Principles of plant breeding Lecture note.pdf

lecture note

1. 1Dr. Zekeria Yusuf (PhD)
Principles of Plant Breeding
(Biol 3092)

2. Introduction
•Plantbreedingisasciencebasedonprinciplesof
geneticsandcytogenetics.Itaimsatimprovingthe
geneticmakeupofthecropplants.
•Improvedvarietiesaredevelopedthroughplant
breeding.
•Itsobjectivesaretoimproveyield,quality,disease-
resistance,droughtandfrost-toleranceandimportant
characteristicsofthecrops.
•Plantbreedinghasbeencrucialinincreasing
productionofcropstomeettheeverincreasing
demandforfood.Somewellknownachievementsare
developmentofsemi-dwarfwheatandricevarieties,
noblizationofcanes(sugarcanes),andproductionof
hybridandcompositevarietiesofmaize…..
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3. Introduction….
•Cropimprovementmeanscombiningdesirable
characteristicsinoneplantandthenmultiplyingit.The
jobofaplantbreederistoselectplantswithdesired
characters,crossthemandthenidentifytheoffspring
thatcombinetheattributesofbothparents.Then
multiplytheprogenytosupplytofarmers,growersor
planters.
•Themodernageofplantbreedingbeganintheearly
partofthe20thC,afterMendel’sworkwas
rediscovered.Today
•plantbreedingisaspecializedtechnologybasedon
genetics.Itisnowclearlyunderstoodthatwithina
givenenvironment,cropimprovementhastobe
achievedthroughsuperiorheredity.
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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
•Inearlierdaysmandependsonhisskillsandjudgementinselecting
betterplants.Heknewnothingabouttheinheritanceof
characters,roleofenvironmentinproducingthemandthebasis
ofvariationinvariousplantcharacters.Hismethodofselection
wasdesignedwithouttheunderstandingofprinciplesof
inheritance.
•Thereforeduringprimitivetimeplantbreedingwaslargelyanart
andverylesssciencewasinvolvedinthat.
Eventodaysuccessofselectiondependsuponabilityoftheperson
involvedintheselection.
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5. Introduction….
2.Science
••Plantbreedingisconsideredasthecurrentphaseofcropevolution.
Astheknowledgeofgeneticsandotherrelatedscienceprogresses
plantbreedingbecomelessartandmorescience.
•Especially,discoveryofMendel̕sworkin1900addedalottothe
knowledgeofscience.
•Selectionofdesirableplanteventodayisanartitdependsontheskill
ofaperson.Butaloneskillisnotenough,modernplantbreedingisa
combinedeffortofartandunderstandinganduseofgenetic
principles.
3.Technology
•Productofallplantbreedingactivities,whetherdependentonthe
artorscience,isimprovedvariety,hybrids,syntheticsand
composites.Thisproductisutilizedbyfarmersforcommercial
cultivation.
•Therefore,plantbreedingcanberightlyviewedasatechnology
sinceitgeneratesausefulproduct.
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6. Role of Plant Breeding:
Humanbeingsaredependentontheplantsfor:
1.Food:-Breedingoffieldcropsprovidesusfoodeitherdirectly(foodgrains)
orindirectly(meatandmilk).
2.Shelter:-Inadditiontofoodbyproduceofagriculturefarmsareusedin
makingshelterbyfarmersofruralareas.
3.Clothing:-Breedingforfibrecropslikecottonprovidesclothesforthehuman
population.
4.Fuels:-CropslikeEuphorbiaandJatrophaareusedforBiofuelproduction.
Breedingofsuchcropstacklestheproblemsofenergyproductionforrapidly
increasinghumanpopulation.Nowadays,Maizeisalsousedasanimportant
sourceofEthanolproduction.
5.Drugs:-Breedingofmedicinalplantsplaysanimportantroleinproductionof
manyimportantdrugs.Thesedrugsareusedfortreatmentofvarioushuman
andanimaldiseases.
6.Entertainment:-Flowersplayanessentialroleinpeoplescelebrationsand
everydayliveslikeweddings,Christmasetc.mostofthemedicinalplantsare
seasonalinnature.Shiftingtheseasonaltimingofreproductionisamajor
goalofplantbreedingeffortstoproducenovelvarietiesthatarebetter
adaptedtolocalenvironmentsandchangingclimaticconditions.
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7. ➢Plant breeding is the process by which humans change certain aspects of
plants over time in order to introduce desired characteristics
PlantBreedingConcept
Increasecropproductivity

