Molecular Breeding in Plants is an introduction to the fundamental techniques associated with plant breeding a

This slide describe the process of molecular breeding in plants which involves the application of molecular markers for Marker Assisted Selection and Marker Assisted Breeding.

1. MOLECULAR BREEDING PLANT MOLECULAR GENETICS

2. The Model Plant: Arabidopsis thaliana

3. The Genome One of the first genomes to be completely sequence was that of Arabidopsis thaliana ( Thale cress). The primary reason for sequencing the genome of a ‘weed’ was its small genome size. Genome size was a limiting factor in sequencing using BACs during the 1990s. The five chromosomes of A. thaliana contain DNA molecules of 115 Mbp and encode 25,498 genes.

4. Genome Structure & Organization The centromeres contain retroelements , transposons , microsatellites and some genes. Gene families are organized into tandem arrays. Duplicated segments comprise 58 – 60% of the genome (24 segments of more than 100 kb in size). This implies evolution from a tetraploid ancestor. Functional proteins: 69% similar, 30% unknown.

5. Arabidopsis thaliana: Chromosomes

6. Chloroplast DNA Large Single Copy (LSC) Small Single Copy (SSC) Inverted Repeats (IR)

7. Mitochondrial DNA The mitochondrial genome in plant cells is the result of duplication events.

8. INTRODUCTION TO PLANT GENETICS The concept of ‘Breeding’. Hybrids: Determinate and Indeterminate. Quantitative Traits. Linkage: equilibrium and disequilibrium. Linkage drag. Gene Stacking. Back-crossing and the recurrent parent. Marker Assisted Selection. Environmental effects and epistasis .

9. Norman Borlaug 1914 - 2009

10. INTRODUCTION Plant breeders have developed thousands of new varieties by selection of parental genotypes with desirable phenotypes followed by controlled 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.

11. Determinate and Indeterminate 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.

12. 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”.

13. LINKAGE EQUILIBRIUM Genomic loci are subject to genetic rearrangement via the twin processes of recombination and 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’.

14. 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 back-crossing or in genetic modification using horizontal gene transfer into inbred lines.

15. 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. What are the three forces which drive the evolution of the genome?

16. DNA MARKERS, QTL & MAS Population Development QTL Mapping: Linkage Mapping QTL Validation Marker Validation Marker Assisted Selection

17. A B X Y A B X Y Traits designated as A, B, X and Y segregate independently of each other. Green indicated pollen and red indicates ovules.

18. A B X Y A X A Y B X B Y F1 CZ F2 A C A Z X C X Z A C A Z Y C Y Z B C B Z X C X Z B C B Z Y C Y Z Recruit

19. 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.

20. rA = 5 cM rB = 4 cM rA = 5 cM rB = 4 cM Marker Locus expressing a specific trait

21. 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.

22. APPLICATIONS OF MAS Marker assisted evaluation of breeding material. Marker assisted backcrossing. Marker assisted pyramiding. Early generation MAS. Combined MAS.

23. MARKER ASSISTED EVALUATION Evaluation of cultivars for purity of breeding material. Assessment of genetic diversity and parental selection. Study of heterosis (hybrid vigour ) : do a majority of hybrids exhibit high levels of genetic polymorphism? Allelic diversity, rare genotypes.

24. 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.

25. A B X Y A X A Y B X B Y F1 X Y F2 A X A Y XX X Y A X A Y Y Y X Y B X B Y XX X Y B X B Y X Y Y Y Marker Assisted Backcrossing: Schematic Recurrent Parent

26. F0 F1 F2 F3 X Wild Type X 50 :50 Recurrent Parent X 75 : 25 Recurrent Parent 87.5 : 12.5

27. 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.

28. Elite variety should technically exhibit traits from all the 4 parents. DNA Markers can be developed for each of the specific QTLs and tracked over successive generations.

29. 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.

30. 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.

31. REASONS FOR LOW IMPACT OF MAS MAS results are not published. Reliability and accuracy. Insufficient linkage between marker and QTL. Limited markers and limited polymorphism. Effects of genetic background. QTLs and environmental effects. High cost of MAS. Application gap. Knowledge gap.

