79 o C ) Low temperature deep freezers (at - 80 o C ) In liquid nitrogen (at - 196 o C ) •Among these the most commonly used cryopreservation is by employing liquid nitrogen. At the temperature of liquid nitrogen (- 196 o C ), the cell stay in a completely inactive state and thus can be conserved for longer period. In fact cryopreservation has been successfully applied for germplasm conservation . Plant species e.g. rice, wheat, peanut, sugarcane ,coconut. 136 Dr. Zekeria Yusuf (PhD)

137. Principle of Cryopreservation Cryopreservation is the only technique that ensures the safe and cost-efficient long-term conservation of various categories of plants, including non-orthodox seed species, vegetatively propagated plants, rare and endangered species and biotechnology products. Storage of Biomaterial at ultra low temperature by means of slow freezing. In all cryopreservation processes, water removal plays a central role in preventing freezing injury and in maintaining post-thaw viability of cryopreserved material. There are two types of cryopreservation protocols that basically differ in their physical mechanisms: 1. Classical cryopreservation 2. Vitrification 137 Dr. Zekeria Yusuf (PhD)

138. Technique of cryopreservation The cryopreservation of plant cell culture followed the regeneration of plants broadly involves the following stages 1. Development of sterile tissue culture. 2. Addition of cryoprotectant and pretreatment 3. Freezing 4. Storage 5. Thawing 6. Reculture 7. Measurement of survival/viability 8. Plant regeneration 138 Dr. Zekeria Yusuf (PhD)

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141. Classical cryopreservation • In this procedure, cooling is performed in the presence of ice. • It involves cryoprotection by using different cryoprotective solutions combined or not with pregrowth of material and followed by slow cooling (0.5– 2.0°C /min) to a determined prefreezing temperature (usually around − 40°C ),rapid immersion of samples in liquid nitrogen, storage, rapid thawing and recovery. • They are generally operationally complex, since they require the use of sophisticated and expensive programmable freezers. • Cryopreservation following classical protocols induces a freeze dehydration process using a slow freezing regime. As temperature decrease slowly, ice is initially formed in the extracellular solution and this external crystallization promotes the efflux of water from the cytoplasm and vacuoles to the outside of the cells where it finally freezes. • Therefore, cell dehydration will depend on the cooling rate and the prefreezing temperature set up before immersion of samples to liquid nitrogen. • Classical cryopreservation techniques have been successfully applied to undifferentiated culture systems of different plant species, such as cell suspensions and calluses. 141 Dr. Zekeria Yusuf (PhD)

142. Vitrification • In this procedure, cooling normally takes place without ice formation • The process where formation of ice cannot take place because of the Concentrated aqueous solution which permit ice crystal nucleation. Instead, water solidifies into an amorphous ‘glassy’ state. • The vitrification -based procedures involve cell dehydration prior to cooling by exposure of samples to highly concentrated cryoprotective media (usually called plant vitrification solutions, PVS ) and/or by air desiccation. 142 Dr. Zekeria Yusuf (PhD)

143. Vitrification … Cooling rate may be rapid or ultra-rapid, depending on how samples are immersed into liquid nitrogen. • Vitrification per se is a physical process, defined as the transition of the liquid phase to an amorphous glassy solid at the glass transition ( Tg ) temperature . • This glass may contribute to preventing tissue collapse, solute concentration and pH alterations during dehydration. • Therefore, the freeze-induced dehydration step characteristic of classical procedures is eliminated and the slow freezing regime is replaced by a rapid or ultra-rapid cooling process, characteristic of the vitrification -based protocols. 143 Dr. Zekeria Yusuf (PhD)

144. 1. Development of sterile tissue culture The selection of plant species and the tissue with particular reference to the morphological and physiological characters largely influence the ability of the explants to survive in cryopreservation . Any tissue from a plant can be used for cryopreservation e.g. meristems , embryos, endosperm, ovules, seeds, culture plants. 144 Dr. Zekeria Yusuf (PhD)

145. 2. Addition of cryoprotectant Cryoprotectant are the compound that can be prevent the damage caused to cells by freezing or thawing. There are several cryoprotectant which include ( DMSO ), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide . Among these DMSO , sucrose & glycerol are most widely used. 145 Dr. Zekeria Yusuf (PhD)

146. 3. Freezing The sensitivity of the cell to low temperature is variable and largely depends on the plant species. Four different types of freezing method are used: 1. Slow freezing method: the tissue is slowly frozen at 0.5- 5°C /min from 0°C to - 100°C , and then transferred to liquid nitrogen. 2. Rapid freezing method: decrease in temperature up to -300 to - 1000°C . 3. Stepwise freezing method: intermediate temperature for 30 min. and rapidly cool. 4. Dry freezing method: reported that non-germinated dry seeds can survive freezing at low temperature in contrast to water imbibing seeds which are susceptible to cryogenic injuries. 146 Dr. Zekeria Yusuf (PhD)

147. 4 : Storage Maintenance of the frozen cultures at the specific temperature is as important as freezing . In general the frozen cells/tissues are kept for storage at temperatures in the range of -72 to - 196°C . Storage is ideally done in liquid nitrogen refrigerator – at 150°C in the vapour phase, or at - 196°C in the liquid phase. The ultimate objective of storage is to stop all the cellular metabolic activities and maintain their viability. For long term storage temperature at - 196°C in liquid nitrogen is ideal. 147 Dr. Zekeria Yusuf (PhD)

148. 5 : Thawing Thawing is usually carried out by plunging the frozen samples in ampoules into a warm water (temp 37 – 45°C ) bath with vigorous swirling. By this approach, rapid thawing (at the rate of 500- 750°C min¯¹ ) occurs, and this protects the cells from the damaging effects ice crystal formation. As the thawing occurs (ice completely melts) the ampoules are quickly transferred to a water bath at temperature 20- 25°C . This transfer is necessary since the cells get damaged if left for long in warm (37- 45°C ) water bath. 148 Dr. Zekeria Yusuf (PhD)

149. 6. Reculture : In general thawned germplasm is washed several times to remove cryoprotectant . The material is then recultured in a fresh media. 7. Plant regeneration: The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant. For appropriate plant growth and regeneration, the cryopreserved cell/tissues have to be carefully nursed, grown. Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is often necessary for successful plant regeneration. 149 Dr. Zekeria Yusuf (PhD)

150. Applications of germplasm conservation Plant materials (cell/tissue) of several species can be cryopreserved and maintained for several years, and used as and when needed. Cryopreservation is an ideal method for long term conservation of cell culture which produce secondary metabolites e.g. medicines Disease (pathogen) free plant material can be frozen and propagated whenever required. Recalcitrant seeds can be maintained for long. Conservation of somaclonal and gametoclonal variation in culture. Plant material from endangered species can be conserved. Cryopreservation is a good method for the selection of cold resistant mutant cell lines which could develop into frost resistant plant . • Disease free plants can be conserved and propagated. • Recalcitrant seeds can be maintained for long time. • Endangered species can be maintained. • Pollens can be maintained to increase longitivity . • Rare germplasm and other genetic manipulations can be stored. 150 Dr. Zekeria Yusuf (PhD)

151. Limitations of germplasm conservation The expensive equipment needed to provide controlled and variable rates of cooling/warming temperatures can however be a limitation in the application of in vitro technology for large scale germplasm conservation. Formation of ice crystal inside the cell should be prevented as they cause injury to the cell. Sometimes certain solutes from the cell leak out during freezing. Cryoprotectant also effect the viability of cells . 151 Dr. Zekeria Yusuf (PhD)

152. Concept of gene pools of cultivated crops Harlan and de Wet proposed a categorization of gene pools of cultivated crops according to the feasibility of gene transfer or gene flow from those species to the crop species. Three categories were defined, primary, secondary, and tertiary gene pools: 1. Primary gene pool (GP1). GP1 consists of biological species that can be intercrossed easily ( interfertile ) without any problems with fertility of the progeny. That is, there is no restriction to gene exchange between members of the group. This group may contain both cultivated and wild progenitors of the species. 2. Secondary gene pool (GP2). Members of this gene pool include both cultivated and wild relatives of the crop species. They are more distantly related and have crossability problems. Nonetheless, crossing produces hybrids and derivatives that are sufficiently fertile to allow gene flow. GP2 species can cross with those in GP1, with some fertility of the F1, but more difficulty with success. 3. Tertiary gene pool (GP3). GP3 involves the outer limits of potential genetic resources. Gene transfer by hybridization between GP1 and GP3 is very problematic, resulting in lethality, sterility, and other abnormalities. To exploit germplasm from distant relatives, tools such as embryo rescue and bridge crossing may be used to nurture an embryo from a wide cross to a full plant and to obtain fertile plants. 152 Dr. Zekeria Yusuf (PhD)

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155. Seed Technology Seed technology is the creation and application of the knowledge on seed for its better usage in agriculture. Seed technology refers to methods or techniques used to maintain the quality of seed from harvest till it is germinated. Scope of Seed Technology 1. Seed technology encompasses all activities carried out to enhance storability, germinability , vigour and health of the seed. 2. Activities include harvesting, transporting, handling, storage, testing, grading, documentation, processing of seeds and germination of seeds. Classes of seed Nucleus or Basic Seed Breeder seed Foundation seed Certified seed 155 Haramaya University

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157. Nucleus or Basic Seed Nucleus seed (or basic seed) is the original or first seed (= propagating material ) of a variety available with the producing breeder or any other recognized breeder of the crop. This seed has 100% genetic & physical purity along with high standards of all other seed quality parameters . When a new variety is released there is very little seed. There may be only a handful of seed selected by the breeder from individual plants. This seed is the basis of a variety and is known as the Nucleus Stock. This nucleus stock must be managed with great care so that all seed produced from it remains true to the new variety. This is a most important step and is the responsibility of the plant breeder who developed the variety. The nucleus stock seed is not available to farmers. The next step in the chain from plant breeder to farmer is that the plant breeder develops Breeder Seed. 157 Haramaya University

158. BREEDER SEED Breeder seed is the seed of the highest purity of the new variety. It is produced by the breeder and provided by the breeder’s institution to agencies for further multiplication. If you are from a non-governmental organization (NGO) seed business or a private company that is producing seed, you may need to purchase breeder seed from a research institution. Breeder seed is the most expensive seed to buy. Breeder seed : seed or vegetative propagating material directly produced or controlled by the originating plant breeder or institution. Breeder seed provides the source for the increase of foundation seed. It is usually limited in quantity. Breeder seed is the progeny of the nucleus seed and is the source for foundation seed. Its production is directly controlled by the originating plant breeder who developed the variety, or any other institution or qualified breeder recognized by the authorities . 158 Haramaya University

159. FOUNDATION SEED Foundation seed is the seed produced from growing breeder seed. It is produced by trained officers of an agricultural station to national standards and handled to maintain the genetic purity of the variety. It may be produced by a government seed production farm or a private organization – this will depend on the regulations of the country. Foundation seed is less expensive than breeder seed. Also know as elite or basic seed. It is the direct increase form breeder seed. The genetic identity and purity of the variety is carefully maintained in foundation seed. Foundation seed is the source of certified seed. 159 Haramaya University

160. REGISTERED SEED Registered seed is produced from growing foundation seed. It is grown by selected farmers in a way that maintains genetic purity. Production has undergone field and seed inspections by Seed Inspectors to ensure conformity with standards. 160 Haramaya University

161. CERTIFIED SEED Certified seed is produced from growing foundation, registered or certified seed. It is grown by selected farmers to maintain sufficient varietal purity.  Production is subject to field and seed inspections prior to approval by the certifying agency. Harvest from this class is used for producing again. Certified seed is the seed, which is certified by a Seed Certification Agency Generally, it is known as the progeny of foundation seed and its production is so handled as to maintain specified genetic identity and purity standards as prescribed for the crop being certified . 161 Haramaya University

162. Requirements of Certified seed Seed has to meet certain rigid requirements before it can be certified for distribution. Seed must be of an improved variety released by either Central or State Variety Release Committee for general cultivation and notified by the Ministry of Agriculture. Genetic purity. Physical purity. Germination . Freedom from Weed seeds. Freedom from Diseases. Optimum Moisture Content. 162 Haramaya University

163. QUALITY DECLARED SEED Truthfully labeled seed or Quality Declared Seed  is produced from foundation, registered or certified seed. It is not subject to inspection by a certifying agency. As this seed is not inspected, its quality is dependent on the good reputation of the farmer who has grown the seed. His good name in the village is important. 163 Haramaya University

164. MODE OF REPRODUCTION Reproduction refers to the process by which living organisms produces offsprings of similar kind (species). In crop plants, the mode of reproduction is of two types: viz. 1) asexual reproduction & 2) sexual reproduction. Asexual reproduction refers to the multiplication of plants without the fusion of male and female gametes .It can occur either by vegetative plant parts or by vegetative embryos which develop without sexual fusion ( apomixis ). Thus asexual reproduction is of two types, viz., a) vegetative reproduction and (b) apomixis . Vegetative reproduction refers to multiplication of plants by means of various vegetative plant parts. Vegetative reproduction is again of two types, viz., a) natural vegetative reproduction and (b) artificial vegetative reproduction. Natural vegetative reproduction is the multiplication of certain plants by underground stems, sub aerial stems, roots and bulbils naturally. In some crop species, underground stems (a modified group of stems) give rise to new plants. 164 Dr. Zekeria Yusuf (PhD)

165. Artificial vegetative reproduction occurs by cuttings of stem and roots, and by layering, grafting etc. Stem cuttings : Sugarcane , grapes , roses etc. Root cuttings: Sweet potato, citrus, lemon, etc. Layering, grafting: Fruit and ornamental crops. Example for underground stems Rhizome: Turmeric , Ginger Tuber: Potato Corm: Colocasia Bulb: Garlic , onion Example for sub aerial stems Runner: Sweet potato , Strawberry Sucker: Banana Stolon : taro, passion flower Bulbils: Garlic 165 Dr. Zekeria Yusuf (PhD)

166. Apomixis refers to the development of seed without fertilization.The embryos are developed without fertilization. Apomixis is found in many crop species and is of two types based on nature. Reproduction in some species occurs only by apomixis . This apomixis is termed as obligate apomixis . But in some species sexual reproduction also occurs in addition to apomixis . Such apomixis is known as facultative apomixis . There are four types of apomixis based on origin: viz. 1) parthenogenesis, 2) apogamy , 3) apospory and 4) adventive embryony . Parthenogenesis refers to development of embryo from the egg cell without fertilization. Apogamy –when the embryo originates from either synergids or antipodal cells of the embryo sac it is called as apogamy . Apospory - In apospory , some diploid cells of ovule lying outside the embryo sac develops into another unreduced embryo sac through a series of mitotic divisions and without meiosis. The embryo then develops directly from the diploid egg cell of such an embryo sac without fertilization. Adventive embryony - The development of embryo directly from the diploid cells of ovule lying outside the embryo sac belonging to either nucellus or integuments is referred to as adventive embryony . It does not involve the production of another embryo sac. 166 Dr. Zekeria Yusuf (PhD)

167. Sexual reproduction: Reproduction by which embryo is developed by the fusion of male and female gamete is known as sexual reproduction. All the seed propagating species belong to this group. MODE OF POLLINATION The process by which pollen grains are transferred from anthers to stigma is referred to as pollination . Pollination is of two types, viz., 1) Autogamy or self pollination and 2) Allogamy or cross pollination. A. Autogamy Transfer of pollen grains from the anther to the stigma of same flower is known as autogamy or self pollination. Autogamy is the closest form of inbreeding. Autogamy leads to homozygosity . Such species develop homozygous balance & do not exhibit significant inbreeding depression. 167 Dr. Zekeria Yusuf (PhD)

168. There are several mechanisms which promote autogamy . 1. Bisexuality is the presence of male and female organs in the same flower. The presence of bisexual flowers is a must for self pollination. All the self pollinated plants have hermaphrodite flowers. Eg . Rice 2 . Homogamy Maturation of anthers and stigma of a flower at the same time is called homogamy which is essential for self-pollination Eg . Bhindi 3. Cleistogamy is when pollination and fertilization occur in an unopened flower bud. It ensures self pollination and prevents cross pollination. Eg . cowpea 4. Chasmogamy is the condition when flower opening occurs only after the completion of pollination. This also promotes self pollination. Eg . sesame. 5. Position of Anthers : When stigmas are surrounded by anthers self pollination is ensured. Eg . tomato and brinjal . In some legumes, the stamens and stigma are enclosed by the petals in such a way that self pollination is ensured. Eg . greengram , blackgram , soybean, chickpea and pea. 168 Dr. Zekeria Yusuf (PhD)

169. B. Allogamy The transfer of pollen grains from the anther of one plant to the stigma of another plant (cross pollination). Allogamy is the common form of out-breeding and leads to heterozygosity . Such species develop heterozygous balance and exhibit significant inbreeding depression on selfing . The conditions which promote allogamy are as follows : 1. Dicliny refers to unisexual flowers . This is of two types: viz. i ) monoecy and ii) dioecy . When male and female flowers are separate but present in the same plant, it is known as monoecy . In some crops, the male & female flowers are present in the same inflorescence such as in coconut, mango, castor & banana . In some cases, they are on separate inflorescence as in maize, cucurbits, cassava and rubber. When staminate and pistillate flowers are present on different plants, it is called dioecy as in papaya, nutmeg and date palm 169 Dr. Zekeria Yusuf (PhD)

170. 2. Dichogamy refers to the maturation of anthers and stigma of the same flowers at different times. Dichogamy promotes cross pollination even in the hermaphrodite species. Dichogamy is of two types: viz. i ) protogyny and ii) protandry . When pistil matures before anthers, it is called protogyny such as in black pepper and pearl millet. When anthers mature before pistil, it is known as protandry as in coconut and several other species. 170 Dr. Zekeria Yusuf (PhD)

171. 3. Heterostyly : When styles and filaments in a flower are of different lengths, it is called heterostyly . It promotes cross pollination, such as linseed. 4. Herkogamy : Hinderance to self-pollination due to some physical barriers such as presence of hyline membrane around the anther is known as herkogamy . Such membrane does not allow the dehiscence of pollen and prevents self-pollination such as in alfalfa. 5. Self incompatibility is the inability of fertile pollens to fertilize the same flower. It prevents self-pollination and promotes cross pollination. Self incompatibility is found in several crop species like Brassica , Radish, Nicotiana , and many grass species. It is of two types sporophytic and gametophytic . 171 Dr. Zekeria Yusuf (PhD)