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.
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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 asedentarylifestyle
Increaseplantyield
Increasenumberofedibleplants
(reducetoxicity)

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13. Geography of crop domestication
•Vavilov’seightcentersoforiginwherecropswere
firsttamed.
•Turnsoutthatcentersofdiversitydonotcoincide
withVavilov’scentersoforigin.
•Areaswithlotsofwildrelativesandprimitive
versionsofmoderncropscanbeinvaluable
sourcesofgenesforplantbreedersand
geneticists.
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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
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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
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19. Tomato - Fewer and Larger Fruits
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20. Sunflowers - reduced branching, larger seeds,
increased seed set per head
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21. Wheat - reduced seed shattering, increased seed size
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22. Squash – larger, fleshier fruits
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23. Corn – reduced fruitcase, softer glume, more kernels per cob, no
dispersal, reduced branching, apical dominance
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24. Lettuce – leaf size/shape, fewer secondary compounds
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25. Rice –no shattering,
larger grains
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26. Crop consequences of domestication:
•More‘yield’ofdesirablepart.
•Non-shattering-seedareeasiertoharvest.
•Bigseeds-domesticatedbeanseedare5-8timesaslargeas
theirwildrelatives.
•Improvedquality-removeorlowertoxicsubstances.
•Increasedprotein,oil,sugarconcentration,whichmeans
improvedflavor,storageability.
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29. Methods for identifying domestication genes
1.BiparentalQTLmapping
2.AssociationMappingUsingUnrelatedIndividuals
3.QTLMappingUsingAdvancedIntercross
Populations
4.Genomicscans
5.GenomeResequencingandScreeningforSelection
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30. Classical Examples…
Teosintebranched 1 (tb1) QTLof 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
Teosinteglumearchitecture1(tga1) was identified as a QTLcontrolling the
formation of the casing that surrounds the kernels of the maize ancestor,
teosinte
tga1is a member of the squamosa-promoter binding protein (SBP) family of
transcriptional regulators
Fruitweight2.2(fw2.2) was identified as a large effect QTLcontrolling 30%
of the difference in fruit mass between wild and cultivated tomato
fw2.2acts 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
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31. •shattering4(sh4)isamajorQTLcontrollingwhethertheseedfalloffthe
plant(shatter)asinwildriceoradheretotheplantasincultivatedrice
•sh4encodesagenewithhomologytoMyb3transcriptionfactors.
•AsingleaminoacidchangeinthepredictedDNAbindingdomainconverts
plantsfromshatteringtonon-shattering
•Rcisadomestication-relatedgenerequiredforredpericarpinrice
•TwoindependentgeneticstocksofRcrevealedthatthedominantredallele
differedfromtherecessivewhiteallelebya14-bpdeletionwithinexon6-
originatedinjaponicacultivarandspreadintoindicacultivars.
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32. Super-domestication
•Theprocessesthatleadtoadomesticatewith
dramaticallyincreasedyieldthatcouldnotbeselected
innaturalenvironmentswithoutnewtechnologies.
•Thearrayofgenomemanipulationsenablebarriersto
geneexchangetobeovercomeandhaveleadtosuper-
domesticateswith
–dramaticallyincreasedyields,
–resistancestobioticandabioticstresses,andwith
–newcharactersforthemarketplace.
•Hybridricecanbeconsideredasuper-domesticate
•ConversionofacropfromC3toC4photosynthesis
wouldcertainlybeasuper-domesticate.
Plantbreeders+GenomicScientists⇨Superdomestication
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33. 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.
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37. selective sweep
•Aselectivesweepisthereductionoreliminationof
variationamongthenucleotidesinneighboring
DNAofamutationastheresultofrecentand
strongpositivenaturalselection
•Astrongselectivesweepresultsinaregionofthe
genomewherethepositivelyselectedhaplotype
(themutatedalleleanditsneighbours)is
essentiallytheonlyonethatexistsinthe
population,resultinginalargereductionofthe
totalgeneticvariationinthatchromosomeregion.
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38. selective sweep
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41. 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?
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42. • 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”
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43. • 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
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44. Domestication of Maize
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45. The evolution of non-shattering
in the archaeological record
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46. 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
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47. 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).
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48. The role of polyploidy in domestication
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49. Towards resolving the genetic basis of
domestication in the Compositae
Artificial selection
through domestication
but HOW ?
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50. 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)?
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51. 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
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52. 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
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53. 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
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54. What to do with candidate genes?
Applications:
breeding / improvement
conservation of genetic diversity
identification of taxon boundaries
understanding adaptation/domestication
comparative analysis –other taxa
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55. 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
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56. 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
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57. 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
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58. Types of crops:
•Graincrops-wheat,rice,corn,sorghum,barley,oats.
•Oilcrops-olive,linseed,sesame,sunflower,soybean,
coconut,palm,corn,peanut,canola.
•Fibercrops-cotton,flax,hemp,jute,kenaf,sisal.
•Foragecrops-alfalfa,clovers,otherlegumes,many
grasses,includingtallfescue.
•Spice/drugcrops-tobacco,blackpepper,cinnamon.
•Fruitcrops,vegetablecrops,ornamentals,forest
trees,etc.
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59. 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
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60. Landmarksin PlantBreeding
1694 1866 1953
Camerarius
crossing as a method
to obtain new plant
types
Mendel
Empirical evidence
on heredity
Watson, Crick,
Wilkins &
Rosalind Franklin
model for DNA
structure
1923
Wallace
Firstcommercial
hybridcorn