32. 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.

33. 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.

34. INSUFFICIENT LINKAGE The loss of linkage may arise from a recombination event between the markers and the QTL. QTL Gene Recombination Site Case I: Tightly linked Case II: Insufficient linkage

35. LIMITED MARKERS & POLYPMORPHISM The complete genomes of many commercially cultivated crops are not currently available at public databases. In cases where genomes are available, there has been no established QTLs between genotype and phenotype. Markers have to be highly polymorphic in order to serve as tools for linkage analysis.

36. 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.

37. 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.

38. HIGH COST OF MAS The 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.

39. 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.

40. 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.

41. GENE PYRAMIDING Gene pyramiding. Marker assisted backcrossing. Efficiency of gene pyramiding. Qualitative improvement through pyramiding. Polygenic trait improvement by gene pyramiding.

42. H1,2 H3,4 H5,6 H 1,2,3,4 (Node) F0 F1 F2 F3 Gene Pyramiding Scheme P1 P2 P3 P4 P5 P6 Founder Parents H 1,2,3,4,5,6 (Root genotype) Pedigree H (1,2,3,4,5,6)(1,2,3,4,5,6) Ideotype

43. Molecular Breeding The objective of molecular breeding is to develop plant varieties with desired traits. Unlike conventional breeding which is founded on selection of the basis of ‘Phenotypes only’, molecular breeding is based on the genetic engineering of plants with specific genes which will result in the desired ‘Phenotype’.

44. Molecular Breeding Strategies Introgression of specific trait encoding genes

45. Steps in Molecular Breeding Identification of the desired ‘trait(s)’. Characterization of the pathway. Identifying genes involved in the pathway. Gene isolation The construct The transformation and delivery system. Transformation . Screening and commercialization.

46. Step 1: The trait Let us consider a hypothetical case in which a protein “DRR” is linked to drought tolerance in Oryza sativa. This protein is found only in wild type O. sativa variety WT-6. DRR

47. Step 2: Pathway Characterization Protein DRR is not the product of a single gene, rather, it is the end product of a pathway, the enzymes of which are encoded by the genes Drase1 , Drbase1 , Drcase4 and Drrase4. DRa DRb DRc DRd DRR Drase1 Drbase1 Drcase4 Drrase4

48. Step 3: Gene / Gene Cluster Each of these genes is encoded on a different chromosome in WT-6 1 3 7 8 Drase1 Drbase1 Drcase4 Drrase4

49. Step 4: Gene Isolation Have you already learnt to isolate the genes? Yes, using PCR. Drase1 Drbase1 Drcase4 Drrase4

50. Step 5: The Construct The genes encoding each of the enzymes involved in the biosynthetic pathway are isolated and linked together to form a single gene construct. A single promoter or multiple promoters may regulate the expression of the genes. Drase1 Drbase1 Drcase4 Drrase4 Promoter

51. Step 6: T & D System We have to develop a suitable vector or transformation process in order to deliver our genetic construct to its intended target chromosome. Inducible promoter Recombination site Recombination site Selectable Marker Reporter gene Construct

52. Step 7: Transformation Transformation can be carried out using Biolistics, Agrobacterim containing Ti plasmid, CaMV or other viral mediated transformations.

53. Plant Tissue Culture System Callus transformed using Biolistics Screening Regeneration Shoot regeneration Root regeneration Grow-out and seed harvesting

54. Step 8: Screening / Commercializing Finally, we screen for transformants carrying our genetic construct and breed commercial lines based on the molecular breeding strategy.

55. What can go wrong ? When a ‘foreign’ or ‘engineered gene’ is introduced into a plant, the following situations can be encountered: Gene does not integrate into the host genome / expresses transiently. Gene integrates but is not expressed. Gene is expressed only in the first generation.

56. What makes GE difficult? Fate of RNAs. Promoter functionality. Loss of introduced gene(s) as a result of recombination. Lethal introductions. Interference in regulatory pathways.

57. Transgene Escape

58. QUESTION BANK What is the meaning of the term ‘linkage drag’ ? If you were to choose between conventional breeding and molecular breeding, which one would you select and why?

59. Thank You!