172. 6 . Male sterility : i n some species, the pollen grains are non functional. Such condition is known as male sterility. It prevents self-pollination & promotes cross pollination. It is of three types: viz. genetic, cytoplasmic and cytoplasmic genetic. It is a useful tool in hybrid seed production. Self incompatibility and male sterility are the two genetic mechanisms favouring cross pollination. The mode of pollination plays an important role in plant breeding. It has impact on five important aspects : 1) gene action 2) genetic constitution 3) adaptability 4) genetic purity 5) transfer of genes. 172 Dr. Zekeria Yusuf (PhD)

173. Estimation of Genetic variability parameters 173 Dr. Zekeria Yusuf (PhD)

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175. VARIANCES AND COVARIANCES The variability present in a population is of polygenic nature and this polygenic variation is of three types 1) Phenotypic 2) Genotypic 3) Environmental The statistical procedure which separates (or) splits the total variation into different components is called analysis of variance (or) ANOVA. ANOVA is useful in estimating the different components of variance. It provides basis for the test of significance and it is carried out only with replicated data obtained from standard statistical experimental results. 175 Dr. Zekeria Yusuf (PhD)

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177. F (calculated) is compared with F(Table) value by looking at the F table for replication df (r-1) and error df values(r-1)(t-1). If the calculated F value is greater than F(Table value) then it is significant. Genotypic variance: It is the inherent variation which remains unaltered by the environment. It is the variation due to genotypes. It is denoted by VG and is calculated using the formula: 177 Dr. Zekeria Yusuf (PhD)

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183. Heritability and genetic advance are important selection parameters and heritability estimate along with genetic advance are interpreted as follows 1) High heritability accompanied with high genetic advance indicates heritability is due to additive (or) fixable variation and selection may be effective. 2) High heritability accompanied with low genetic advance indicates non additive gene action and selection for such characters may not be rewarding. 3) Low heritability accompanied with high genetic advance reveals that characters are governed by fixable gene effects and low heritability is due to high environmental influence and selection may be effective. 4) Low heritability accompanied with low genetic advance indicates that character is highly influenced by environment and selection is ineffective. 183 Dr. Zekeria Yusuf (PhD)

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185. ESTIMATION OF HETEROSIS AND INBREEDING DEPRESSION refers to the superiority of F1 in one or more characters over its parents. It is also defined as increase in fitness and yield over its parental values. Heterosis , or hybrid vigor, or outbreeding enhancement, is the improved or increased function of any biological quality in a hybrid offspring. It is the occurrence of a genetically superior offspring from mixing the genes of its parents. It is also called as hybrid vigour . The three main causes of heterosis are over dominance, dominance and epistasis , of this dominance is the widely accepted one. In crop plants there are three main ways for fixation of heterosis i.e. asexual reproduction, polyploidy and apomixis 185 Dr. Zekeria Yusuf (PhD)

186. Manifestations of Heterosis 1. increased heterozygosity 2. increased size and productivity in plants 3. Greater resistance to diseases, insects and environmental factors 4. Early maturity when compared to either of the parents. 186 Dr. Zekeria Yusuf (PhD)

187. . Four different methods are used to estimate heterosis . 187 Dr. Zekeria Yusuf (PhD)

188. Giving the mean yield of two inbred strains A=80kg , B= 50 and F1 is 90kg, calculate i . Hmp ; ii. Hbp Solution: 1. mp =(80+50)/2= 65 Hmp = (F – mp)/mp= (90 – 65)/65 =0.3846 This implies that the hybrid vigour is 38.46% 2. Hbp = (F – bp )/ bp = (90 – 80)/80 =0.125 Herobeltiosis is 12.5% The better parent heterosis is more significant as far as breeding is concerned because individual progenies are more superior to the better parent. 188 Dr. Zekeria Yusuf (PhD)

189. Gene action Alleles may interact with one another in a number of ways to produce variability in their phenotypic expression. The following models may help us understand various modes of gene action. Additive gene action: absence of DOMINANCE in case of single locus. Therefore, the breeding procedure chosen for a crop genotype will depend on the prevalence of gene action e.g additive gene action will be effective in accumulating favourable alleles in breeding materials especially in self-pollinating crops. Heritability in this narrower sense is the ratio of the additive genetic variance to the phenotypic variance: 189 Dr. Zekeria Yusuf (PhD)

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191. Additive gene action When both alleles contribute for the phenotype. Thus, offspring phenotype resemble their parents It can be directly inherited from parents to their offsprings as phenotype is effect of each alleles from both parents being added together. It can be fixable Dominance gene action: In which one allele contributes more or less to the final phenotype. Dominance variance is not directly inherited from parents to their offsprings . Since it is due to interaction of genes from both parents within individuals, and of course only one allele is passed from each parent to offsprings . 191 Dr. Zekeria Yusuf (PhD)

192. Dominance variance has two components : variance due to homozygous alleles (w/c is additive) and variance due to heterozygous genotypic values. Dominance effects are deviations from additivity that make heterozygote resemble one parent more than the other. When dominance is complete heterozygote is equal to homozygotes in effects (i.e. Aa =AA). Breeder can’t distinguish between heterozygous and homozygous phenotypes as a result both Aa and AA will be selected. Thus fixing superiour genes will be less effective with dominance gene action since Aa will segregate in next generation. 192 Dr. Zekeria Yusuf (PhD)

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197. The variance component method of estimating heritability uses the statistical procedure of analysis of variance (ANOVA). Variance estimates depend on the types of populations in the experiment. Estimating genetic components suffers from certain statistical weaknesses. Variances are less accurately estimated than means. Also, variances are unrobost and sensitive to departure from normality. An example of a heritability estimate using F2 and backcross populations is as follows: 197 Dr. Zekeria Yusuf (PhD)

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199. Overdominance gene action The hetrozygote is more valuable than either homozygotes . Exists when each allele at a locus produces a separate effect on the phenotype and either combined effect exceeds independent effect of the alleles. breeder can fix overdominance effects only in F1 generation through apomixis or through chromosome doulbling of the product of wide cross. Epistasis gene action ( nonalleleic interaction) -interaction between alleles at different loci. VG = VA + VD + VI Vp = VG + VE For inbreeding population, Vp = Ve , since Vg= 0 Vp = VA + VD + VI + VE + Vgxe Narrow sense heritability = 199 Dr. Zekeria Yusuf (PhD)

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202. Path interpretation In the path analysis for genetic correlations, the direct effect of NBP trait on O/L ratio was smaller than the indirect effect of AGBP , NSPOD and oleic acid traits on O/L ratio; in this case, the significant correlation value between NBP and O/L ratio was attributed to the indirect effects of AGBP , NSPOD and oleic acid traits. That is breeding for high oil quality can be effective through indirect selection for high AGBP . In the path analysis of AGBP on O/L ratio, the direct effect of AGBP on O/L ratio was smaller than the indirect effects of NSPOD and oleic acid on O/L ratio; in this case, the significant and positive correlation value between AGBP and O/L ratio is attributed to the indirect effects of NSPOD and oleic acid traits. The path analysis has shown that breeding for high O/L ratio can be conducted through selection for AGBP trait. The coefficient of determination () in the path analysis for genotypic correlation indicates that 92% of the O/L ratio variability was explained by the variables which is a good fit for the model and shows the importance of the explaining variables in the O/L ratio. Dr. Zekeria Yusuf (PhD) 202

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209. Detection of G×E interaction Dr. Zekeria Yusuf (PhD) 209

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227. Multiplicative Models 1. AMMI model 2. GGE biplot 3. Shifted Multiplicative Model 4. Genotype Regression Model 5. Completely Multiplicative Model Dr. Zekeria Yusuf (PhD) 227

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242. Mating Designs in Plant Breeding DEFINITION: Schematic cross between two groups or strains of plants made to produce progenies in plant breeding that is concerned in agriculture and bio sciences. OBJECTIVES: i . To obtain information and understand genetic control of a trait or behavior that is observed ii. To get base population for development of plant cultivars NEED FOR MATING DESIGN: ANOVA in offspring plants resulted from a mating design was used to evaluate effects of additive genetic, dominance level, epistasis and heritability value equal to value of genetic expectations. Dr. Zekeria Yusuf (PhD) 242

243. Factors affecting choice of Mating Design i . The type of pollination (self- or cross-pollinated) ii. The type of crossing to be used (artificial or natural) iii. The type of pollen dissemination (wind or insect) iv. The presence of a male-sterility system v. The purpose of the project (for breeding or genetic studies) vi. The size of the population required Keen interest to discover i . How significant is genetic variation? ii. How much of the variation is heritable? A main interest of the breeder is identifying plants with superior genotypes as judged by the performance of their progeny. Dr. Zekeria Yusuf (PhD) 243

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245. MAJOR MATING DESIGNS IN Plant Breeding and Genetics Several studies described and contrasted different mating designs and six mating designs are considered more beneficial and helpful. 1. Biparental Mating: • Also called paired crossing design • Large no of plants are selected at random and then crossed in pairs. They produce 1/ 2n full sib families Normally, individual pairs of plants can be crossed reciprocally to produce progenies which can be bulked for evaluation across environments. • Progenies are tested by simply analysis of variances into between and within the families • Most limitation of this design is its inability to provide sufficient information about all parameters required by the model. Dr. Zekeria Yusuf (PhD) 245

246. 1. Biparental Mating (Full-Sib Families) This is one of the simplest mating designs used for estimation of genetic variance in a reference population as was termed by Mather in 1949. The mating design provides opportunity for creating variability with minimum effort and cost (e.g., cross-pollinated species) and also provides information needed to determine whether the variation within a population is significant for a long term selection program. However, the design cannot give information on the type of genetic variation. Many crosses are required to allow accurate measurements and adequate interpretations relative to the reference population. If n parents are used the total number of crosses = n/2. Dr. Zekeria Yusuf (PhD) 246

247. 1. Biparental Mating (Full-Sib Families)… Dr. Zekeria Yusuf (PhD) 247

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250. 2. Triple testcross ( TTC ) Triple testcross design was developed by Kearsey and Jinks (1968) as an extension of Comstock and Robinson’s (1952) NC Design III. The design is able to detect epistatic effects (additive × additive, additive × dominance, dominance × dominance) for quantitative traits, and also provides estimates of additive and dominance genetic variances in the absence of epistasis . In TTC , a random sample of male individuals from the F2 generation obtained by crossing two inbred lines P1 and P2 are backcrossed to three testers: P1 , P2 and F1 . This will generate 3 families where P1 X F2 = L1i , P2 x F2 = L2i , and F1 x F2 = L3i (Figure 2). Each family is tested in replicated trials and individual family means obtained. The means are used to calculate the epistatic deviation from the means of the parental lines. The design is effective for predicting the properties of recombinant inbred lines is the triple test cross. Triple testcross has been used intensively used in the area of genetic components and correlation studies in various crops [16]. Statistical model for detection of epistasis is as described by Kearsey and Jinks (1968) and is given by: Dr. Zekeria Yusuf (PhD) 250

251. 3. Pure line progenies Pure lines are the basis of most breeding programs where homozygous lines such as DH (doubled haploid), RILs (recombinant inbred lines) and NILs (near isogenic lines) are used. The concept ( Johanssons 1903) is based on production of progenies from crosses between two parents and advance to later stage of inbreeding ( e.g at S8 ) through selfing or backcrossing, followed by selection of highly homozygous progenies (Figure 3). At this stage the reference population is assumed to compose of complete homozygous inbred lines containing additive and additive-by-additive types of epistatic variances. Dr. Zekeria Yusuf (PhD) 251

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254. 4. Parent-offspring regression analysis The resemblance between a parent and its offspring was employed by Robinson (1949) for estimation of genetic variance and heritability [1]. A set of male parents randomly chosen from a reference population ( S0 ) are mated to a set of females randomly chosen from another reference population ( S0 ). The reference population can be S0 population obtained from open-pollinated crops or the F2 population derived from a cross of two inbred lines [5]. The progenies produced are used to determine estimates of parent-offspring regressions by (1) regression of offspring on one parent (half-sib method), (2) regression of offspring on the mean of two parents (full-sib method), and (3) regression of selfed progeny on parents ( selfing method). Measurements of traits of interests are made on individual plants from the reference population and on the offspring. This allows good determination of the degree of association between the traits measured in the parents and in their respective offspring [5]. Progenies from the crosses are evaluated in trials and the same traits are measured in the replicated progeny trials (Table 3). Dr. Zekeria Yusuf (PhD) 254

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258. 5. North Carolina designs This mating design was developed by Comstock and Robinson (1948) and has since been one of the most useful mating designs for estimation of genetic variance and crop selection. North Carolina design has three different mating schemes and these include NC Design I, NC Design II and NC Design III respectively. Dr. Zekeria Yusuf (PhD) 258

259. 5.1 North Carolina (NC Design I) NC Design I is adequate only for estimating genetic variance of a reference population which is assumed to be a random mating population and is in linkage equilibrium. The S0 plants are chosen from the reference population, then plants are divided into two groups of males (m) and females (f). Each male is crossed to a different set of females (independent sample) to produce progenies for evaluation. The genetic structure of the progenies will include full-sibs that have both parents in common and half-sibs that have a male parent in common. As a result, expected mean squares are expressed in covariance of relatives. Dr. Zekeria Yusuf (PhD) 259

260. 5.2. North Caroline (NC Design II) Is one of the useful mating design also known as factorial design , in which parents are divided into one group (males) and the other group (females). Each member of a group has equal chance to cross with a member from the other group. For example in a two-factor design if 1, 2 and 3 are male parents and 4 and 5 are female parents then factorial design will be: ( 1x4 ), ( 1x5 ), ( 2x4 ), ( 2x5 ), ( 3x4 ), and ( 3x5 ) respectively. Depending on the capacity and availability of resources, the breeder can use more than 2 factorial schemes where crosses among three or more groups of parents are involved. Although the assumptions for NC Design II are similar as those for NC Design I, NC Design II has greater precision, it is more applicable to self-pollinated crops, and has a direct estimate of the level of dominance. Dr. Zekeria Yusuf (PhD) 260

261. 5.3. North Caroline (NC Design III) • In this design, a random sample of F2 plants is backcrossed to the two inbred lines from which the F2 was descended. • It is considered the most powerful of all the three NC designs. • A modification called the triple test cross has also been introduced i.e added a third tester not just the two inbreds . • The parents being progenitors of the F2 , are very special testers. • It is capable of testing non-allelic ( epistatic ) interactions, also capable of estimating additive and dominance variance. • Moreover, both in its original and extended form design 3 has a general utility for investigating any population, irrespective of gene frequency or mating system. Dr. Zekeria Yusuf (PhD) 261

262. 6. Diallel mating designs Diallel mating design first presented by Schmidt (1919) became an important tool used to produce crosses for evaluation of genetic variances . The diallel cross is defined as making all possible crosses among a group of genotypes. Crosses are generated from parents ranging from inbred lines to broad genetic base varieties where progenies are developed from all possible combinations of parents involved. Analysis of diallel progenies allows inference about heterosis (Gardner & Eberhart , 1966), estimation of general and specific combining ability ( Griffing , 1956) and study of genetic control of traits. Two models designated as model I and model II by Eisenhart (1947) are available and have been equally used in diallel mating with each having its own assumptions. Model I is a fixed model based on the assumption that the parents used have undergone selection for a period of time and have become a complete population. The model measures only GCA and SCA effects because the parents are fixed. Fixed Effect Model: Also known as model no. 1 which is used with diallel set of crosses made among fixed sample of inbred lines. In this model, the experimental material includes a set of fixed genotypes, say a set of inbreds or varieties. Such a set of genotypes is considered as a population and inferences are drawn about individual line/variety. Random Effect Model: Also known as model no. 2 which is used with diallel set of crosses made among random samples of individuals from random mating population. In this case, inferences cannot be drawn about individual line but about the parent population as a whole. Dr. Zekeria Yusuf (PhD) 262

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264. Full Diallel (Method I) In this design, all possible matings among the selected parents are made in both directions, i.e. direct and reciprocals. In a full diallel , each parent is used as male and female for each mating. Main features of full diallel : • Total number of single crosses in a full diallel is equal to P(P-1), where p is the number of parents used. • Full diallel is used when, a) reciprocal differences are significant and b) parents do not have male sterility or self incompatibility. • Permits estimation of maternal effects. • Each parent is used as male and female in the mating. • There are two methods for evaluation of full diallel crosses i.e. with parents and without parents as described below: a. Full diallel with Parents ( F1s , Reciprocals and Parents) This includes both way crosses and parents. The total number of entries to be evaluated is equal to P2 where P = number of parents. This used when do not have self incompatibility or male sterility. The inclusion of parents permits the estimation of heterosis . Dr. Zekeria Yusuf (PhD) 264

265. b. Full diallel without Parents ( F1s and Reciprocals): This includes all possible single crosses made among P parents in both the directions i.e. direct or reciprocal crosses. The total number of single crosses is equal to P(P-1). This method can be used when the presence of self incompatibility or male sterility prevent the inclusion of parents in the experiment and does not permit estimation of heterosis . Advantage of Full Diallel 1. Each parent has equal chance of mating as male and female with every other parent. 2. It measures the maternal effects. Disadvantage of Full Diallel 1. Double mating are required i.e. P(P-1), which limits the number of parents for evaluation. 2. More experimental area is required for evaluation. Dr. Zekeria Yusuf (PhD) 265

266. Partial/half diallel Complete diallel mating designs have been instrumental in progeny generation for genetic analysis. Kempthorne and Curnow (1961) had introduced partial dialel mating design as a modification of complete diallel . However, breeders find the use of the design cumbersome especially for big number of parents in which all possible combinations are expected. In partial diallel the number of parents is increased but the parents are not mated in all possible combinations Dr. Zekeria Yusuf (PhD) 266