61. “TheGreen 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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63. Future Challenges
Challenge: Increaseofhuman
populationby60-80%, requiringto
nearlydoubletheglobal food
production
MultidisciplinaryField
Biometry/
Statistics
Pathology

64. Challenges before Plant Breeder :
1.Increasingpopulation:atpresent,theworldpopulation
standat6.3billionandwillreachat10-12billionduring
thenext50-70years.
Themainproblemfrombreedingrespectisthatthe
populationisgrowingfasterthanincreasesinfood
productivity,toreducetheuseofharmfulagrochemicals
andtoproducenutritiousandhealthfulfoodisgreater
today.
2.Squeezingarableland:Day-by-daythetotalarableland
foragricultureisdecreasingduetourbanizationand
industrialdevelopment.
Breedershavetotacklethisproblembyreleasing
improvedvarietiesofmajorcropswhichgivesbetter
productionperunitarea.
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65. Challenges before Plant Breeder…
3.Erraticrainfall:espintropicsrainfalliserratic,
unpredictableandunevenlydistributed.Over80%ofthe
annualrainfallisreceivedinthefourrainymonthsofJuneto
September.Therefore,varietieswhichcantoleratedryspells
andperformbetteratlowwateravailabilityareneededtobe
developbyIndianBreeders.
4.Mechanization:-Thevarietydevelopedbyplantbreeders
shouldgiveresponsetoapplicationoffertilizers,manures,
irrigationandshouldbesuitableformechanicalcultivation.
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66. 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-HomeMessage