267. Half Diallel • In this design, all possible crosses among the selected parents are made in one direction only i.e. direct crosses. Main features of half diallel • In half diallel , parent is used either as male or as female in the mating. • The number of single crosses required is equal to P(P-1)/2, where P is the number of parents used. • Half diallel is used when reciprocal differences are not significant. • It can be used when parents have male sterility or self incompatibility. • It can be evaluated in two ways i.e. with parents and without parents as given below: a. Half Diallel with Parents ( F1s and Parents) This includes one way crosses and parents. The total number of entries to be evaluated is equal to P( P+1 )/2, where P = number of parents. This can be used when parents do not have self incompatibility or male sterility. The inclusion of parents in the experiment permits the estimates of heterosis . b. Half Diallel without Parents ( F1s only) This includes all possible single crosses made in one direction only. The single crosses required is equal to P(P-1)/2. This design can be used when the presence of self incompatibility or male sterility prevents the inclusion of parents in the experiment. Thus estimates of heterosis are not possible in this method. Dr. Zekeria Yusuf (PhD) 267

268. Advantage of Half Diallel 1. Each parent has equal opportunity to mate and recombine with every other parent. 2. It requires half mating than full diallel . 3. Requires less experimental area for evaluation of material. Disadvantage of Half Diallel 1. Maternal effect cannot be measured. Dr. Zekeria Yusuf (PhD) 268

269. Assumptions of Diallel Analysis • Normal diploid segregation • Lack of maternal effects • Absence of multiple alleles • Homozygosity of parents • Absence of linkage • Lack of epistasis • Random mating Approaches of Diallel Analysis There are two approaches of diallel analysis Hayman’s graphical approach Griffing’s numerical approach Dr. Zekeria Yusuf (PhD) 269

270. Hayman’s graphical approach It was initially developed by Jinks and Hayman in 1954. This approach is based on the estimation of components of variation. Main features of Hayman’s approach: • This is a graphical approach which involves Vr-Wr graph • The analysis is based on the estimation of components of variance • The following six components of variation are estimated: Dr. Zekeria Yusuf (PhD) 270

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272. Hayman’s graphical approach…. But the formula for proportions of genes with positive and negative effects in the parents = ( H2 / 4H1 ), and the number of groups of genes which control the character and exhibit dominance are the same in F2 as those in F1 . (2) Vr – Wr Graph Vr = the variance of the rth array, and Wr = the covariance between the parents and the offspring on the rth array Inferences from the Vr – Wr graph Dr. Zekeria Yusuf (PhD) 272

273. As for the case of numerical approach this approach uses the following components of variance as explained earlier, to perform graphical analysis: D = additive variance, H1 = dominance variance, H2 = Variation due to dominance effects of genes correlation, E= expected environmental variance, F= mean of Fr over the arrays where Fr is the covariance of additive and dominance effects in a single array, and h2 = dominance effects of all loci in heterozygous phase in all the crosses. The analysis involves Vr-Wr graph constructed with the help of variances of arrays ( Vr ) and covariances ( Wr ) between parents and their offspring. The array refers to the crosses in which a particular parent is common. The Vr-Wr distribution is used to simultaneously study the genetical properties of homogeneous parents. Dr. Zekeria Yusuf (PhD) 273

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275. Hayman’s graphical approach…. The Vr - Wr regression is used to explain the graphical relationship between the offspring and the parents in which main inferences are mentioned below. 1. when regression line passes through the origin, there is complete dominance (D = H1 ); 2. when regression line passes above the origin and cutting the Wr axis, there is partial dominance (D> H1 ); 3. when regression line passes above the origin, cutting Wr axis and touching the limiting parabola, there is no dominance; and 4. when regression line passes below the origin and cutting the Vr axis, there is over dominance. 5. The position of parental points on the regression line indicating the dominance order of the parents. The parents with more dominant genes are located nearer to the origin, while those with more recessive genes fall farther from the origin. The parents with equal frequencies of dominant and recessive genes occupy an intermediate position. The above genetic estimates can be represented by a Vr-Wr graph where Vr is the variance of the rth array, and Wr is the he covariance between the parents and the offspring on the rth array (Figure 6). The points Vr and Wr are distributed along the corresponding straight line inside the limiting parabola, based on the value of the ratio H1 /D. Dr. Zekeria Yusuf (PhD) 275

276. Hayman’s graphical approach…. If a sloping straight line cuts Wr at A, and another parallel tangent to the parabola cuts it at B, then the line is determined by AB/OB = H1 /D. The line marked A in the diagram represent diallel cross with H1 /D = 4. The position of Vr , Wr on the line indicates the relative proportion of dominant and recessive genes in the rth parent. For any diallel cross, the point corresponding to a parent containing p% dominants and q% recessive lies on the curve labeled p : q. Completely recessive parents correspond to points at the upper ends of the sloping lines on the part of the limiting parabola labeled 0:100, and completely dominant parents to points at the lower ends on the part labeled 1:0. In an experiment where there is no dominance, the points coincide at (1/ 4D , 1/ 2D ) where H1 = 0 (Figure 6). Dr. Zekeria Yusuf (PhD) 276

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278. Griffing’s Numerical Approach: This is based on the estimation of general combining ability ( GCA ) and specific combining ability ( SCA ) variances and effects. Griffing (1956) gave four different methods of diallel analysis depending on the material (s) included in the experimentation. Dr. Zekeria Yusuf (PhD) 278

279. In their approach, gene action is deduced through the estimates of GCA and SCA variances and effects. The GCA component is primarily a function of additive genetic variance. However, if epistasis is present, the GCA will include the additive x additive interaction as well. On the other hand, the SCA variance is mainly a function of dominance variance but it would include all the three types of epistatic interaction component; if epistasis were present. Dr. Zekeria Yusuf (PhD) 279

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284. Diallel mating designs are frequently used in plant breeding due to their capacity to test general and specific combining abilities and other secondary genetic parameters. They are also used to reduce the costs and the complexity of experiments. Dr. Zekeria Yusuf (PhD) 284

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288. Interpretation of Gene Action • If gca variances are higher than sca variances, it means that there is preponderance of additive gene action and progeny selection will be effective for the genetic improvement of such traits. • If sca variances are higher than gca variances, it indicates that there is preponderance of nonadditive gene action (dominance & epistasis ) and therefore, heterosis breeding may be rewarding. • If both gca & sca variances are of equal magnitude, it shows that additive and non-additive genes are equally important in the expression of character then we use reciprocal recurrent selection. Dr. Zekeria Yusuf (PhD) 288

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290. Summary of Diallel mating Method 1 or full diallel design • The method F1 or full diallel design consists of parents, one set of F1’s and reciprocal F1’s . • This system gives n2 genotypes. Method 2 • This method includes parents and one set of F1’s without reciprocal F1’s . • This design gives p( p+1 )/2 genotypes. Method 3 • In this method one set of F1’s and the reciprocals are included. • This type of mating designs give rise to a=p(p-1) different number of genotypes. Method 4 • In this method only one set of F1’s are included. • Most common of diallel crossing systems. • There are a=p(p-1)/2 different genotypes evaluated. Dr. Zekeria Yusuf (PhD) 290

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292. Combining ability analysis Dr. Zekeria Yusuf (PhD) 292

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296. Parents showing a high average combining ability in crosses are considered to have good GCA while if their potential to combine well is bounded to a particular cross, they are considered to have good SCA . From a statistical point of view, the GCA is a main effect and the SCA is an interaction effect [6]. Based on Sprague and Tatum [5], GCA is owing to the activity of genes which are largely additive in their effects as well as additive × additive interactions. Specific combining ability is regarded as an indication of loci with dominance variance (non-additive effects) and all the three types of epistatic interaction components if epistasis were present. They include additive × dominance and dominance × dominance interactions. Dr. Zekeria Yusuf (PhD) 296

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303. 7. Triallel and quadralle Triallel and quadrallel Cockerham (1961) produced triallel (three-way crosses) and quadrallel (double-cross hybrids) crosses from a group of parents that originated from the same population. He determined the variances and covariances between all possible pairs of the hybrid relatives among single cross, threeway , and double cross hybrids. Population size is important for example if we have n lines, then number of possible three-way combinations will be n(n - 1)(n - 2)/6, and assuming no reciprocal crosses, three possible arrangement of the three-way crosses will be 3*[ n(n - 1)(n - 2)/6]. Similarly, if we have n parents the possible number of quadrallel (double-crosses) is n(n - 1)(n - 2)(n - 3)/24, and three possible arrangement of double-crosses will be 3*[n(n - 1)(n - 2)(n - 3)/24] respectively. Dr. Zekeria Yusuf (PhD) 303

304. 8. Top cross The top cross design was proposed by Jenkins and Brunsen (1932) for testing inbred lines of maize in cross-bred combinations and later renamed top cross by Tysdal and Grandall (1948). The cross is made between a plant (line, clone, etc) selected as female and a common male tester of a known performance (variety, inbred line or single cross). Possible number of crosses that can be made is n x 1, where n is number of inbreds . Top cross scheme is effective for testing big number of elite lines especially when crossed to a tester with wide or narrow genetic base. However, the most appropriate tester used for top cross is single cross hybrid ( F1 ) because of its uniformity. Top cross, sometimes referred to as inbred variety cross , is mostly suitable for preliminary evaluation of combining ability of new inbred lines before pairing them into single cross hybrids ( Abrha , 2014). The parental pair-wise combinations are estimated based on: i ) parental performance in pairwise combinations; ii) direct contribution of each parent to the progeny mean through additive gene action; and iii) reliability of the results being obtained is independent of the quantity of the data. Dr. Zekeria Yusuf (PhD) 304

305. 9. Ploy cross Term polycross was coined by Tysdal and Kiesselbach (1942), to indicate progeny from seed of a line that was subject to outcrossing with other selected lines growing in the same nursery. The design allows a group of cultivars to undergo natural crossing in isolated block. The main aim of polycross is to improve homozygosity of open pollinated variety while at the same time maintaining the high level of heterozygosity . It is most suited to species that are obligate cross- pollinaters (e.g., vegetables, forage grasses and legumes, sugarcane, sweet potato). Dr. Zekeria Yusuf (PhD) 305

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335. 335 Breeding Self Pollinated Crops

336. 336 Cultivar Is a group of genetically similar plants, which may be identified (by some means) from other groups of genetically similar plants Essential Characteristics: Identity: cultivar must be distinguishable from other cultivars Reproducibility: the distinguishing characteristic(s) need to be reproduced in the progeny faithfully Cultivars

337. 337 Types of Cultivars Open-Pollinated cultivars O.P. seeds are a result of either natural or human selection for specific traits which are then reselected in every crop. The seed is kept true to type through selection and isolation; the flowers of open-pollinated or O.P. seed varieties are pollinated by bees or wind.

338. 338 Types of Cultivars Synthetic cultivars A population developed by inter-crossing a set of good combiner inbred lines with subsequent maintenance through open-pollination. The components of synthetics are inbreds or clones so the cultivar can be periodically reconstituted.

339. 339 Multi-line cultivars A mixture of isolines each of which is different for a single gene controlling different forms of the same character (e.g., for different races of pathogens) F1 cultivars The first generation of offspring from a cross of genetically different plants Pure-line cultivars The progeny of a single homozygous individual produced through self-pollination Types of Cultivars

340. 340 Cultivars and Self-pollinated Crops In self-pollinated species: Homozygous loci will remain homozygous following self-pollination Heterozygous loci will segregate producing half homozygous progeny and half heterozygous progeny Plants selected from mixed populations after 5-8 self generations will normally have reached a practical level of homozygosity

341. 341 In general, a mixed population of self-pollinated plants is composed of plants with different homozygous genotypes (i.e., a heterogeneous population of homozygotes If single plants are selected from this population and seed increased, each plant will produce a ‘pure’ population, but each population will be different, based on the parental selection Cultivars and Self-pollinated Crops

342. Genetic Basis of Self pollinated crops In self-pollinated species: Homozygous loci will remain homozygous following self-pollination Heterozygous loci will segregate producing half homozygous progeny and half heterozygous progeny Plants selected from mixed populations after 5-8 self generations will normally have reached a practical level of homozygosity . In general, a mixed population of self-pollinated plants is composed of plants with different homozygous genotypes If single plants are selected from this population and seed increased, each plant will produce a ‘pure’ population, but each population will be different, based on the parental selection Dr. Zekeria Yusuf (PhD) 342

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344. Johanson’s Pure line theory Dr. Zekeria Yusuf (PhD) 344

345. 345 Selection involves the ID and propagation of individual genotypes from a land race population, or following designed hybridizations Genetic variation must be identified and distinguished from environment-based variation Selection procedures practiced in mixed populations of self-pollinated crops can be divided into two selection procedures Breeding Self-pollinated Crops

346. 346 Breeding Methods of Self Pollinated Crops Pure line Mass Bulk Pedigree Single Seed Descent ( modified pedigree) Backcross

347. 347 Pure Line: (Recount Johannsen. 1903) usually no hybridization Initial parents (IPs) selected from a heterogenous population (i.e. genetically variable) procedure continues until homogeneity is achieved last phase is field testing

348. 348 A pure line consists of progeny descended solely by self-pollination from a single homozygous plant Pure line selection is therefore a procedure for isolating pure line(s) from a mixed population Pure-line Selection

349. 349 Pure line cultivars are more uniform than cultivars developed through mass selection (by definition, a pure line cultivar will be composed of plants with a single genotype) Progeny testing is an essential component of pure line selection Improvement using pure line breeding is limited to the isolation of the ‘best’ genotypes present in the mixed population Pure-line Selection

350. 350 Pure-line Selection More effective than MS in development of self-pollinated cultivars However, leads to rapid depletion of genetic variation Genetic variability can be managed through directed cross hybridizations Essential to progeny test selections

351. 351 Pure-line Selection-Steps Select desirable plants Number depends on variation of original population, space and resources for following year progeny tests Selecting too few plants may risk losing superior genetic variation A genotype missed early is lost forever Seed from each selection is harvested individually

352. 352 Pure-line Selection-Steps Single plant progeny rows grown out Evaluate for desirable traits and uniformity Should use severe selection criteria (rogue out all poor, unpromising and variable progenies) Selected progenies are harvested individually In subsequent years, run replicated yield trials with selection of highest yielding plants After 4-6 rounds, highest yielding plant is put forward as a new cultivar

353. 353 Advantages ID of best pure line reflects maximum genetic advance from a variable population; no ‘poor’ plants maintained Higher degree of uniformity Selection based on progeny performance is effective for characters with relatively low h 2

354. 354 Requires relatively more time, space, and resources for progeny testing than MS to develop new cultivar High degree of genetic uniformity; more genetically vulnerable and less adaptable to fluctuating environments ID and multiplication of one outstanding pure-line depletes available genetic variation; leads to fast genetic erosion Disadvantages

355. 355 How long will a cultivar remain pure? As long as the commercial life of the cultivar, unless: Seed becomes contaminated with seed from other sources (e.g. from harvesting and seed cleaning equipment) Natural out-crossing occurs (amount varies by species but seldom exceeds 1-2% in self-pollinated crops) Mutations occur To maintain purity, off-types arising from mutation or out-crossing must be rogued out

356. 356 May or may not include hybridization Make IP selections based on single, ideal or desirable phenotype and BULK seed May repeat or go directly to performance testing Mass Selection has 2 important functions: Rapid improvement in land-race or mixed cultivars Maintenance of existing cultivars (sometimes purification) * Many pb’ers of self pollinated crops believe that combining closely related pure lines imparts “genetic flexibility” or buffering capacity and so are careful to eliminate only obvious off types Mass Selection

357. 357 Success depends on extent of variation and h 2 of the traits of interest Land races make an ideal starting source High genetic variability accumulated over generations of mutation and natural hybridization

358. 358 Mass Selection Initial selection Can be either a positive or a negative selection Negative screening: A screening technique designed to identify and eliminate the least desirable plants. positive screening: which involves identifying and preserving the most desirable plants.

359. 359 Mass Selection - 1 st Year Select plants with respect to height, maturity, grain size, and any other traits that have ‘production’ or ‘acceptability’ issues Bulk seed (may ‘block’ these bulks if wide variation is present for certain traits; e.g. height) May be able to use machines to select Harvest only tall plants, or save only large seed passed through a sieve

360. 360 Mass Selection - 2 nd Year MS really only takes 1 yr because selected seed represents a mixture of only the superior pure lines that existed in the original population However, additional rounds of selection and bulking will allow for evaluation under different environments, disease and pest pressures. Also, multiple years will allow you to compare performance with established cultivars over years and environments.

361. 361 Objectives of Mass Selection: To increase the frequency of superior genotypes from a genetically variable population Purify a mixed population with differing phenotypes Develop a new cultivar by improving the average performance of the population

362. 362 Selection based on phenotypic performance; not effective with low h 2 traits Without progeny testing, heterozygotes can be inadvertently selected Population cannot realize maximum potential displayed by the ‘best’ pure line, due to bulking Final population is not as uniform as those developed through pure-line selection Disadvantages

363. 363 Mass selection vs pure line selection Line mixture Bulk of phenotypically similar plants Cultivar register and marketing Single plant offsprings L1 L2 L3……. LN Register and market the best pure lines Mass selection Pure line selection Heterogenous cultivars Homogenous cultivars Line mixture Bulk of phenotypically similar plants Cultivar register and marketing Single plant offsprings L1 L2 L3……. LN Register and market the best pure lines Mass selection Pure line selection Heterogenous cultivars Homogenous cultivars Line mixture Bulk of phenotypically similar plants Cultivar register and marketing Single plant offsprings L1 L2 L3……. LN Register and market the best pure lines Mass selection Pure line selection Heterogenous cultivars Homogenous cultivars

364. 364 Bulk Method

365. 365 Bulk Inbreed in bulk to have homozygous lines Select superior lines after F6 Crosses with no high heritability traits segregating

366. 366 Natural selection changes gene freq. via natural survival Breeder may assist nature and discard obviously poor types Relieves breeder of most record keeping Most of us treat bulks with extremely low inputs and low expectations. Points to consider in Bulk Method

367. 367 The bulk method is a procedure for inbreeding a segregating population until a desired level of homozygosity is reached. Seed used to grow each selfed generation is a sample of the seed harvested in bulk from the previous generation. In the bulk method, seeds harvested in the F 1 through F 4 generations are bulked without selection; selection is delayed until advanced generations (F 5 -F 8 ). By this time, most segregation has stopped.

368. 368 Advantages Less record keeping than pedigree Inexpensive Easy to handle more crosses Natural selection is primarily for competitive ability More useful than pedigree method with lower h 2 traits Large numbers of genotypes can be maintained Works well with unadapted germplasm Can be carried on for many years with little effort by the breeder

369. 369 Environmental changes from season to season so adaptive advantages shift Most grow bulk seed lots in area of adaptation Less efficient than pedigree method on highly heritable traits (because can purge non-selections in early generations) Not useful in selecting plant types at a competitive disadvantage (dwarf types) Final genotypes may be able to withstand environmental stress, but may not be highest yielding If used with a cross pollinated species, inbreeding depression may be a problem Disadvantages

370. 370 Most popular Essentially a plant to row system to develop near pure lines Followed by performance testing of resulting strains This method and its variants require a lot of record keeping Pedigree Method

371. PROCEDURE OF PEDIGREE METHOD First year: Hybridization of selected parents 2nd year (F1): 10 – 30 seed spaced planted, harvested in bulk. 3rd year (F2): (i) 2000 – 10,000 plant space plated (ii) 100 – 500 superior plant selected 4th and 5th year: (i) Individual plant progeny space-planted (ii) Superior plant selected 6th year: (i) Individual plant progeny planted in multi-row plot (ii) Superior plant selected from superior progenies 7th year: (i) As in (i) and (ii) for F5 (ii) Preliminary yield trial (PYT) may be conducted. 8th year: (i) PYT (ii) Quality test 9th – 13th year: (i) Coordinated yield trial (ii) Disease and Quality test 14th year: (i) Seed increase for distribution 371

372. 372 Pedigree Selection during inbreeding Early generations: High heritability traits Late generations: low heritability traits

373. 373 Genetic Considerations: Additive genetic variability decreases within lines and increases among lines, assuming no selection recall the movement toward homozygosity following the hybridization of unlike and homozygous parents Dominant genetic variability complicates pedigree selection homozygous and heterozygous individuals look alike and therefore you may continually select the heterozygote THUS, selection can be discontinued with phenotypic uniformity within a line is obtained

374. 374 Advantages Eliminates unpromising material at early stages; Multi-year records allow good overview of inheritance, and more effective selection through trials in different environments; Multiple families (from different F 2 individuals) are maintained yielding different gene combinations with common phenotype Allows for comparison to other breeding strategies

375. 375 Disadvantages Most labor, time and resource intensive method; usually compromise between # crosses and population sizes; Very dependent on skill of breeder in recognizing promising material; Not very effective with low h 2 traits; Slow; can usually put through only one generation per year, and the right environmental conditions must be at hand for accurate selection. Upper ceiling set by allelic contents of F 2 ; can not purge selections of undesirable alleles once ‘fixed’.

376. 376 Single Seed Descent

377. 377 Single Seed Descent Inbreed with one seed from each plant in each generation Select superior line after F6 Crosses with no high heritability traits segregating

378. 378 Advantages Rapid generation advance; 2-4 generations/yr Requires less space,time and resources in early stages, therefore accommodates higher # crosses; Superior to bulk/mass selection if the desired genotype is at a competitive disadvantage; natural selection usually has little impact on population. Delayed selection eliminated confusing effects of heterozygosity; more effective than pedigree breeding when dealing with low h 2 traits; Highly amenable to modifications and can be combined with any method of selection.

379. 379 Disadvantages May carry inferior material forward Fewer field evaluations, so you lose the advantage of natural selection Need appropriate facilities to allow controlled environment manipulation of plants for rapid seed production cycles (day length, moisture and nutrient control)

380. 380 Backcross

381. 381 Same form whether self or cross pollinated species Only difference is pollination control With backcross we approach homozygosity at the same rate as with selfing Goal is to move 1 to a few traits from a donor parent (deficient in other traits) to a recurrent parent (deficient in the trait of interest) Backcross

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383. 383 Limited use of BC to create a population for selection that fosters wider genetic variance and modest introgression is a separate issue than a repeated BC to derive a new cultivar Jensen suggested that a 3-way (a backcross to another recurrent or superior parent following he single cross of a desirable and an undesirable parent) was superior to single cross followed by pedigree or other selection methodology Backcross

384. 384 BC must be used with other, more exploratory procedures; otherwise G s =0 Must have a suitable recurrent parent # of BCs to make? usually 4 Use several RP plants! WHY? To incorporate > 1 trait, use parallel programs and then converge Evaluation phase can be less stringent because you should already know the utility of the recurrent parent! Backcross

385. 385 Backcross Breeding Recovery of the recurrent parent genotype follows this pattern: % recurrent % donor F 1 50 50 BC 1 75 25 BC 2 87.5 12.5 BC 3 93.7 6.3 BC 4 96.9 3.1 BC m 1-(1/2) m+1 (1/2) m+1

386. Basic breeding schemes for prevalently allogamous species (the large majority of forage and turf species) - Recurrent selection (to obtain advanced basic populations) -- Mass selection Phenotypic selection Simple recurrent selection Recurrent selection for General Combining Ability (GCA) Progeny tests: -selfed progenies -open pollinated progenies - topcross,polycross,single cross, -Synthetics, Hybrids Backcross breeding 386

387. Breeding cross pollinated crops… Populations of cross pollinated crops are highly heterozygous. When inbreeding is practiced they show severe inbreeding depression. So to avoid inbreeding depression and its undesirable effects, the breeding methods in the crop is designed in such a way that there will be a minimum inbreeding. 387

388. I. Population improvement A. Selection a) Mass selection b) Modified mass selection Detasseling Panmixis Stratified or grid or unit selection Contiguous control. B. Progeny testing or selection (more effective): selection based progeny performance. a) Half sib family selection i ) Ear to row ii) Modified ear to row. b) Full sib family selection. c) Inbred or selfed family selection. i ) Sl self family selection ii) S2 self family selection. 388

389. C. Recurrent selection 1) Simple recurrent selection 2) Reciprocal recurrent selection for GCA 3) Reciprocal recurrent selection SCA 4) Reciprocal recurrent selection. D. Hybrids E. Synthetics and Composites. 389

390. Mass selection This is similar to the one, which is practiced, in self-pollinated crops. A number of plants are selected based on their phenotype and open pollinated seed from them are bulked together to raise the next generation. The selection cycle is repeated one or more times to increase the frequency of favorable alleles. Such a selection is known as phenotypic recurrent selection. Merits i) Simple and less time consuming ii) Highly effective for character that are easily heritable. Eg. Plant height, duration. iii) It will have high adaptability because the base population is locally adapted one. Demerits 1. Selection is based on phenotype only which is influenced by environment 2. The selected plants are pollinated both by superior & inferior pollens present in the population. 3. High intensity of selection may lead reduction in population there by leading to inbreeding. 390

391. To over come these defects modified mass selection is proposed they are a) Detasseling-i s practiced in maize. The inferior plants will be detasseled there by inferior pollen from base population is eliminated. b) Panmixis: f rom the selected plants pollen will be collected and mixed together. This will be used to pollinate the selected plants. This ensures full control on pollen source. c) Stratified mass selection Unit selection Here the field from which plants are to be selected will be divided into smaller units or plots having 40 to 50 plants / plot. From each plot equal number of plants will be selected. The seeds from selected plants will be harvested and bulked to raise the next generation, by dividing the field into smaller plots, the environmental variation is minimized. This method is followed to improve maize crop. It is also known as Grid method of mass selection 391

392. a) Half sib family selection Half sibs are those, which have one parent in common. Here only superior progenies are planted and allowed to open pollinate. 1. Ear to row method It is the simplest form of progeny selection, w/c extensively used in maize. a) A number of plants are selected on the basis of their phenotype. They are allowed to open pollinate and seeds are harvested on single plant basis. b) A single row of say 50 plants i.e. progeny row is raised from seeds harvested on single plant basis. The progeny rows are evaluated for desirable characters and superior progenies are identified. c) Several phenotypically superior plants are selected from progeny rows. There is no control on pollination and plants are permitted to open pollinate. Though this scheme is simple, there is no control over pollination of of selected plants. Inferior pollen may pollinate the plants in the progeny row. B) Progeny Testing and Selection 392

393. To overcome this defect, the following method is suggested. a) At the time of harvest of selected plants from base population on single plant basis, part of the seed is reserved. b) While raising progeny rows, after reserving part of the seeds, the rest are sown in smaller progeny rows. c) Study the performance of progenies in rows and identify the best ones. d) After identifying the best progenies, the reserve seeds of the best progenies may be raised in progeny rows. e) The progenies will be allowed for open pollination and best ones are selected. There are a number of other modifications made in the ear to row selection. For example, i . The selected progenies may be selfed instead of open pollination ii. The selected plants may be crossed to a tester parent. The tester parent may be a open pollinated variety, or inbred iii. The progeny test may be conducted in replicated trial. 393

394. Half-sib Selection With Test Cross or Ear to Row Selection: In this method, phenotypically superior ears are selected from superior plants from a source or base population grown in isolation. Seeds from harvested ears (half-sib-seeds, one parent in common) are kept separate unlike mass selection where the seeds from different ears are bulked. Seed from each selected ear is divided into two halves. In 2nd year, half of seeds from each ear is used to grow progeny row in isolation for evaluation . Remnant seeds are kept separately. In 3rd year, a new population is reconstituted from by bulking equal quantity of seeds either from harvested superior progenies or from the remnant seeds which gave rise to superior progenies. 394

395. Modified Ear to Row Selection (MER- Lonnquist Method): a type of half-sib selection in which half-sib families are planted in different environments for evaluation . The experiment in one environment is sown as an isolated evaluation-cum-recombination block. In this block, ear-to-rows are inter-planted with a balanced male (BM) composite developed by mixing an equal number of seeds of all ears under evaluation. The ear to- rows are detasselled and are thus pollinated by the pollen of BM composites. Selection is carried out among &within families, selection among ear-to-rows being based on performance across the target environments & that within ear-to-rows being mass selection in isolated block. MER selection is a unique method of family selection as one cycle of selection is completed in one season but it provides no control over pollen parent. 395

396. Modified Ear to Row Selection…. Season I. 100 or more ears are selected from a base population based on grain & agronomic characters. Each ear is shelled separately. Season II. Seeds of each selected ear are grown as progeny row in single replication at different locations of the environment. Season II. Replicate at the main station is handled as a crossing block. A balanced mixture of seed is used as pollen parent. For creating balanced mixture, equal number of seeds are taken from each progeny and mixed. Season II. At the main location, five phenotypically superior plants in each progeny row are marked. Five ears from the marked plants are kept in a bag after each row is harvested & weighed (II season). Season III. Top 20% of the progenies are selected on the basis of average performance over locations and five ears/cobs from each of these 20% of selected progenies (100 cobs, if number of planted progenies is 100) are grown in next season in ear-to-row manner (III season) and above cycle may be repeated. 396

397. Half-sib Selection With Test Cross/Half-sib Progeny Selection Individuals having one parent in common are called half-sibs. In half-sib breeding, the selected plants are always test crossed. Two different terms are used in this method depending upon as to how the next population is reconstituted. These terms are: a. Half-sib progeny test: Under this scheme, males are selfed and crossed to females to produce half-sib families. Selfed seeds from selected males are composited to reconstitute the next population. b. Half-sib test: It is similar to half-sib progeny test except that compositing is done from open-pollinated seeds. The steps are as follows: Season I : Selection of superior plants (50-100) in a source population, crossing to a tester, and selfing under half-sib progeny test and crossing only to a tester under half-sib test. Season II : Evaluation of testcross progenies. Season III : Compositing selfed seeds from plants with superior testcross progenies under half-sib progeny test and compositing open-pollinated seeds from plants with superior testcross progenies under half-sib test. Under halfsib progeny test if tester is broad based, the procedure is similar to recurrent selection for general combining ability(GCA) & if the tester is an inbred line, then procedure is equivalent to recurrent selection for specific combining ability (SCA). 397

398. Full-sib Selection Full-sib Selection: Individuals having both parents in common are known as full-sibs and are derived from crossing of two selected plants from the base population. The crosses are made between selected pairs of plants in the source/base population. Crossed seeds are used for progeny test and for reconstituting the improved new base population. Full sibs are those which are produced by mating between selected plants in pairs. Here the progenies will have a common ancestry. The crossed progenies are tested. A x B B x A The basic procedure is depicted in Fig 1. 398

399. 399

400. Inbred line development: c) Inbred or selfed family selection Families produced by selfing . S1 family selection Families produced by one generation of selfing . These are used for evaluation and superior families are intermated (Simple recurrent selection). S2 family selection Families obtained by two generations of selfing and used for evaluation. Superior families are intermated . Merits of progeny testing and selection 1. Selection based on progeny test and not on phenotype of individual plants. 2. In breeding can be avoided if care is taken raising a larger population for selection. 3. Selection scheme is simple. Demerits 1. No control over pollen source. Selection is based only on maternal parent only. 2. Compared to mass selection, the cycle requires 2-3 years which is time consuming. 400

401. Dr. Zekeria Yusuf (PhD) 401

402. Recurrent Selection: • The initial idea of recurrent selection was independently given by Hayes and Garber in 1919 and East and Jones in 1920. • But the term recurrent selection was first coined by Hull in 1945. Definition: Recurrent selection is defined as reselection generation after generation, with intermating of selected plant to produce the population for the next cycle of selection. The idea of this method was to ensure the isolation of superior inbreds from the population subjected to recurrent selection. Dr. Zekeria Yusuf (PhD) 402

403. Genetic basis of recurrent selection Recurrent for GCA is more effective when additive gene effects are more important. Recurrent for SCA is more effective when overdominance gene effects ( heterosis ) are more important. Reciprocal recurrent selection is more effective when both additive and overdominance gene effects are important. Dr. Zekeria Yusuf (PhD) 403

404. Recurrent selection Here single plants are selected based on their phenotype or by progeny testing. The selected single plants are selfed. In the next generation they are intermated (cross in all possible combinations) to produce population for next cycle of selection. The recurrent selection schemes are modified forms of progeny selection programmes. The main difference between progeny selection and recurrent selection. i) The manner in which progenies are obtained for evaluation. ii) Instead of open pollination, making all possible inter crosses among the selected lines. 404

405. Dr. Zekeria Yusuf (PhD) 405

406. 1. Simple recurrent selection • A type of recurrent selection that does not include tester is referred as simple recurrent selection. It is also known as phenotypic recurrent selection. In this method a number of desirable plants are selected and self pollinated. Separate progeny rows are grown from the selected plants in next generation. The progenies are intercrossed in all possible combination by hand. Equal amount of seed from each cross is mixed to raise next generation. This completes original selection cycle. From this, several desirable plants are selected and self pollinated. Progeny rows are grown and inter crosses made. Equal amount of seeds are composited to raise next generation. This forms the first recurrent selection cycle. 406

407. Simple recurrent selection 407

408. 2) Recurrent selection for general combining ability A form of recurrent selection used to important the general combining ability of a population for a character and the heterozygous tester (broad genetic base) is referred as RSGCA . It is also known as half sib recurrent selection. A broad genetic base tester parent is a common parent mated to a number of lines. A tester with broad genetic base means an open pollinated variety, a synthetic variety or segregating generation of a multiple cross. Recurrent selection for GCA can be used for two basically different purposes. 1. It may be used to improve the yielding ability and the agronomic characteristics of a population. In this case the end product will be a synthetic variety. 2. It may be used to concentrate genes for superior GCA . Here the end product will be superior inbreds . Such inbreds can be developed after a few cycles of RSGCA 408

409. Dr. Zekeria Yusuf (PhD) 409

410. Recurrent Selection for SCA : • It was originally proposed by Hull in 1945. • Its a form of recurrent selection that is used to improve the SCA of a population for a character by using homozygous tester is referred as ( RSSCA ) recurrent selection for specific combining ability. • It is also known as half sib recurrent selection with homozygous tester. The selection procedure of this method is same as for RSGCA , except that the tester is an inbred line which has narrow genetic base i.e tester used must be an outstanding inbred. The differences in the performance of test cross are due to difference in their specific combining ability. Dr. Zekeria Yusuf (PhD) 410

411. Dr. Zekeria Yusuf (PhD) 411

412. Reciprocal Recurrent Selection/Half-sib Reciprocal Recurrent Selection: aims at simultaneous improvement of two heterozygous & heterogeneous populations (designated as A & B) in their ability to combine well with each other. where popn A serves as tester for popn B & vice-versa. In this method we can make selection for both GCA and SCA. This method is as effective as recurrent selection for general combining ability when additive gene action predominates, and is as effective as recurrent selection for specific combining ability when non-additive gene action are of major importance in deciding the genetics of a particular trait. 412

413. Dr. Zekeria Yusuf (PhD) 413

414. Half sib reciprocal recurrent selection methods 1st year 1. Several plants selected in population A and B. 2. Selected plants are self pollinated 3. Selected plant from A is test crossed with plants in B and Vice versa. Harvest crossed plant on S.P.(single plant) basis each. 2nd year Separate yield trials conducted from test cross progenies of A and B Superior progenies identified 3rd year 1. Selfed seed from plants producing superior test cross progenies planted. 2. All possible inter crosses made to reconstitute two new populations which will now be called as A’ and B’. 3. Seeds harvested and composited This completes one cycle of reciprocal recurrent selection & additional cycle(s) may be initiated depending upon the variability in the original population & the improvements achieved in the new populations. 414

415. Application of recurrent selection Establish broad genetic base, add new germplasm It break linkage blocks Dr. Zekeria Yusuf (PhD) 415

416. Dr. Zekeria Yusuf (PhD) 416

417. 2. Evaluation of inbred lines: After an inbred line is developed, it is crossed with other inbreds and its productiveness in single and double cross combination is evaluated. The ability or an inbred to transmit desirable performance to its hybrid progenies is referred as its combining ability . GCA : the average performance of an inbred line in a series of crosses with other inbred lines is known as GCA . E.g. Ax B, C, D, E,F . SCA : The excessive performance of a cross over (progeny) and above the excepted performance based on GCA of the parents is known as specific combining ability. Thus GCA is the characteristic of parents and SCA is characteristic of crosses or hybrids. 417