67. Scientific disciplines and technologies
of plant breeding
•Genetics
•Botany
•Plant physiology
•Agronomy
•Pathology and entomology
•Statistics
•Biochemistry
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68. Importance of Plant breeding
•Plantbreedingallowedcivilizationtoformand
itscontinualsuccessiscriticaltomaintaining
ourwayoflife
•Problem:Feeding9billion(+)peoplewiththe
same(orfewer)inputs
Sameorlessacreage
Sameorlessfertilizer,pesticides,water
Adaptingtoclimateandenvironmental
change
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69. Goals of Plant breeding
•Plantbreedingaimstoimprovethecharacteristicsofplantssothat
theybecomemoredesirableagronomicallyandeconomically.The
specificobjectivesmayvarygreatlydependingonthecropunder
consideration.
Increasethefrequencyoffavorablealleleswithinaline(favoring
additiveeffects)
•Increasethefrequencyoffavorablegenotypeswithinaline(with
dominanceandinteractioneffects)
•Betteradaptcropstospecificenvironments
–Region-specificcultivars(highlocationGxE)
–Stabilityacrossyearswithinaregion(lowyearto-yearGxE)
Food(yieldandnutritionalvalue),feed,fibre,
pharmaceuticals(antibodies),landscape,industrialneed(eg.
Cropsarebeingproducedinregionstowhichtheyarenot
native).
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70. Objectives of Plant Breeding
•Developmentofpure(i.e.highlyinbred)lineswithhigh
perseperformance,
•Developmentofpurelineswithhighhybridperformance
(eitherwitheachotherorwithatestcross),
•Lessemphasisondevelopingoutbred(random-mating)
populationswithimprovedperformance
•DevelopmentoflineswithhighregionalGxE,lowyearG
xE.
Note:Detailsamongplantspeciesvarybecauseof
origin,modeofreproduction,ploidylevels,and
traitsofgreaterimportanceandadjustments
weremadetoadapttospecificsituations.
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71. Objectives of Plant Breeding...
•Theprimeobjectiveofplantbreedingistodevelopsuperiorplants
overtheexistingonesinrelationtotheireconomicuse.The
objectivesofplantbreedingdifferfromcroptocrop.Abriefaccount
ofsomeimportantobjectivesare:
1.Higherproductivity/yield-Increasedyieldhasbeentheultimate
aimofmostplantbreeders.Thiscanbeachievedbydeveloping
moreefficientgenotypeshavinggreaterphysiologicalefficiency.
2.Improvedquality-Improvedqualityofagriculturalproductshas
contributedalottothehumanwell-being.Qualitycharactersvary
fromonecroptoanothercrop.Forexample,Grainsize,colour,
milling,andbakingqualitiesinwheat(Triticumaestivum).
3.DiseaseandInsectResistance-Resistancevarietiesofferthe
cheapestandmostconvenientmethodofdiseaseandinsect
management.Insomecases,theyofferonlyfeasiblemeansof
control.eg.RustinWheat.
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72. Objectives of Plant Breeding...
4.Varietiesfornewseasons-Thevarietiesfornewseasons
havebeendevelopedbyadjustingthegrowthcycleofthe
varietytosuitbettertotheavailablegrowingseason.
5.Modificationofagronomiccharacteristics-modificationof
agronomiccharacteristicssuchasplantheight,tillering,
branching,erectortrailinghabitetc.isoftendesirable.For
example,dwarfnessincerealsisgenerallyassociatedwith
lodgingresistanceandfertilizerresponsiveness.
6.Changeinmaturityduration-itpermitsnewcrop
rotationsandoftenextendsthecroparea.Developmentof
wheatvarietiessuitableforlateplantinghaspermittedrice-
wheatrotation.Thisobjectiveismoredesirableespeciallyin
thoseareaswheremultiplecroppingsystemhasbeen
followed.
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73. Objectives of Plant Breeding...
7.Photoandthermoinsensitivity-Developmentofphoto
andthermoinsensitivewheatandphotoinsensitiverice
varietieshaspermittedtheircultivationinnewareas.
8.Synchronousmaturity-Synchronousmaturityishighly
desirableincropswhereseveralpickingsarenecessary.Eg.
Mungbean,pigeonpea,cottonetc.
9.Non-shatteringcharacteristics-Itwouldbeofgreatvalue
incropslikemung,castor,soybeanetc.whereshatteringis
amajorproblemincaseofmanycommercialvarieties.
10.Determinategrowth-Developmentofvarietieswith
determinategrowthisdesirableincropslikemung,pigeon
pea,cotton,etc.
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74. Objectives of Plant Breeding...
11.Dormancy-Dormancyplaysbothbeneficialandharmful
roleaccordingtotheneedofgrower.
Forexample,ifwewantnextcropimmediateafterharvesting
ofpreviouscrop,insuchcasedormancyisnotrequired.But
ifwewanttostoretheseedforitsfuturepurpose,aperiod
ofdormancyisessential.
12.Eliminationoftoxicsubstances:somecropshavetoxic
substanceswhichmustbeeliminatedtomakethemsafefor
consumption.
Forexample,•Khesari(Lathyrusodoratus)seedshavea
neurotoxin,β-N-oxalyl-α-β-diaminopropionicacid(BOAA)
thatcausesparalysisinhumans.
•Similarly,eliminationofErusicacidfromBrassicaoiland
Gossypolfromseedcottonisnecessarytomakethemfitfor
consumption.
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75. Objectives of Plant Breeding...
13.MoistureStressandSaltTolerance:
developmentofvarietiesforarainfedareaand
salinesoilswouldhelptoincreasecrop
production.
14.WiderAdaptability:ithelpsinstabilizingthe
cropproductionoverregionandseasons.
15.UsefulforMechanicalCultivation:thevariety
developedshouldgiveresponsetoapplicationof
fertilizers,manuresandirrigation,suitablefor
mechanicalcultivationetc.
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76. Animal and tree breeding
•Similargoals,butsincemostlyoutcrossing,the
goalistocreatehigh-performingpopulations,
notinbredlines
•Generallyspeaking,inbreedingisbadin
animalsandmanytrees
•Focusonfindingthoseparentswiththebest
transmittingabilities(highestbreedingvalues)
•LessofaGxEfocuswithanimals,lessofa
focusonlineandhybridbreeding
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77. Special features exploited by plant breeders
•Selfingallowsforthecaptureofspecificgenotypes,
andhencethecaptureofinteractionsbetween
allelesandloci(dominanceandepistasis)
–Homozygousforselfedlines
–Heterozygousforcrossedlines
•Oftenhighreproductiveoutput(relativetoanimal
breeding)
•Seedsallowformultigenerationprogenytesting,
whereinindividualsarechosenonthe
performanceoftheirprogeny,oroftheirsibs
–AllowsforbettercontroloverGxEbytestingover
multiplesites/years
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78. 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
–Usingcrossestopredictaverageperformance
underinbreedingorcrossingorresponseto
selection
–Better management of G x E
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79. ModernBreedingTools
Increaseofbreedingeffectivenessandefficiency
In vitro culture Genomictools Genomicengineering