418. Inbreds are evaluated in following way a. Phenotypic evaluation : It is based on phenotypic performance of inbreds themselves. It is effective for characters, which are highly heritable i.e. high GCA . Poorly performing inbreds are rejected. The performance of inbreds is tested in replicated yield trials and the inbreds showing poor performance are discarded. b. Top Cross test: The inbreds , which are selected on phenotypic evaluation, are crossed to a tester with wide genetic eg . An OPV , a synthetic variety or a double cross. A simple way of producing top cross seed in maize is to plant alternate rows of the tester and the inbred line and the inbred line has to be detasselled . The seed from the inbreds is harvested and it represents the top cross seed. The performance of top cross progeny is evaluated in replicated yield trains preferably over locations and years. c. Single cross evaluation: Out standing single cross combinations can be identified only by testing the performance of single cross. The remaining inbred lines after top cross test are generally crossed in diallel or line x tester mating design to test for SCA . A single cross plants are completely heterozygous and homogenous and they are uniform. A superior single cross regains the vigour and roductivity that was lost during inbreeding and can be more vigorous and productive than the original open pollinated variety. 418

419. GCA and SCA Certain inbred lines will display hybrid vigour when crossed. These vigorous lines are said to have favorable combining ability.  Certain inbreds have the ability to combine well with testers--these have general combining ability ( GCA ). When the inbred combines well only in certain crosses, it has specific combining ability ( SCA ). The only way to select for combining ability is to grow and examine the progeny. An astute breeder can recognize the potential for hybrid vigour by identifying the dominant traits of the parents and deducing which lines may combine favorably.  Predicting the combining ability of recessive traits can only be determined through progeny testing.  The breeder is interested in single crosses (also known as F1 generations) that outperform other single crosses. If the breeder has multiple IBLs to work with, she could select first for GCA , then for SCA among the lines with GCA , then identify the best parental gene donors. 419

420. σ 2 gca/ σ 2 sca= 1 (equal proportion) σ 2 gca/ σ 2 sca >1 (additive) σ 2 gca/ σ 2 sca <1( dominance or non-additive) -If gca variances are higher than sca variances-preponderance of additive gene action and progeny selection will be effective -If sca variances are higher-preponderance of non-additive gene action (dominance and epistasis ), heterosis breeding will be effecitve . -If both are equal-both are equally important, and reciprocal recurrent selection may be used. 420

421. Use of RRS (reciprocal recurrent selection) 1. Two populations are developed by this method 2. They may be intermated to produce a superior population with broad genetic base. This is similar to a varietal cross but in this case the populations have been subjected to selection for combining ability (GCA and SCA) 3. Inbreds may be developed from populations A and B. These inbreds may be crossed to produce a single cross or double cross hybrids. 421

422. Full-sib Reciprocal Recurrent Selection: Two heterotic populations are taken and S0 plants in these populations are simultaneously selfed and crossed in inter-population pairs to produce S1 and full-sib (FS) families, respectively. The FS families are evaluated and S1 seeds of S0 plants that have produced superior FS families are used to reconstitute the two populations separately. 422

423. Synthetic variety: A synthetic variety consists of the advanced generations of open pollinated seed mixtures of a group of strains, clones, inbreds or hybrids. The lines or genotypes are selected for uniformity and high GCA and the variety is maintained by open pollination. As a consequence, a synthetic variety is theoretically a variety in Hardy-Weinberg equilibrium. Hybrid variety: A variety produced from the cross fertilization of inbred lines with favourable SCA. The progeny is homogeneous and highly heterozygous. The inbred parent lines of the hybrid variety are derived from continous selfing of selected plants in normally cross pollinated species. At the moment, hybrid varieties are not very interesting for forage and turf grasses. Backcross breeding: A plant breeding system in which recurrent backcrosses are made to one of the parents of a hybrid, accompanied by specific selection for a particular character or set of characters. In backcross breeding the recurrent parent is the parent to which a hybrid is crossed in a backcross while the non recurrent parent ( donor parent ) is the parent of a hybrid which is not used for further crossing in a backcross. BC 1 , BC 2 …. BC n are symbols indicating the first, second and n th backcross generation, respectively. 423

424. Recurrent parent Donor parent aa AA Aa F 1 aa Aa BC 1 F 1 BC 2 F 1 aa Aa BC 3 F 1 BC 2 F 1 aa AA aa Aa Aa aa BC 4 F 1 Removed Removed Removed Removed Aa Removed Selfing (1) (2) (1) maintained (2) Removed Backcross for a dominant allele (1/2) n = (1/2) 5 = 0,031 Progeny test 424

425. Donor parent Recurrent parent Backcross for a recessive allele AA aa F 1 - Aa BC 1 F 1 S * * S = selfing BC 1 F 2 AA removed Aa removed aa maintained BC 2 F 1 - Aa BC 3 F 1 S BC 3 F 2 AA removed Aa removed aa maintained BC 4 F 1 - Aa BC 5 F 1 - Aa S BC 5 F 2 AA removed Aa removed aa maintained 425

426. Dr. Zekeria Yusuf (PhD) 426

427. BREEDING OF ASEXUALLY PROPAGATED CROPS Selection is straight forward in asexually propagated crops since any genotype may be perpetuated intact. Obtaining segregating populations from which superior genotypes may be found is the problem in breeding asexually propagated materials. Clone : A clone is a group of plants produced from a single plant through asexual reproduction. The crop plants can either be propogated by seeds or by vegetative parts. The vegetative propogation is resorted due to : 1. Lack of seed : Eg . Ginger, termiric 2. There is short viability of seed : Eg . Sugarcane 3. The seed production is very rare : Eg . Banana 4. Seeds are produced under special conditions only : Eg . Sugarcane, potato 427

428. Methods of Breeding Asexually Propagated Species Important breeding methods applicable to asexually propagated species are (1) Plant Introduction (2) Clonal selection (3) Mass selection (4) Heterosis breeding (5) Mutation breeding (6) Polyploidy breeding (7) Distant hybridization (8) Transgenic breeding. Mass selection is rarely used in asexually propagated species. 428

429. Characteristics of Asexually propagated crops : 1. Majority of them are perennials : Eg . Sugarcane, fruit trees. The annual crops are mostly tuber crops : Eg. Potato, cassava, Sweet potato 2. Many of them show reduced flowering & seed set 3. They are invariably cross pollinated 4. These crops are highly heterozygous and show severe inbreeding depression upon selfing. 5. Majority of asexually propagated crops are polyploids : Eg. Sugarcane, Potato, Sweet, Potato 6. Many species are interspecific hybrids. Eg. Banana, Sugarcane 429

430. Characteristics of a clones : 1. All the individual belonging to a single clone are identical in genetype 2. The phenotypic variation within a clone in due to environment only 3. The phenotype of a clone is due to the effects of genotype(g), the environment(e) and the genotype x environment interaction ( GxE ), over the pop.mean (M) 4. Theoratically clones are immortal. They deteriorate due to viral/bacterial infection and mutations. 5. Clones are highly heterozygous and stable 6. They can be propagated generation after generation without any change. Importance of a clone 1. Owing to heterozygosity and sterility in many crops clones are the only means of propagation. 2. Clones are used to produce new varieties. 3. Clones are very useful tools to preserve the heterozygosity once obtained. In many crops the superior plants are maintained. (Mango, orange, apple, sugarcane) Sources of clonal selection : 1. Local varieties 2. Introduced material 3. Hybrids and 4. Segregating populations 430

431. Clonal selection : The various steps involved in clonal selection are briefly mentioned below. First year : From a mixed variable population, few hundred to few thousand desirable plants are selected. Rigid selection can be done for simply inherited characters with high heritability. Plants with obvious weakness are eliminated. Second year : Clones from the selected plants are grown separately, generally without replication. This is because of the limited supply of propagating material for each clone, and because of the large number of the clones involved. Characteristics of the clones will be more clear now than in the previous generation. Based on the observations the inferior clones are eliminated. 431

432. observations and on judgement of the breeder on the value of clones. Fifty to one hundred clones are selected on the basis of clonal characteristics. Third year : Replicated preliminary yield trial is conducted. A suitable check is included for comparison few superior performing clones with desirable characteristics are selected for multilocation trials. At this stage, selection for quality in done. If necessary, separate disease nurseries may be planted to evaluate disease resistance of the clone s. Fourth to eighth years : Replicated yield trials are conducted at several locations along with suitable check. The yielding ability, quality and disease resistance etc. of the clones are rigidly evaluated. The best clones that are superior to the check in one or more characteristics are identified for release as varieties. Ninth year : The superior clones are multiplied and released as varieties. 432

433. Problems in Breeding asexually propagated crops: 1. Reduced flowering and fertility 2. Difficulties in genetic analysis 3. Perennial life cycle. Genetic variation within a clone may arise due to : 1. Mutation 2. Mechanical mixture 3. Sexual Reproduction 433

434. P lant breeding for Biotic stresses Dr. Zekeria Yusuf (PhD) 434

435. I. Breeding for Insect Resistance Most important because many crops are affected by insects. For e.g. Cotton is attacked by more than 160 species of insects of these a dozen are major pests . The necessity for resistance breeding are: i ) Environmental pollution prevention ii) Reducing Higher costs iii) Death of Beneficial Predators and Parasites. iv) Building up of Resistance - e.g. Pyrethroid . Mechanism of Insect Resistance: Non preference , Antibiosis , Tolerance, Avoidance .

436. Non preference : Non acceptance or Antixenosis Un attractive or unsuitable for colonization, Oviposition or both by an insect pest. Aphid resistance in raspberry. It involves various morphological and biochemical features of host plants. Antibiosis: adverse effects caused by the host to an insect feeding on it . It may hinder the development, reproduction or in some cases death also. The antibiosis may be either. i ) Morphological, ii) Physiological, iii) Biochemical features of the host plant. e.g. Gossypol content in cotton. Tolerance: Able to tolerate the attack, withstand and give yield. Avoidance: Insects avoid certain plants. Early maturing cotton varieties escape pink bollworm. Sorghum early lines escape shoot fly attack.

437. Nature of Insect Resistance 1. Hairiness: Hairiness of leaves is associated with resistance. Eg . Jassid resistance – cotton and cereal leaf beetle. 2. Colour of Plant: Induces non-preference for oviposition . Red cabbage - Lepidopteran . Red colour Cotton - Boll worms. 3. Thickness of plant Tissue: Cotton - Jassid resistance. Dense thick leaves - It is more of mechanical obstruction. 4. Presence of Silica in Plant Body: Shoot fly resistance in sorghum - Damage to mandibles. 5. Biochemical Factor: Gossypol content, DIMBOA content in leaves, (Bio chemical) – Stem borer in maize. 6. Physiological Factors: Osmotic concentration of cell sap, cell exudaters etc. Solanum sp - Gum exudate - Aphids are trapped in it. Genetics of Insect resistance : 1 . Oligogenic-Monogene 3 : 1. e.g. Jassid resistance , Cotton Wheat rust resistance Green bug resistance. 2. Polygenic More durable Wheat cereal leaf beetle resistance. 3. Cytoplasmic Plasmogenes : European corn borer in maize.

438. Sources of Resistance 1. Cultivated variety 2. Germplasm Collection. 3. Related Wild species - S.nitidum - shoot fly resistance – Sorghum, G.anamalum – Jassid resistance - Cotton. Screening Technique a) Field condition : i ) Infector rows are planted at regular intervals. ii) Testing in areas where ever the pest is recorded as endemic area . iii) Seasonal testing when insect population is most. iv) Rearing the insect in lab and releasing them in fields or by transferring equal no. of eggs or larvae to each plant. b) Glass House Screening Raised in cages and definite number of larvae are released in the cage.

439. II. Breeding for Disease Resistance Need for Disease Resistance Breeding i ) To prevent yield loss. ii) High cost of reduction . iii) Prevention of environmental pollution. Kinds of Disease Reaction i ) Susceptible reaction : Disease reaction is profuse, if unchecked it may lead to total yield loss . ii) Immune reaction : Host does not show the symptoms of a disease. iii) Resistance reaction : Infection and establishment takes place but growth of the pathogen in the host is restricted. iv) Tolerance : Host is attacked by the pathogen in the same manner as the susceptible variety but there may not be yield loss.

440. Mechanism of Disease Resistance a) Mechanical : Certain mechanical or anatomical features of host may prevent infection e.g . Closed flowering habit of wheat and barley prevents infection by spores of ovary infecting fungi. b) Hypersensitivity : Immediately after infection several host cells surrounding the point of infection die. This leads to death of pathogen also. Phytoalexins present in plant body is responsible for hypersensitivity reaction. c) Antibiosis : Presence of some toxic substance. This is more correct for insect resistance . e.g . Gossypol content in cotton. d) Nutritional factors : The reduction in growth and spore formation may be due to nutritional factors of the host. Genetics of Disease Resistance a) Oligogenic Resistance: Resistance is governed by one or few major genes and resistance is generally dominant. The action of major genes may be altered by modifiers.

441. Methods of Disease Resistance Breeding 1. Plant introduction: Resistant varieties from other can be directly introduced for cultivation . e.g. IR 20 rice resistant to blast. 2. Selection: This may be from local land races or from introduced cultivars. 3. Hybridisation and Selection: depending on gene action the selection procedure may vary. If the resistance is governed by polygenes , then pedigree method of selection is to be followed. If the resistance is governed by major genes linked with other undesirable characters we have to go for back cross method of breeding . 3. Mutation Breeding 4. Polyploidy Breeding: Nicotiana crosses for resistance against leaf spot . 5. Tissue Culture Method: Resistance reaction can be screened easily in test tubes and resistant lines can be mass multiplied. e.g . Banana

442. Screening Techniques for Disease Resistance Depending on mode of spread of disease the screening technique may differ. The screening can be done both at screen or glass house level and field level. The different screening techniques are as follows. 1. Soil Borne Diseases Wilt, root rot are produced by soil borne fungi. In this case sick plot technique is followed. Susceptible varieties can be grown and infected plants can be ploughed insitu to maintain optimum condition for infection. 2. Air Borne Diseases e.g . Rust, Smut, mildews, blights. For ground nut rust, infestor rows can be sown 15 days earlier as border rows and the disease will infest the susceptible infestor rows. After 15 days the varieties tested to be are to be sown. Spraying the spore suspension from affected leaves will also increase the load .

443. Seed Borne Disease Smut, bunt etc. Artificial inoculation can be done by soaking the seeds in solution of pathogen under vaccum condition. Insect transmitted Diseases e.g. Virus Diseases, Red gram sterility mosaic virus. Sap transmitted. Here the stapling technique is used. Leaves from affected plants can be stapled to the entries to be tested. The insect feeding in susceptible leaf will transmit virus to test entries.

444. Breeding for Abiotic Stress Resistance (Drought, Cold, Salinity & alkalinity) Dr. Zekeria Yusuf (PhD) 444

445. Dr. Zekeria Yusuf (PhD) 445

446. Dr. Zekeria Yusuf (PhD) 446

447. 1. Temperature Stress a. Cold resistance/Tolerance b. High Temperature : Due to high temperature seed set may be affected. In case of male sterile lines, the sterility may be broken down. In this case also testing single plants for high temperature resistance is time consuming and skill is required. Tests like heat test with leaf discs and desiccation tolerance test are followed. 2.Water Stress a. Low water i.e., Drought resistance: This is more important for all the dry land crops.

448. 3. Chemical Stress Salinity and alkalinity : Screening for salinity and alkalinity can be done more successfully by in vitro techniques. Raising the seedling in test tube containing different concentration of salt. This is followed in case of pesticide and herbicide tolerance also. Difficulties in Abiotic Stress Breeding i . Screening techniques require high skill and they are time consuming. ii. Creation of artifical conditions is expensive. iii. Under field screening, nature may or may not provide optimum condition for screening. iv. In many cases in vitro techniques are to be followed which is expensive. v. Abiotic stress breeding depends mostly on physiological traits which are often not stable.

449. Drought resistance in crop plants can be divided in to three categories. Drought escape - ability of a plant to complete its life cycle before serious soil and plant water deficit occurs. ii. Drought tolerance with high tissue water potential. iii. Drought tolerance with low tissue water potential . Drought resistance in crop plants are more due to physiological conditions of plant like stomatal aperture & photosynthetic rates, root characteristics.

450. C. Breeding for Drought Resistance 1. Breeder search for a source for Drought resistance. 2. Yield should be a secondary character economic Parts. 3. Partitioning of Photosynthates Vegetative Parts Total Dry matter should be taken as a criterion for selection. Drought Resistance Drought avoidance & Drought tolerance 1. Xeromorphic traits 2 . Root Growth 3 . Stomal control , 4. Cuticular resistance(water permeability of leaf cuticle) 4. Stomatal number (transpiration low, low stomatal frequency & high photosynthetic rate) 5. Cell turgor (Inhibit plant growth) (root water absorption & stomatal water loss)

451. Breeding for Drought resistance variety -High yield x High cuticular wax content (Poor cuticular Transpiration). -F1 (F1 tested under moisture stress condition). -F2 1. Progeny rows screened in moisture stress nursery in two locations. 2. Selection based on cuticular wax & a number of agronomic characters are considered. F3 Selected single plants - Screened under normal conditions for yield and then associated characters . F4 Selected single plants - Screened under stress situation. F5 Normal condition - yield. F7/F8 1. Homogeneity with relative resistance to drought and with considerable yield. 2. Converge genes for yield and drought resistance.

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453. Biotechnology Biotechnology: the use of biological organisms, biological cells, biological materials and bioprocess(: fermentation) for manufacturing and industry. The application of biotechnological methods in plant breeding Cell and tissue culture Genetic engineering and recombinant DNA technology Dr. Zekeria Yusuf (PhD) 488

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566. Seed Technology Seed technology is the creation and application of the knowledge on seed for its better usage in agriculture. Seed technology refers to methods or techniques used to maintain the quality of seed from harvest till it is germinated. Scope of Seed Technology 1. Seed technology encompasses all activities carried out to enhance storability, germinability , vigour and health of the seed. 2. Activities include harvesting, transporting, handling, storage, testing, grading, documentation, processing of seeds and germination of seeds. Classes of seed Nucleus or Basic Seed Breeder seed Foundation seed Certified seed 603 Haramaya University

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568. Nucleus or Basic Seed Nucleus seed (or basic seed) is the original or first seed (= propagating material ) of a variety available with the producing breeder or any other recognized breeder of the crop. This seed has 100% genetic & physical purity along with high standards of all other seed quality parameters . When a new variety is released there is very little seed. There may be only a handful of seed selected by the breeder from individual plants. This seed is the basis of a variety and is known as the Nucleus Stock. This nucleus stock must be managed with great care so that all seed produced from it remains true to the new variety. This is a most important step and is the responsibility of the plant breeder who developed the variety. The nucleus stock seed is not available to farmers. The next step in the chain from plant breeder to farmer is that the plant breeder develops Breeder Seed. 605 Haramaya University

569. BREEDER SEED Breeder seed is the seed of the highest purity of the new variety. It is produced by the breeder and provided by the breeder’s institution to agencies for further multiplication. If you are from a non-governmental organization (NGO) seed business or a private company that is producing seed, you may need to purchase breeder seed from a research institution. Breeder seed is the most expensive seed to buy. Breeder seed : seed or vegetative propagating material directly produced or controlled by the originating plant breeder or institution. Breeder seed provides the source for the increase of foundation seed. It is usually limited in quantity. Breeder seed is the progeny of the nucleus seed and is the source for foundation seed. Its production is directly controlled by the originating plant breeder who developed the variety, or any other institution or qualified breeder recognized by the authorities . 606 Haramaya University

570. FOUNDATION SEED Foundation seed is the seed produced from growing breeder seed. It is produced by trained officers of an agricultural station to national standards and handled to maintain the genetic purity of the variety. It may be produced by a government seed production farm or a private organization – this will depend on the regulations of the country. Foundation seed is less expensive than breeder seed. Also know as elite or basic seed. It is the direct increase form breeder seed. The genetic identity and purity of the variety is carefully maintained in foundation seed. Foundation seed is the source of certified seed. 607 Haramaya University

571. REGISTERED SEED Registered seed is produced from growing foundation seed. It is grown by selected farmers in a way that maintains genetic purity. Production has undergone field and seed inspections by Seed Inspectors to ensure conformity with standards. 608 Haramaya University

572. CERTIFIED SEED Certified seed is produced from growing foundation, registered or certified seed. It is grown by selected farmers to maintain sufficient varietal purity.  Production is subject to field and seed inspections prior to approval by the certifying agency. Harvest from this class is used for producing again. Certified seed is the seed, which is certified by a Seed Certification Agency Generally, it is known as the progeny of foundation seed and its production is so handled as to maintain specified genetic identity and purity standards as prescribed for the crop being certified . 609 Haramaya University

573. Requirements of Certified seed Seed has to meet certain rigid requirements before it can be certified for distribution. Seed must be of an improved variety released by either Central or State Variety Release Committee for general cultivation and notified by the Ministry of Agriculture. Genetic purity. Physical purity. Germination . Freedom from Weed seeds. Freedom from Diseases. Optimum Moisture Content. 610 Haramaya University

574. QUALITY DECLARED SEED Truthfully labeled seed or Quality Declared Seed  is produced from foundation, registered or certified seed. It is not subject to inspection by a certifying agency. As this seed is not inspected, its quality is dependent on the good reputation of the farmer who has grown the seed. His good name in the village is important. 611 Haramaya University

575. Genetic Diversity : the differences that distinguish one plant from another are encoded in the plant’s genetic material, the DNA. DNA is packaged in chromosome pairs, one coming from each parent. The genes, which control a plant’s characteristics, are located on specific segments of each chromosome. 612 Haramaya University Measurement of Genetic diversity

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577. Why genetic diversity is important in populations... genetic diversity required to evolve or to adapt to new environment or environmental modifications. genetic diversity reflects evolutionary potential Loss of genetic diversity often associated with inbreeding, reduction of reproductive fitness and extinction risk example 1 - habitat selection: peppered moth ( Biston betularia ) in UK - dark and light forms - night: active / day: resting on trees ➡ camouflage critical for survival - light form: camouflaged on lichen-covered tree trunks - Industrialisation : kill lichen by sulphur pollution ➡ light form: visible / dark form: camouflaged Haramaya University 614

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579. Variation from recombination linkage equilibrium —repeated recombination between genes, randomizing combinations of alleles of different genes Example: if frequency of allele a is 0.2 and frequency of b is 0.4, under linkage equilibrium: frequency of ab = 0.2*0.4 = 0.08 616 Haramaya University

580. Variation from recombination linkage disequilibrium - original nonrandom association between alleles of different genes on same chromosome linkage disequilibrium changes slowly through time, at rate proportional to amount of recombination between genes genetic variation through recombination can be much faster than through mutation 617 Haramaya University

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591. Haramaya University 628 Methods used to measure genetic variation at molecular level:

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593. Genetic Mapping Genetic mapping is based on the principle that genes (markers or loci) segregate via chromosome recombination during meiosis (i.e. sexual reproduction), thus allowing their analysis in the progeny (Paterson, 1996). What is mean genetic markers? Genetic markers represent genetic differences between individual organisms or species They do not represent the target genes themselves but act as ‘ signs’ or ‘ flags’ Genetic markers that are located in close proximity to genes may be referred to as ‘ gene tags’ All genetic markers occupy specific genomic positions within chromosomes called ‘ loci’ 630 Haramaya University

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595. Genetic Markers A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP ), or a long one, like minisatellites . Haramaya University 632

596. Genetic Markers Genetic markers are the biological features that are determined by allelic forms of genes or genetic loci and can be transmitted from one generation to another, and thus they can be used as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene. Genetic markers used in genetics and plant breeding can be classified into two categories: classical markers and DNA markers. Classical markers include morphological markers , cytological markers and biochemical markers. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods ( southern blotting–nuclear acid hybridization, PCR –polymerase chain reaction, & DNA sequencing ) such as RFLP , AFLP , RAPD , SSR , SNP , etc. 633 Haramaya University

597. Morphological markers During the early history of plant breeding, the markers used mainly included visible traits, such as leaf shape, flower color, pubescence color, pod color, seed color, seed shape, hilum color, awn type & length , fruit shape, rind ( exocarp ) color and stripe, flesh color, stem length, etc. These morphological markers generally represent genetic polymorphisms which are easily identified & manipulated. Therefore, they are usually used in construction of linkage maps by classical two- and/or three point tests . Some of these markers are linked with other agronomic traits and thus can be used as indirect selection criteria in practical breeding. 634 Haramaya University

598. Cytological markers In cytology, the structural features of chromosomes can be shown by chromosome karyotype and bands. The banding patterns, displayed in color, width, order and position, reveal the difference in distributions of euchromatin and heterochromatin. For instance , Q bands are produced by quinacrine hydrochloride, G bands are produced by Giemsa stain, and R bands are the reversed G bands. These chromosome landmarks are used not only for characterization of normal chromosomes and detection of chromosome mutation, but also widely used in physical mapping and linkage group identification. The physical maps based on morphological and cytological markers lay a foundation for genetic linkage mapping with the aid of molecular techniques. 635 Haramaya University

599. Biochemical/protein markers- Allozymes & Isozyme Biochemical/protein markers: Protein markers may also be categorized into molecular markers. Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution. Therefore , iso‐ zyme markers can be genetically mapped onto chromosomes and then used as genetic markers to map other genes . They are also used in seed purity test and occasionally in plant breeding . There are only a small number of isozymes in most crop species and some of them can be identified only with a specific stain . Therefore, the use of enzyme markers is limited . Another example of biochemical markers used in plant breeding is high molecular weight glutenin subunit ( HMW -GS) in wheat . 636 Haramaya University

600. Biochemical Marker - Allozymes ( Isozyme )…. Allozymes are allelic variants of enzymes encoded by structural genes. Because of changes in electric charge and conformation can affect the migration rate of proteins in an electric field, allelic variation can be detected by gel electrophoresis and subsequent enzyme-specific stains that contain substrate for the enzyme, cofactors and an oxidized salt (e.g. nitro-blue tetrazolium ). Usually two, or sometimes even more loci can be distinguished for an enzyme and these are termed isoloci . Therefore, allozyme variation is often also referred to as isozyme variation . Although protein markers circumvent the effects of environment, they have the drawbacks of a limitation in the number of detectable isozymes as well as tissue and development stage specificity. 637 Haramaya University

601. Biochemical Marker - Allozymes ( Isozyme )…. Advantages: The strength of allozymes is simplicity. Because allozyme analysis does not require DNA extraction or the availability of sequence information, primers or probes, they are quick and easy to use. Simple analytical procedures, allow some allozymes to be applied at relatively low costs, depending on the enzyme staining reagents used. Allozymes are codominant markers that have high reproducibility. Zymograms (the banding pattern of isozymes ) can be readily interpreted in terms of loci and alleles, or they may require segregation analysis of progeny of known parental crosses for interpretation. Sometimes, however, zymograms present complex banding profiles arising from polyploidy or duplicated genes and the formation of intergenic heterodimers , which may complicate interpretation. 638 Haramaya University

602. Biochemical Marker - Allozymes ( Isozyme )…. Disadvantages: relatively low abundance and low level of polymorphism. Moreover, proteins with identical electrophoretic mobility (co-migration) may not be homologous for distantly related germplasm . In addition, their selective neutrality may be in question. Lastly, often allozymes are considered molecular markers since they represent enzyme variants, and enzymes are molecules. However, allozymes are in fact phenotypic markers , and as such they may be affected by environmental conditions. For example, the banding profile obtained for a particular allozyme marker may change depending on the type of tissue used for the analysis (e.g. root vs. leaf). This is because a gene that is being expressed in one tissue might not be expressed in other tissues . On the contrary, molecular markers, because they are based on differences in the DNA sequence, are not environmentally influenced, which means that the same banding profiles can be expected at all times for the same genotype. 639 Haramaya University

603. DNA markers Most widely used type of marker predominantly due to their abundance Selectively neutral because they are non-coding sequences Not affected by environmental factors and/or the developmental stage of the plant Numerous applications in plant breeding such as assessing the level of genetic diversity within germplasm and cultivar identity 640 Haramaya University

604. DNA Makers/ Molecular markers DNA markers are defined as a fragment of DNA revealing mutations/variations, which can be used to detect polymorphism between different genotypes or alleles of a gene for a particular sequence of DNA in a population or gene pool. A molecular marker is segment of DNA whose characteristics can be measured and make inference to the ecology and evolution of individuals, populations, and species There are three methods to detect the polymorphism: Southern blotting , a nuclear acid hybridization technique (Southern 1975 ), PCR , a polymerase chain reaction technique ( Mullis, 1990), as well as microarray chip techniques use DNA hybridization combined with labeled nucleotides, and new sequencing techniques detect polymorphism by sequencing. Using PCR and/or molecular hybridization followed by electrophoresis (e.g. PAGE – polyacrylamide gel electrophoresis, AGE – agarose gel electrophoresis, CE – capillary electrophoresis ), the variation in DNA samples or polymorphism for a specific region of DNA sequence can be identified based on the product features, such as band size and mobility. In addition to Sothern blotting and PCR , more detection systems have been also developed. 641 Haramaya University

605. Applications of molecular markers DNA marker systems, which were introduced to genetic analysis in the 1980s , have many advantages over the traditional morphological & protein markers that are used in genetic & ecological analyses of plant populations : 1. an unlimited number of DNA markers can be generated ; 2. DNA marker profiles are not affected by the environment , & DNA markers , unlike isozyme markers, are not constrained by tissue or developmental stage specificity . 4. Genetic Diversity Measurements -Selecting what genotypes to use in breeding -Narrowing germplasmsearches (only if less costly then phenotyping !) -Managing germplasmcollections 5. Intellectual Property Protection -Preventing others from using your proprietary technology 6. Food Safety -Detecting transgenes -Detecting pathogens 7. QTL Mapping -We will discuss today 8. Marker-Assisted Selection -Backcrossing in a transgene -Maintaining or crossing in a QTL 9. Genomic Selection 642 Haramaya University

606. Properties of ideal molecular markers An ideal molecular marker must have some desirable properties which are enlisted as follows: 1. Highly polymorphic/ hypervariable nature : It must be polymorphic as it is polymorphism that is measured for genetic diversity studies. 2. Codominant inheritance : discrimination of homozygous and heterozygous states of diploid organisms . 3. Frequent occurrence in genome : a marker should be evenly and frequently distributed throughout the genome. 4. Selective neutral behaviours : the DNA sequences of any organism are neutral to environmental conditions or management practices. 4. High reproducibility: giving same result across labs. 5. Even distribution across the whole genome (not clustered in certain regions) 6. Clear distinct allelic features (so that the different alleles can be easily identified ) 7. Low cost to use (or cost-efficient marker development and genotyping) 8. Easy assay/detection & automation 9. High availability (un-restricted use) and suitability to be duplicated/multiplexed (so that the data can be accumulated and shared between laboratories) 10. Single copy & Genome-specific in nature (especially with polyploids ) 11. No detrimental effect on phenotype 643 Haramaya University

607. Properties of ideal molecular markers… 12. Freedom from environmental and pleiotropic effects . Molecular markers do not exhibit phenotypic plasticity, while morphological and biochemical markers can vary in different environments. DNA characters have a much better chance of providing homologous traits. Most morphological or biochemical markers, in contrast, are under polygenic control, and subject to epistatic control & environmental modification (plasticity ); 13. Potentially unlimited number of independent markers are available , unlike morphological or biochemical data; 14. DNA characters can be more easily scored as discrete states of alleles or DNA base pairs , while some morphological, biochemical and field evaluation data must be scored as continuously variable characters that are less amenable to robust analytical methods; These advantages do not imply that other more traditional data used to characterize biodiversity are not valuable. On the contrary, morphological , ecological and other “traditional” data will continue to provide practical and often critical information needed to characterize genetic resources. 644 Haramaya University

608. Properties of ideal molecular markers… It is extremely difficult to find a molecular marker, which would meet all the above criteria. A wide range of molecular techniques is available that detects polymorphism at the DNA level. Depending on the type of study to be undertaken, a marker system can be identified that would fulfill at least a few of the above characteristics. Various types of molecular markers are utilized to evaluate DNA polymorphism and are generally classified as hybridization-based markers and polymerase chain reaction ( PCR )-based markers. In the former, DNA profiles are visualized by hybridizing the restriction enzyme-digested DNA, to a labeled probe(which is a DNA fragment of known origin or sequence). PCR based markers involve in vitro amplification of particular DNA sequences or loci, with the help of specifically or arbitrarily chosen oligonucleotide sequences (primers) and a thermos table DNA polymerase enzyme . The amplified fragments are separated electrophoretically and banding patterns are detected by different methods such as staining and autoradiography. 645 Haramaya University

609. Genetic markers: dominance / co-dominance principle dominance: when heterozygotes are not distinguishable from homozygotes ‣ AA with PCR product, aa without PCR product, Aa with PCR product co-dominance: when heterozygotes are distinguishable from homozygotes ‣ AA with low mobility, aa with high mobility, Aa with a medium mobility → difficulties in the analyses Haramaya University 646

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613. Applications of molecular markers Assessment of genetic diversity and characterization of germplasm Identification & fingerprinting of genotypes Estimation of genetic distance between populations, inbreds & breeding materials Detection of monogenic / qualitative & quantitative trait loci( QTL ) Marker assisted breeding Identification of sequences of useful candidate genes etc. Haramaya University 650

614. CLASSIFICATION OF DNA MARKERS Based on the method of their detection broadly divided into Hybridization-based PCR based DNA sequence-based Visualises the genetic differences by gel electrophoresis Staining with chemicals ( ethidium bromide or silver) Detection with radioactive or colourimetric probes Polymorphic DNA markers : helps in revealing the differences between individuals of the same or different species. Monomorphic DNA markers: Markers that do not differentiate between genotypes are called monomorphic markers Haramaya University 651

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618. First generation molecular markers The first generation of DNA marker systems employed Southern blot based markers. RFLPs (restriction fragment length polymorphisms ): result from point mutations in restriction enzyme recognition sites. Chromosomal mutations such as insertions, deletions, inversions, and translocations can also cause restriction fragment size polymorphisms. The RFLP technique employs molecular hybridization of cDNA or genomic DNA probes with genomic DNA fragmented by restriction enzymes . Another Southern blot based marker system relied on minisatellite probes for ‘fingerprinting ’ individual specific human DNA . Minisatellite DNAs are short stretches of DNA that are present in tandem repeats in eukaryotes. They are highly abundant, and individuals often carry different numbers of tandem repeats which can be detected as VNTRs (variable number of tandem repeat) by PCR amplification. 655 Haramaya University

619. Minisatellites , Variable Number of Tandem Repeats ( VNTR ) The term DNA fingerprinting was introduced for minisatellites , though DNA fingerprinting is now used in a more general way to refer to a DNA-based assay to uniquely identify individuals. Minisatellites are particularly useful in studies involving genetic identity, parentage, clonal growth and structure, and identification of varieties and cultivars , and for population-level studies. Minisatellites are of reduced value for taxonomic studies because of hypervariability . DNA fingerprints ( minisatellites ) ‣ core repeat sequences of 10-100 bp highly variable, high quantity of DNA, difficult to set-up / old method 656 Haramaya University

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624. Second Generation DNA Markers The second generation DNA markers for genetic analysis were those derived from PCR polymerase chain reaction . PCR revolutionized genetic and ecological analyses of populations in several ways because it had two major advantages over Southern blot based markers: it requires only small DNA amounts to allow analysis at very early stages, thus reducing the need for plant nurseries. it is inexpensive, and simple enough that large scale experiments can be carried out rapidly on a large scale. Of the many PCR -marker techniques that have been developed, RAPD , AFLP ISSR , SSR and SNP are the major systems, with the other systems being modifications of these three. 661 Haramaya University

625. Inter Simple Sequence Repeats ( ISSR ) ISSR involves amplification of DNA segments present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction. The technique uses microsatellites as primers in a single primer PCR reaction targeting multiple genomic loci to amplify mainly inter simple sequence repeats of different sizes. The microsatellite repeats used as primers for ISSRs can be di -nucleotide, trinucleotide , tetranucleotide or penta -nucleotide. ISSRs use longer primers (15–30 mers ) as compared to RAPD primers (10 mers ), which permit the subsequent use of high annealing temperature leading to higher stringency. In contrast to the SSR marker technique that amplifies with primers located on the flanking single copy DNA, microsatellites anchored primers that anneal to an SSR region can amplify regions between adjacent SSRs . The ISSR technique uses primers that are complimentary to a single SSR and anchored at either the 5' or 3' end with a one- to three-base extension. The amplicons generated consist of regions between neighbouring and inverted SSRs . As a result, the high complex banding pattern obtained will often differ greatly between genotypes of the same species. Haramaya University 662