80. 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
80Dr. Zekeria Yusuf (PhD)

81. PlantBreedingMethods
Conventionalbreeding
•Mutationorcrossingto introducevariability
•Selectionbasedonmorphologicalcharacteres
•Growthofselectedseeds
Challenge: reducethetime neededto complete a breedingprogram

82. Plant Breeding Methods
Conventional Methods:
1.Plantintroduction
2. Purelineselection
3. Mass selection
4. Pedigree method
5. Bulk method
6. Single Seed descent
method
7. Back cross method
8. Hetrosisbreeding
Modern Methods
1. Mutation breeding
2. Polyploidy breeding
3. Transgenic breeding
4. Molecular breeding
82Dr. Zekeria Yusuf (PhD)

83. 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.
83Dr. Zekeria Yusuf (PhD)

84. Modern tools Used in Plant Breeding
•Molecularmarkers:
–InitiallylowdensityforQTLmapping,introgressionofmajor
genesintoelitegermplasm
–Withhigh-densitymarkers,associationmappingand
MAS/genomicselection
•Newstatisticaltools:
–Mixedmodelmethods
–Bayesianapproachestohandlehigh-dimensionaldatasets
–NewmethodstodealwithGxE
•Othertechnologies:
–Betterstandardizationoffieldsites(laser-tilledfields,GPS,
bettermicro-andmacro-environmentalmeasurements)
–Highthroughputphenotypicscoring
–DH(doublehaploid)lines
84Dr. Zekeria Yusuf (PhD)