626. Microsatellites or Simple sequence Repeat ( SSR ) SSRs , also called microsatellites, Simple Sequence Repeats ( SSRs ), short tandem repeats ( STRs ) or sequence-tagged microsatellite sites ( STMS ) , are sections of DNA, consisting of tandem repeats of short nucleotide motifs (1-6 bp /nucleotides long), mono-, di -, tri-,tetra- or penta -nucleotide units that are randomly arranged throughout the genomes of most eukaryotic species. e.g. (GT)n, ( AAT )n and ( GATA )n. Microsatellite markers, developed from genomic libraries, can belong to either the transcribed region or the non transcribed region of the genome, and rarely is there information available regarding their functions. Because the DNA sequences flanking microsatellite regions are usually conserved, primers specific for these regions are designed for use in the PCR reaction. SSR loci are individually amplified by PCR using pairs of oligonucleotide primers specific to unique/conserved DNA sequences flanking the SSR sequence. In addition, primers may be used that have already been designed for closely related species. 663 Haramaya University

627. SSR … The PCR -amplified products can be separated in high-resolution electrophoresis systems (e.g. AGE and PAGE) and the bands can be visually recorded by fluorescent labeling or silver-staining. Microsatellites, like minisatellites , represent tandem repeats, but their repeat motifs are shorter. Polymerase slippage during DNA replication, or slipped strand mispairing , is considered to be the main cause of variation in the number of repeat units of a microsatellite, resulting in length polymorphisms that can be detected by gel electrophoresis. Microsatellite sequences are especially suited to distinguish closely related genotypes; because of their high degree of variability/polymorphism, they are, therefore, favoured in population studies & for the identification of closely related cultivars. 664 Haramaya University

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629. The third generation of molecular markers The third generation of molecular markers is the system that utilizes SNPs (single nucleotide polymorphisms ) and microarrays. Compared to the gel-based molecular marker systems, SNP detection and analysis can be carried out with non-gel based assays . The polymorphism of a single base difference can be assessed by through-put analysis: hybridization with allele-specific oligonucleotides ( ASO ) , primer extension, oligonucleotide ligation assay (OLA) , & invasive cleavage. The frequency of the SNPs can range from approximately one per 30 bp to one per 500 bp in other plant species. Although these new generation marker systems are powerful tools in linkage disequilibrium analysis, germplasm assay by haplotyping , QTL (quantitative trait loci) analysis and a few others, they are only amenable to use in those species for which extensive nucleotide sequence information is available in major crops. 666 Haramaya University

630. SNP markers An SNP is a single nucleotide base difference between two DNA sequences or individuals . SNPs can be categorized according to nucleotide substitutions either as transitions (C/T or G/A) or transversions (C/G, A/T, C/A or T/G ). In practice, single base variants in cDNA (mRNA) are considered to be SNPs as are single base insertions and deletions ( indels ) in the genome. The fact that in many organisms most polymorphisms result from changes in a single nucleotide position (point mutations), has led to the development of techniques to study single nucleotide polymorphisms ( SNPs ). SNPs provide the ultimate/simplest form of molecular markers as a single nucleotide base is the smallest unit of inheritance, and thus they can provide maximum markers . SNPs occur very commonly in animals and plants. Typically , SNP frequencies are in a range of one SNP every 100-300 bp in plants. SNPs may present within coding sequences of genes, non-coding regions of genes or in the intergenic regions between genes at different frequencies in different chromosome regions . 667 Haramaya University

631. Retrotransposon -based markers Retrotransposons consist of long terminal repeats ( LTR ) with a highly conserved terminus, which is exploited for primer design in the development of retrotransposon -based markers . Retrotransposons have been found to comprise the most common class of transposable elements in eukaryotes , and to occur in high copy number in plant genomes. Several of these elements have been sequenced and were found to display a high degree of heterogeneity and insertional polymorphism, both within and between species. Because retrotransposon insertions are irreversible , they are considered particularly useful in phylogenetic studies. In addition , their widespread occurrence throughout the genome can be exploited in gene mapping studies, and they are frequently observed in regions adjacent to known plant genes. 668 Haramaya University

632. Several variations of retrotransposon -based markers exist: 1. Sequence-Specific Amplified Polymorphism (S-SAP) is a dominant , multiplex marker system for the detection of variation in DNA flanking the retrotransposon insertion site. 2. Retrotransposon containing fragments are amplified by PCR , using one primer designed from the conserved terminus of the LTR and one based on the presence of a nearby restriction endonucleases site. Experimental procedures resemble those used for AFLP analysis and they are usually dominant markers . Interretrotransposon Amplified Polymorphism ( IRAP ) and Retrotransposon - Microsatellite Amplified Polymorphism (REMAP ) are dominant, multiplex marker systems that examine variation in retrotransposon insertion sites. Retrotransposon - Based Insertional Polymorphism ( RBIP ) is a codominant marker system that uses PCR primers designed from the retrotransposon and its flanking DNA to examine insertional polymorphisms for individual retrotransposons . A drawback of the method is that sequence data of the flanking regions is required for primer design. 669 Haramaya University

633. Gene microarrays Gene microarrays enable researchers to study all of the genes expressed in a tissue very fast Microarray (gene chip) is created with use of small glass microscope slide Single stranded DNA molecules are spotted on the slide using an arrayer (computer controlled robotic arm) which fixes DNA (multiple copies of cDNA ) at different spots on the slide which is recorded by a computer. Haramaya University 670

634. Gene microarrays… Procedure to do array: Extract mRNA from a tissue of interest and cDNA is synthesized from mRNA and labeled with fluorescent dye Labeled cDNA is incubated overnight with the array where it hybridizes with different spot on the array that contain complementary DNA sequences Can have over 10,000 spots of DNA Array is washed and scanned by a laser that causes cDNA hybridized to array to fluoresce Fluorescent spots reveal which genes were regulated and Intensity of fluorescence indicates relative amount of gene expression. Haramaya University 671

635. Haramaya University 672 In this example, mRNA levels are compared between the green and red stages of fruit development. First, mRNA is isolated from each tissue and reverse-transcribed in the presence of different fluorescent dyes resulting in labelled cDNA . Next, the two cDNA populations are mixed and hybridized to a cDNA microarray. Each array element contains DNA representing a different gene. The specific cDNAs from both populations, representing individual transcripts, will hybridize specifically with the probe on the corresponding array element.

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714. The real genetic and genomic world is not A’s and peas: Human genome: about 3 billion nucleotides, with about 3 million of them variable among any two random humans (99.9% identity ); most variants probably have no phenotypic effects (are ‘neutral’) Human Genome Project has provided the sequence (all online) of one human, but the most interesting and important data as regards health is the variation among humans , analyzed using the: HapMap ( Haplotype Map) project has characterized genetic variation among three major populations, one African, one Asian, one Caucasian (one or more common SNP genotyped at least every 5000 base pairs); > 1 million SNPs overall 1000 Genomes project : full sequences of 1000 humans -> rare variants SNP - single nucleotide polymorphism (2 or more bases at a locus) Haplotype - linear combination of SNPs or other markers on a chromosome such as C...C.... A.T ( haplotype 1), C...G.... A.T ( haplotype 2); sets of linked bases tend to be inherited together -- form flanked ‘blocks’ Microsatellites - repetitive elements with variable numbers of short repeats such as CAGCAGCAG ...or ATATAT - used as markers, and underly some diseases Copy number variation - variation in number of copies of large sections of genome , including one or more genes (large deletions, duplications) 751 Haramaya University

715. Some important findings from HapMap project (and earlier studies using other genetic markers) About 10-15% of total human genetic variation is among populations; rest is within populations Africa harbours substantially higher levels of human genetic variation than other regions Patterns of natural selection ‘for’ given alleles (positive selection) vary substantially among populations -> local adaptation 752 Haramaya University

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741. Molecular Breeding Molecular breeding (MB) may be defined in a broad-sense as the use of genetic manipulation performed at DNA molecular levels to improve characters of interest in plants and animals ( MAB+GMO ). Marker-assisted breeding ( MAB ) and is defined as the application of molecular biotechnologies, specifically molecular markers, in combination with linkage maps and genomics, to improve plant or animal traits on the basis of genotypic assays this term is covered several modern breeding strategies, including marker-assisted selection (MAS), marker-assisted backcrossing ( MABC ), marker-assisted recurrent selection (MARS), and genome-wide selection ( GWS ) or genomic selection (GS) ( Ribaut et al., 2010). Dr. Zekeria Yusuf (PhD) 778

742. Dr. Zekeria Yusuf (PhD) 779

743. Dr. Zekeria Yusuf (PhD) 780

744. Dr. Zekeria Yusuf (PhD) 781

745. Dr. Zekeria Yusuf (PhD) 782

746. Molecular breeding Molecular markers linked to specific phenotypic traits (Quantitative Trait Loci) are now being applied to screen for varieties with the desired traits prior to selection of their genetic material for incorporation into breeding programs. In the case where both the parental genotypes (donor and recipient) are known, the F1 progeny is designated as ‘determinate’. When F1 hybrids are „open pollinated‟ in the field, the resultant F2 generation is designated as ‘indeterminate’. The commercial seed industry is based on the development of determinate hybrids. Dr. Zekeria Yusuf (PhD) 783

747. Molecular Breeding Methods Marker Assisted Selection (MAS) Marker Assisted Backcross ( MABC ) Marker Assisted Pyramiding Marker Assisted Recurrent Selection (MARS) Quantitative Trait Loci ( QTL ) Genomic Selection Dr. Zekeria Yusuf (PhD) 784

748. QUANTITATIVE TRAITS Phenotypic characteristics are designated as „traits‟. Some traits such as flower color can be observed physically, whereas others such as „grain yield‟, „disease tolerance‟ and „herbicide resistance‟ need to be evaluated by subjecting the plant to specific challenges. DISCRETE and CONTINUOUS: When a DNA marker can be linked to a specific trait, it is referred to as a “Quantitative Trait Locus”. Dr. Zekeria Yusuf (PhD) 785

749. Linkage equilibrium Genomic loci are subject to genetic rearrangement via the twin processes of recombination & transposition. In some cases successive recombination events may result in two or more loci that appear to be linked to each other and are designated to be in „Linkage disequilibrium‟. The converse of the above phenomenon is „Linkage equilibrium‟. Dr. Zekeria Yusuf (PhD) 786

750. Variation from recombination Linkage equilibrium—repeated recombination between genes, randomizing combinations of alleles of different genes. Example: if frequency of allele a is 0.2 and frequency of b is 0.4, under linkage equilibrium: frequency of ab = 0.2*0.4 = 0.08. Linkage disequilibrium: original nonrandom association between alleles of different genes on same chromosome. Linkage disequilibrium changes slowly through time, at rate proportional to amount of recombination between genes. Genetic variation through recombination can be much faster than through mutation. Dr. Zekeria Yusuf (PhD) 787

751. Linkage Drag When two varieties of a specific crop plant, the „wild type‟ and „inbred line‟ are crossed to develop a novel F1 hybrid, the desirable traits from the „wild type‟ are acquired by the F1 generation, however the process may also result in the acquisition of some undesired traits. This phenomenon is known as ‘linkage drag’. The solution to this problem lies in backcrossing or in genetic modification using horizontal gene transfer into inbred lines. Dr. Zekeria Yusuf (PhD) 788

752. EPISTASIS AND ENVIRONMENTAL EFFECTS Not all F1 hybrids may exhibit the traits contained in the genetic material inherited from their parental genomes. This effect can be attributed to the phenomenon of „ Epistasis ‟ as well as the influence of environmental factors on the expression of specific genes. Dr. Zekeria Yusuf (PhD) 789

753. Dr. Zekeria Yusuf (PhD) 790

754. GENETIC MARKERS Marker Assisted Selection (MAS) relies on markers which are tightly linked to the locus expressing the desired trait. Ideally, two markers will be more accurate at predicting the presence or absence of the trait as compared to a single marker. The distance between the marker and the trait should not be in excess of 5 cM. Dr. Zekeria Yusuf (PhD) 791

755. Dr. Zekeria Yusuf (PhD) 792

756. Dr. Zekeria Yusuf (PhD) 793

757. Dr. Zekeria Yusuf (PhD) 797

758. ADVANTAGES OF MAS Simpler than phenotypic screening especially in the case of complex traits. Selection can be carried out at the seedling stage. Single plants can be selected: both homozygotes and heterozygotes can be identified. Reduction in the space required for breeding as only selected germplasm is propagated. Dr. Zekeria Yusuf (PhD) 798

759. Dr. Zekeria Yusuf (PhD) 799

760. Dr. Zekeria Yusuf (PhD) 800

761. Prerequisites for an efficient marker-assisted selection program High throughput DNA extraction 2. For efficient MAS (morphological, protein, cytological) markers should be: Ease of use Small amount of DNA required Low cost Repeatability of results High rate of polymorphism Occurrence throughout the genome Codominance 803 Haramaya University

762. Prerequisites for an efficient marker-assisted selection program… 3. Genetic maps: Linkage maps provide a framework for detecting marker-trait associations and for choosing markers to employ in MAS. Once a marker is found to be associated with a trait in a given population, a dense molecular marker map in a standard reference population will help identify markers that are closer to, or that flank, the target. 4. The most crucial ingredient for MAS is knowledge of markers that are associated with traits important to a breeding program. 5. Data management system Large numbers of samples are handled in a MAS program, with each sample potentially evaluated for multiple markers. This situation requires an efficient system for labeling, storing, retrieving, and analyzing large data sets, and producing reports useful to the breeder. 804 Haramaya University

763. MARKER ASSISTED BACKCROSSING DNA markers greatly increase the efficiency of selection. Useful in the case of screening for traits which are difficult to detect in the phenotype (e.g. insect resistance). Backcrossing reduced the level of introgression and results in a lower degree of linkage drag. Background selection is used to screen for integrity of the recurrent parent genome. Dr. Zekeria Yusuf (PhD) 808

764. Dr. Zekeria Yusuf (PhD) 809

765. Dr. Zekeria Yusuf (PhD) 810

766. Dr. Zekeria Yusuf (PhD) 812

767. MARKER ASSISTED PYRAMIDING Marker assisted pyramiding involves breeding several different varieties in order to develop a genetically distinct „pedigree‟. Markers can be developed for specific traits on each of the varieties being inbred and then applied to determine the gain of the trait or its subsequent loss over several cycles of breeding. Dr. Zekeria Yusuf (PhD) 814

768. Dr. Zekeria Yusuf (PhD) 815

769. Dr. Zekeria Yusuf (PhD) 816

770. Dr. Zekeria Yusuf (PhD) 818

771. EARLY GENERATION MAS Early generation MAS facilitates the elimination of F1 hybrids which do not carry the desired traits as reflected by their DNA profile. Single large scale ( SLS -MAS) relies on markers which are less than 5cM on either side of a locus. The selection of homozygotes or heterozygotes for a specific locus facilitates the linkage of heterozygosity on fitness. Dr. Zekeria Yusuf (PhD) 820

772. COMBINED MAS Phenotypic screening combined with MAS is essential because not all traits can be identified using molecular genetic approaches. Certain traits may be under the influence of more than one QTL . Dr. Zekeria Yusuf (PhD) 824

773. Dr. Zekeria Yusuf (PhD) 826

774. MAS ARE NOT PUBLISHED Commercial plant breeders do not publish MAS data as it may reveal information related to newly developed plant varieties. Although newly developed plant varieties are protected, release of information relate to DNA markers may compromise commercial interests. Dr. Zekeria Yusuf (PhD) 827

775. RELIABILITY AND ACCURACY Polygenic traits which are linked to more than one QTL are difficult to establish. In the case of small populations, sampling bias can result in loss of accuracy. A large toolbox of markers is required to establish QTLs with a high degree of precision. Dr. Zekeria Yusuf (PhD) 828

776. Dr. Zekeria Yusuf (PhD) 829

777. Dr. Zekeria Yusuf (PhD) 830

778. EFFECT OF GENETIC BACKGROUND Markers developed in one breeding population may not be effective in other breeding populations, a phenomenon which can be attributed to epistatic interaction between gene products. Dr. Zekeria Yusuf (PhD) 831

779. ENVIRONMENTAL EFFECTS In many cases the expression of specific genes is controlled by environmental cues and QTLs linked to these genes may be ineffective in determining the relationship between genotype and phenotype. HIGH COST OF MAS he development of MAS based breeding programs requires a significant investment in the isolation of molecular markers, testing of these markers as well as the establishment of inbred lines. These puts molecular markers beyond the reach of small breeders. Dr. Zekeria Yusuf (PhD) 832

780. APPLICATION GAP There is a lack of knowledge transfer between scientists at research laboratories and breeding stations. This may be the result of the need to protect Intellectual Property (IP). Research scientists are driven by the need to publish rather than to assist breeders in long-term field experiments. KNOWLEDGE GAP Fundamental concepts in plant breeding may not be understood by plant breeders and other plant scientists. Highly specialized equipment for high-throughput analysis is not available to plant breeders. Dr. Zekeria Yusuf (PhD) 833

781. 834 Haramaya University

782. REQUIREMENTS FOR GENETIC MAPPING Genetic linkage map construction requires that the researcher; 1) Develop appropriate mapping population and decide the sample size . 2) Decide the type of molecular marker(s) for genotyping the mapping population. 3) Screen parents for marker polymorphism , and then genotype the mapping population ( parents plus all progenies). 4) Perform linkage analyses (calculate pairwise recombination frequencies between markers, establish linkage groups, estimate map distances, and determine map order ) using statistical programs. 835 Haramaya University

783. Gene Mapping principle Genes and markers segregate via chromosome recombination (called crossing-over) during meiosis Genes or markers that are close together or tightly-linked will be transmitted together from parent to progeny more frequently than genes or markers that are located further apart In a segregating population, there is a mixture of parental and recombinant genotypes The frequency of recombinant genotypes: infer the genetic distance between markers The lower the frequency of recombination between two markers, the closer they are situated on a chromosome and the higher the frequency of recombination between two markers, the further away they are situated on a chromosome). Markers that have a recombination frequency of 50% are described as ‘unlinked’ and assumed to be located far apart on the same chromosome or on different chromosomes 836 Haramaya University