85. 85Dr. Zekeria Yusuf (PhD)

86. Integrated Approaches
•Howdowebestcombinetherichhistoryof
quantitativegeneticsandclassicalplantbreeding
withthenewtoolsfromgenomicsandother
advances?
•Key:Quantitativegeneticshasallofthe
machineryneededtofullyincorporatethesenew
sourcesofinformation
86Dr. Zekeria Yusuf (PhD)

87. Basic steps in plant breeding
•Objective
•Germplasm
•Selection
•Evaluation
87Dr. Zekeria Yusuf (PhD)

88. Activities in plant breeding:
•1.Creationofvariation:variationmeansdifferences
amongindividualsofapopulationorspeciesforaspecific
character.
•Geneticvariationisthesourceofrawmaterialfor
selection.Theseareheritableandaretransmittedfrom
onegenerationtoother.Suchvariationisusefulin
selection.
•Successofabreedingprogramusuallydependsonthe
desiredgeneticvariation.Itcanbedoneinfollowingways
i.e.domestication,germplasmcollection,plant
introduction,hybridization,polyploidy,mutation,
somaclonalvariationandgeneticengineering.
88Dr. Zekeria Yusuf (PhD)

89. Activities in plant breeding….
2. Selection:
•Duringselection,theindividualplantorgroupofplants
havingthedesiredcharactersarepickedupfroma
populationeliminatingtheundesirableones.
•Thoseplantsareselectedwhicharelookingpromisingfor
thecharacterontheebasisofphenotype.
•Theselectedplantsarethenallowedtogrowforsetting
theirseeds.
•Seedsareselectedandagainanewcropisdeveloped.
•Thisprocessisrepeatedagainandagaintillthedesired
resultisachieved.
•Selectionactsonthegeneticvariationpresentina
populationandproducesanewpopulationwithimproved
characters.
89Dr. Zekeria Yusuf (PhD)

90. Activities in plant breeding….
3.Evaluation:
•Thenewlyselectedlines/strains/populationsaretested
foryieldandothertraitsandtheirperformanceis
comparedwithexistingbestvarietiescalledChecks.
Ifthenewlines/strain/populationshowssuperior
performancetothechecks,itisreleasedandnotifiedas
anewvariety.
4.Multiplication:
•Thisstepconcernswithlargescalecertifiedseed
productionofthereleasedandnotifiedvariety.
5.Distribution:
•Certifiedseedisultimatelysoldtothefarmerswhouseit
forcommercialcropcultivation.
90Dr. Zekeria Yusuf (PhD)

91. 91Dr. Zekeria Yusuf (PhD)

92. Scope of plant breeding
•Fromtimesimmemorial,theplantbreedinghasbeenhelpingthemankind.
Withknowledgeofclassicalgenetics,numberofvarietieshavebeenevolvedin
differentcropplants.
•Sincethepopulationisincreasingatanalarmingrate,thereisneedto
strengthenedthefoodproductionwhichisseriouschallengetothosescientists
concernedwithagriculture.
•Advancesinmolecularbiologyhavesharpenedthetoolsofthebreeders,and
brightentheprospectsofconfidencetoservethehumanity.
•Theapplicationofbiotechnologytofieldcrophasalreadyledtothefield
testingofgeneticallymodifiedcropplants.Geneticallyengineeredrice,maize,
soybean,cotton,oilseedsrape,sugarbeetandalfalfacultivarsareexpectedto
becommercializedbeforethecloseof20thcentury.
•Genesfromvariedorganismsmaybeexpectedtoboosttheperformanceof
cropsespeciallywithregardtotheirresistancetobioticandabioticstresses.
•Inaddition,cropplantsarelikelytobecultivatedforrecoveryofvaluable
compoundslikepharmaceuticalsproducedbygenesintroducedintothem
throughgeneticengineering.ItmaybepointedoutthatinEuropehirudin,an
anti-thrombinproteinisalreadybeingproducedfromtransgenicBrassica
92Dr. Zekeria Yusuf (PhD)