784. 837 Haramaya University

785. Gene Mapping principles…. In a segregating population, there is a mixture of parental and recombinant genotypes The frequency of recombinant genotypes: infer the genetic distance between markers The lower the frequency of recombination between two markers, the closer they are situated on a chromosome and the higher the frequency of recombination between two markers, the further away they are situated on a chromosome). Markers that have a recombination frequency of 50% are described as ‘unlinked’ and assumed to be located far apart on the same chromosome or on different chromosomes. 838 Haramaya University

786. QTL Mapping • The process of constructing linkage maps and conducting QTL analysis to identify genomic regions associated with traits with the help of molecular markers is known as QTL mapping; • Also called as genetic, gene or genome mapping ; Many agriculturally important traits such as yield, quality and some forms of disease resistance are controlled by many genes and are known as “quantitative traits or polygenic or multifactorial or complex traits”. These traits show continuous variation in a population. These traits do not fall into discrete classes. They are measurable. 839 Haramaya University

787. 840 Haramaya University

788. Quantitative Trait Loci ( QTL ) The loci controlling quantitative traits are called quantitative trait loci or QTL . Term first coined by Gelderman in 1975. It is the region of the genome that is associated with an effect on a quantitative trait. It can be a single gene or cluster of linked genes that affect the trait. QTLs are controlled by multiple genes, each segregating according to Mendel's laws. These traits can also be affected by the environment to varying degrees. Individual gene effects is small &The genes involved can be dominant, or codominant . The genes involved can be subject to epistasis or pleiotrophic effect. Quantitative Trait Locus ( QTL ): a statistically significant locus(not necessarily a gene) that quantitatively affects a phenotype of interest with physical boundaries defined by linked molecular markers. 841 Haramaya University

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792. 845 Haramaya University

793. Quantitative Trait Loci ( QTL ) Identification of QTLs based only on conventional phenotypic evaluation is not possible In 1980 discovery of molecular markers tremendously increase the identification of QTLs The use of DNA markers in plant breeding has opened a new field in agriculture called molecular breeding Also DNA markers were used in the construction of linkage maps Linkage maps can be utilised for identifying chromosomal regions that contain genes controlling simple traits (controlled by a single gene) and quantitative traits using QTL . 846 Haramaya University

794. QTL Mapping: The process of constructing linkage maps and conducting QTL analysis i.e. to identify genomic regions associated with traits is known as QTL mapping. Identification and location of polygenes or QTL by use of DNA markers. It involves testing DNA markers throughout the genome for the likelihood that they are associated with a QTL . 847 Haramaya University

795. Objectives of QTL Mapping The basic objective is to detect QTL , while minimizing the occurrence of false positives (Type I errors, that is declaring an association between a marker and QTL when in fact one does not exist). To identify the regions of the genome that affects the trait of interest. To analyze the effect of the QTL on the trait. How much of the variation for the trait is caused by a specific region? What is the gene action associated with the QTL – additive effect? Dominant effect? Which allele is associated with the favorable effect? 848 Haramaya University

796. Prerequisites for QTL mapping Availability of a good linkage map (this can be done at the same time the QTL mapping) A segregating population derived from parents that differ for the trait(s) of interest, & which allow for replication of each segregant , so that phenotype can be measured with precision (such as RILs or DHs) A good assay for the trait(s) of interest Software available for analyses Molecular Markers Sophisticated Laboratory 849 Haramaya University

797. Prerequisites for QTL mapping… Selection of parental lines Sufficient polymorphism Parental lines are highly contrasting phenotypically Genetically divergent Selection of molecular markers (dominant/ codominant ) Making crosses Creation of mapping population 850 Haramaya University

798. Steps involved in QTL Mapping: Phenotyping of the progenies Genotyping of the progenies Construction of linkage map Screening the mapping population using polymorphic molecular markers Segregation patterns Data is then analyzed using a statistical package such as MAPMAKER or JOINMAP Assigning them to their linkage groups on the basis of recombination values For practical purposes, in general recombination events considered to be less than 10 recombinations per 100 meiosis, or a map distance of less than 10 centiMorgans ( cM ). 851 Haramaya University

799. Principle QTL analysis Genes and markers segregate via chromosome recombination during meiosis, thus allowing their analysis in the progeny. QTL analysis depends on the linkage disequilibrium. QTL analysis is usually undertaken in segregating mapping populations. • A significant difference between phenotypic means of the groups indicates that the particular marker locus is linked to a QTL controlling the trait. It is based on the principle of detecting an association between phenotype and the genotype of the markers. Markers are used to partition the mapping population into different genotypic groups based on the presence or absence of a particular marker locus and to determine whether significant differences exist between groups with respect to the trait being measured. A significant difference between phenotypic means of the groups, depending on the marker system and type of mapping population, It is not easy to do this analysis manually and so with the help of a computer and a software it is done. 852 Haramaya University

800. Principle of QTL analysis contd … • If, QTL & marker is closely linked, chance of recombination will be less. • So QTL and marker will be inherited together and mean of the group will have significant difference • If loosely linked or unlinked, there is independent segregation of the marker and QTL . Thus, there will be no significant difference between means of the genotype groups. 853 Haramaya University

801. 854 Haramaya University

802. What are linkage maps? It is the ‘road map’ of the chromosomes derived from two different parents Indicate the position and relative genetic distances between markers along chromosomes (signs or landmarks along a highway) Helps to identify chromosomal locations containing genes and QTLs associated with traits of interest; such maps may then be referred to as ‘ QTL ’ /or ‘genetic’ maps 855 Haramaya University

803. 856 Haramaya University

804. 857 Haramaya University

805. How linkage map constructed? The three main steps of linkage map construction are: (1) Production of a mapping population (2) Identification of polymorphism (3) Linkage analysis of markers 858 Haramaya University

806. 1. Production of a mapping population Requires a segregating plant population (i.e. a population derived from sexual reproduction) The parents selected will differ for one or more traits of interest Population sizes: generally range from 50 to 250 individuals If map is used for QTL studies then the mapping population must be phenotypically evaluated (i.e. trait data must be collected) before subsequent QTL mapping In self pollinated species, parents that are both highly homozygous (inbred) In cross pollinating species, the situation is more complicated being heterozygous 859 Haramaya University

807. Mapping population The first step in producing a mapping population is selecting two genetically divergent parents, which show clear genetic differences for one or more traits of interest (e.g., the recipient or recurrent parent can be a highly productive and commercially successful cultivar but lacks disease resistance, which is present in another donor parent). The parents should be genetically divergent enough to exhibit sufficient polymorphism and at the same time they should not be too genetically distant so as to: a) Cause sterility of the progenies and/or, b) Show very high levels of segregation distortion during linkage analysis. 860 Haramaya University

808. Mapping populations developed for self pollinating species. In self-pollinating species, mapping populations originate from parents that are both highly homozygous (inbred). As shown in Figure 2, progenies from the second filial generation ( F2 ), backcross (BC), recombinant inbred lines ( RILs ), double haploids (DHs), and near isogenic lines ( NILs ) can be used for genetic mapping in self pollinating species. Their main advantages are that they are easy to construct and require only a short time to produce. Inbreeding from individual F2 plants allows the construction of recombinant inbred (RI) lines, which consist of a series of homozygous lines, each containing a unique combination of chromosomal segments from the original parents. The length of time needed for producing RI populations is the major disadvantage, because usually six to eight generations are required. Doubled haploid (DH) populations may be produced by regenerating plants by the induction of chromosome doubling from pollen grains, however, the production of DH populations is only possible in species that are amenable to tissue culture (e.g. cereal species such as rice, barley and wheat). The major advantages of RI and DH populations are that they produce homozygous or ‘true-breeding’ lines that can be multiplied and reproduced without genetic change Occurring. Haramaya University 861

809. Haramaya University 862

810. Mapping Population in cross pollinated crops In cross pollinating ( outcrossing ) species, the situation is more complicated since most of these species do not tolerate inbreeding. Two-way pseudo-testcross, half-sib and full-sib families derived from controlled crosses have been proposed for mapping in outcrossing species. F2 populations are developed by selfing F1 hybrids derived by crossing the two parents while BC population is produced by crossing F1 back into one of the parents (the recipient or recurrent parent). RILs are developed by single-seed selections from individual plants of an F2 population; such selections continue for six to eight generations. If backcross selection is repeated at least for six generations, more than 99% of the genome from BC6 and above will be derived from recurrent parent ( Babu et al., 2004). Selfing of selected individuals from BC7F1 will produce BC7F2 lines that are homozygous for the target gene, which is said to be nearly isogenic with the recipient parent ( NILs ). NILs are frequently generated by plant breeders as they transfer major genes between varieties by backcross breeding ( Tanksley et al., 1995). A DH population is produced by doubling the gametes of F1 or F2 population. Plants will be regenerated using tissue culture techniques after induction of chromosome doubling from pollen grains or haploid embryos resulting from species crosses. 863 Haramaya University

811. 2. Identification of polymorphism • Select an appropriate marker that shows differences between parents (polymorphic markers) • It is critical that sufficient polymorphism exists between parents • In Cross pollinating species, higher DNA polymorphism exits compared to inbreeding species, • So in inbreeding species, parents selected should be distantly related, • Then marker should be screened across the entire mapping population, including the parents: marker genotyping. 864 Haramaya University

812. Haramaya University 865

813. Identification of polymorphism contd.. Generally, markers will segregate in a Mendelian fashion although distorted segregation ratios may be encountered Significant deviations from expected ratios can be analysed using chisquare tests In polyploid species, identifying polymorphic markers is more complicated So mapping of diploid relatives of polyploid species is done However, diploid relatives do not exist for all polyploid species 866 Haramaya University

814. 3. Linkage analysis of markers • Code data for each DNA marker on each individual of a population and conduct linkage analysis using computer programs • Linkage between markers is usually calculated using • Odds ratios: the ratio of linkage versus no linkage • LOD value/ LOD score: Logarithm of the odd ratio • An LOD value of 3 between 2 markers: linkage is 1000 times more likely than no linkage (1000:1) • Manual analysis is not feasible, so computer programmes needed • Commonly used software programs • Mapmaker/ EXP, MapManager QTX , JoinMap etc 867 Haramaya University

815. Linkage map… • Linked markers are grouped together into ‘ linkage groups,’ which represent chromosomal segments or entire chromosomes • Polymorphic markers detected are not necessarily evenly distributed over the chromosome, but clustered in some regions and absent in others • The accuracy of measuring the genetic distance and determining marker order is directly related to the number of individuals studied in the mapping population, min 50 individuals 868 Haramaya University

816. Haramaya University 869

817. Genetic distance and mapping functions • Greater the distance between markers, the greater the chance of recombination occurring during meiosis • Distance along a linkage map is measured in terms of the frequency of recombination between genetic markers • Mapping functions are required to convert recombination fractions into centiMorgans ( cM ) • When map distances are small (<10 cM ), the map distance equals the recombination frequency • However, this relationship does not apply for map distances that are greater than 10cM Haramaya University 870

818. Mapping Function • Kosambi mapping function, which assumes that recombination events influence the occurrence of adjacent recombination events • Haldane mapping function, which assumes no interference between crossover events • There are recombination ‘ hot spots’ and ‘cold spots,’ which are chromosomal regions in which recombination occurs more frequently or less frequently, respectively 871 Haramaya University

819. QTL Mapping Five primary types of QTL mapping with increasing complexity and (theoretically) power Single marker analysis Interval mapping (IM) Composite interval mapping ( CIM ) Multiple interval mapping ( MIM ) Bayesian ( Hidden Markov Model) Others that are more rare. 872 Haramaya University

820. 1. Single-Marker Analysis ( SMA ) Also known as single- point analysis. It is the simplest method for detecting QTLs associated with single markers. This method does not require a complete linkage map and can be performed with basic statistical software programs. The statistical methods used for single-marker analysis include t-tests, analysis of variance (ANOVA) and linear regression. Linear regression is most commonly used because the coefficient of determination ( R2 ) from the marker explains the phenotypic variation arising from the QTL linked to the marker. 873 Haramaya University

821. 874 Haramaya University

822. 875 Haramaya University

823. 876 Haramaya University

824. Limitations Likelihood of QTL detection significantly decreases as the distance between the marker and QTL increases. It cannot determine whether the markers are associated with one or more markers QTLs .  The effects of QTL are likely to be underestimated because they are confounded with recombination frequencies. To overcome these limitations the use of large number of segregating DNA markers covering the entire genome may minimize these problems. Qgene and MapManager QTX are commonly used computer programs to perform singlemarker analysis. 877 Haramaya University

825. 2. Simple Interval Mapping ( SIM ) It was first proposed by Lander and Bolstein . It takes full advantages of the linkage map. This method evaluates the target association between the trait values and the genotype of a hypothetical QTL (target QTL ) at multiple analysis points between pair of adjacent marker loci (target interval). Presence of a putative QTL is estimated if the log of odds ratio exceeds a critical threshold. The use of linked markers for analysis compensates for recombination between the markers and the QTL , and is considered statistically more powerful compared to single-point analysis. MapMaker / QTL and QGene are used to conduct SIM . The principle behind interval mapping is to test a model for the presence of a QTL at many positions between two mapped loci. 878 Haramaya University

826. Statistical methods used for SIM i . Maximum Likelihood Approach It is assumed that a QTL is located between two markers, the two loci marker genotypes ( i.e. AABB , AAbb , aaBB , aabb for DH progeny) each contain mixtures of QTL genotypes. Maximum likelihood involves searching for QTL parameters that give the best approximation for quantitative trait distribution that are observed for each marker class. Models are evaluated by comparing the likelihood of the observed distributions with and without finding QTL effect The map position of a QTL is determined as the maximum likelihood from the distribution of likelihood values. 879 Haramaya University

827. ii. Logarithm of the odds ratio ( LOD score): Linkage between markers is usually calculated using odds ratio. This ratio is more conveniently expressed as the logarithm of the ratio, and is called a logarithm of odds ( LOD ) value or LOD score. LOD values of >3 are typically used to construct linkage maps. LOD of 2 means that it is 100 times more likely that a QTL exists in the interval than that there is no QTL . 880 Haramaya University

828. ii. Logarithm of the odds ratio ( LOD score): LOD of 3 between two markers indicates that linkage is 1000 times more likely (i.e. 1000:1) than no linkage. LOD values may be lowered in order to detect a greater level of linkage or to place additional markers within maps constructed at higher LOD values. The LOD score is a measure of the strength of evidence for the presence of a QTL at a particular location. 881 Haramaya University

829. Interval Mapping by Regression It is essentially the same as the method of basic QTL analysis( regression on coded marker genotypes) except that phenotypes are regressed on QTL genotypes. Since QTL genotypes are unknown they are replaced by probabilities estimated from the nearest flanking markers. 882 Haramaya University

830. 3. Composite Interval Mapping ( CIM ) Developed by Jansen and Stam in 1994 It combines interval mapping for a single QTL in a given interval with multiple regression analysis on marker associated with other QTL . It is more precise and effective when linked QTLs are involved. It considers marker interval plus a few other well chosen single markers in each analysis, so that n-1 tests for interval – QTL associations are performed on a chromosome with n markers. 883 Haramaya University

831. Understanding interval mapping results SIM and CIM produce a profile of the likely sites for a QTL between adjacent linked markers The statistical results are typically presented using a logarithmic of odds ( LOD ) score or likelihood ratio statistic ( LRS ) • LRS = 4 .6 × LOD • Position for a QTL : position where the highest LOD value is obtained Haramaya University 884

832. Understanding interval mapping results contd … • Real QTL : The peak must also exceed a specified significance and is determined using permutation tests • The phenotypic values of the population are ‘shuffled’ whilst the marker genotypic values are held constant and QTL analysis is performed some 500-1000 times to assess the level of false positive marker-trait associations and significant levels are determined • Previously, LOD score of between 2.0 to 3.0 (most commonly 3.0) was usually chosen as the significance threshold. Haramaya University 885

833. Haramaya University 886

834. Haramaya University 887

835. 4. Multiple Interval Mapping ( MIM ) It is also a modification of simple interval mapping. It utilizes multiple marker intervals simultaneously to fit multiple putative QTL directly in the model for mapping QTL . It provides information about number and position of QTL in the genome. It also determines interaction of significant QTLs and their contribution to the genetic variance. It is based on Cockerham’s model for interpreting genetic parameters. 888 Haramaya University

836. 5. Bayesian Interval Mapping (BIM) Developed by Satagopan et al. in 1996. It provides information about number and position of QTL and their effects The BIM estimates should agree with MIM estimates and should be similar to CIM estimates. It provides information posterior estimates of multiple QTL in the intervals. It can estimate QTL effect and position separately. 889 Haramaya University

837. Merits of QTL Mapping Identification of novel genes Where mutant approaches fail to detect genes with phenotypic functions, QTL mapping can help Good alternative when mutant screening is laborious and expensive e.g circadium rhythm screens Can identify New functional alleles of known function genes e.g.Flowering time QTL … Natural variation studies provide insight into the origins of plant evolution 890 Haramaya University

838. LIMITATIONS • Mainly identifies loci with large effects. • Less strong ones can be hard to pursue. • No. of QTLs detected, their position and effects are subjected to statistical error. • Small additive effects / epistatic loci are not detected and may require further analyses. 891 Haramaya University

839. Minor and major QTL QTL can be major or minor based on the proportion of the phenotypic variation i.e. R2 value: Major QTLs : >10%), stable QTL Minor QTLs : <10%. environmentally sensitive especially (disease Resistance): (1) Suggestive; (2) Significant; (3) Highly significant to “avoid a flood of false positive claims” and to ensure that “true hints of linkage” were not missed Significant and highly-significant: significance levels of 5 and 0.1%, Respectively Suggestive: Expected to occur once at random in a QTL mapping study. 892 Haramaya University

840. 893 Haramaya University

841. 894 Haramaya University

842. 895 Haramaya University

843. Genomic Selection Genomic selection (GS) is a new approach for improving quantitative traits in large plant breeding populations that uses whole genome molecular markers and combines marker data with phenotypic data in an attempt to increase the accuracy of the prediction of breeding and genotypic values. 896 Haramaya University

844. Genomic Selection… 897 Haramaya University

845. 898 Haramaya University

846. Dr. Zekeria Yusuf (PhD) 899