93. What is Germplasm?
•Germplasmbroadlyrefertothehereditary
material(totalcontentofgene)transmittedtothe
offspringthroughgermcell.
•Itcanalsobedescribedasacollectionofgenetic
resourcesforanorganism.
•Forplants,thegermplasmmaybestoredasa
seed,stem,Callus,Wholeplantinnurseries.
•Incaseofanimals-Genes,Bodypartsstoredin
genebank/cryobank.
•Germplasmprovidetherawmaterial(genes)
whichthebreederusestodevelopcommercial
cropvarieties.
93Dr. Zekeria Yusuf (PhD)

94. Sources of germplasmfor plant breeding
•Germplasmmay be classified into five major types –
1.advanced (elite) germplasm,
2.improved germplasm(cultivars and varieties),
3.landraces,
4.wild or weedy relatives, and
5.Genetic stocks.
Themajorsourcesofvariabilityforplantbreedersmay
alsobecategorizedintothreebroadgroups–
I.domesticated plants,
II.undomesticated plants, and
III.other species or genera.
94Dr. Zekeria Yusuf (PhD)

95. 1. Undomesticated plants
•Whendesiredgenesarenotfoundindomesticatedcultivars,plant
breedersmayseekthemfromwildpopulations.
•Whenwildplantsareusedincrosses,theymayintroducewildtraits
thathaveanadvantageforsurvivalinthewild(e.g.,hardseedcoat,
shattering,indeterminacy)
•butareundesirableinmoderncultivation.Theseundesirabletraits
havebeenselectedagainstthroughtheprocessofdomestication.
•Wildgermplasmshavebeenusedasdonorsofseveralimportant
disease-andinsectresistancegenesandgenesforadaptationto
stressfulenvironments.Thecultivatedtomatohasbenefitedfromsuch
introgressionbycrossingwithavarietyofwildLicopersiconspp.
•Otherspeciessuchaspotato,sunflower,andricehavebenefitedfrom
widecrosses.
•Inhorticulture,variouswildrelativesofcultivatedplantsmaybeused
asrootstockingrafting(e.g.,citrus,grape)toallowcultivationofthe
plantinvariousadversesoilandclimaticconditions.
95Dr. Zekeria Yusuf (PhD)

96. 2. Domesticated plants
•Domesticatedplantsarethoseplantmaterialsthathavebeen
subjectedtosomeformofhumanselectionandaregrownforfoodor
otheruses.Therearevarioustypesofsuchmaterial:
1.Commercialcultivars:
•Therearetwoformsofthismaterial–currentcultivarsandretired
orobsoletecultivars.
•Theseareproductsofformalplantbreedingforspecificobjectives.It
isexpectedthatsuchgenotypeswouldhavesuperiorgene
combinations,beadaptedtoagrowingarea,andhaveagenerally
goodperformance.
•Theobsoletecultivarsweretakenoutofcommercialproduction
becausetheymayhavesufferedasetback(e.g.,susceptibleto
disease)orhigherperformingcultivarsweredevelopedtoreplace
them.
•Ifdesirableparentsarefoundincommercialcultivars,thebreederhas
aheadstartonbreedingsincemostofthegenecombinationswould
alreadybedesirableandadaptedtotheproductionenvironment.
96Dr. Zekeria Yusuf (PhD)

97. 2. Breeding materials
•Ongoingormoreestablishedbreedingprogramsmaintain
variabilityfrompreviousprojects.
•Theseintermediatebreedingproductsareusuallygenetically
narrow-basedbecausetheyoriginatefromasmallnumberof
genotypesorpopulations.
•Forexample,abreedermayreleaseonegenotypeasa
commercialcultivarafteryieldtests.
•Manyofthegenotypesthatmadeittothefinalstageorhave
uniquetraitswillberetainedasbreedingmaterialstobe
consideredinfutureprojects.Similarly,genotypeswithunique
combinationsmayberetained.
97Dr. Zekeria Yusuf (PhD)

98. 3. Landraces
•Landracesarefarmer-developedandmaintainedcultivars.Theyare
developedoververylongperiodsoftimeandhavecoadaptedgene
complexes.
•Theyareadaptedtothegrowingregionandareoftenhighlyheterogeneous.
•Landracesarerobust,havingdevelopedresistancetotheenvironmental
stressesintheirareasofadaptation.
•Theyareadaptedtounfavorableconditionsandproducelowbutrelatively
stableperformance.
•Landraces,hence,characterizesubsistenceagriculture.Theymaybeusedas
startingmaterialinmassselectionorpure-linebreedingprojects.
98Dr. Zekeria Yusuf (PhD)

99. 4. Plant introductions
•Theplantbreedermayimportnew,unadapted
genotypesfromoutsidetheproductionregion,usually
fromanothercountry(calledplantintroductions).
•Thesenewmaterialsmaybeevaluatedandadapted
tonewproductionregionsasnewcultivars,orusedas
parentsforcrossinginbreedingprojects.
99Dr. Zekeria Yusuf (PhD)

100. Plant introductions…
Plantintroductionistheprocessofimportingnewplantsorcultivars
ofwell-establishedplantsfromtheareaoftheiradaptationto
anotherareawheretheirpotentialisevaluatedforsuitabilityfor
agriculturalorhorticulturaluse.
First,thegermplasmtobeintroducedisprocessedthroughaplant
quarantinestationattheentryport,toensurethatnopestand
diseasesareintroducedalongwiththedesiredmaterial.
Oncethisisaccomplished,thematerialisreleasedtotheresearcher
forevaluationinthefieldforadaptation.
Thefundamentalprocessofplantintroductionsasaplantbreeding
approachisacclimatization.
Theinherentgeneticvariationintheintroducedgermplasmservesas
therawmaterialforadaptationtothenewenvironment,enablingthe
breedertoselectsuperiorperformerstoformthenewcultivar.
100Dr. Zekeria Yusuf (PhD)

101. Plant introductions…
•Whentheplantintroductioniscommerciallyusableas
introducedwithoutanymodification,itiscalledaprimary
introduction.
•However,moreoftenthannot,thebreedermakes
selectionsfromthevariablepopulation,orusestheplant
introductionasaparentincrosses.Theproductsofsuch
effortsarecalledsecondaryintroductions.
•Someplantintroductionsmaynotbeusefulascultivars
inthenewenvironment.However,theymaybeusefulin
breedingprogramsforspecificgenestheycarry.
•Manydiseases,plantstature,compositionaltraits,and
genesforenvironmentalstresseshavebeenintroduced
byplantbreeders.
•Asaplantbreedingmethod,plantintroductionshavehad
asignificantimpactonworldfoodandagriculture.
101Dr. Zekeria Yusuf (PhD)

102. 5. Genetic stock
Geneticstock:consistsofproductsofspecializedgenetic
manipulationsbyresearchers(e.g.,byusingmutagenesisto
generatevariouschromosomalandgenomicmutants).
6.Otherspeciesandgenera:
•Genetransferviacrossingrequiresthattheparentsbecross-
compatibleorcross-fertile.
•Crossinginvolvingparentsfromwithinaspeciesisusually
successfulandunproblematic.
•However,astheparentsbecomemoregeneticallydivergent,
crossing(widecrosses)islesssuccessful,oftenrequiringspecial
techniques(e.g.,embryorescue)forinterveningintheprocessin
ordertoobtainaviableplant.
•Sometimes,relatedspeciesmaybecrossedwithlittledifficulty.
102Dr. Zekeria Yusuf (PhD)

103. Germplasmenhancement
•Thereareoccasionswhenbreedersarecompelledtolookbeyondthe
advancedgermplasmpooltofinddesirablegenes.T
•Thedesiredgenesmayresideinunadaptedgenepools.
•Breedersarefrequentlyreluctanttousesuchmaterialsbecausethe
desiredgenesareoftenassociatedwithundesirableeffects(unadapted,
unreproductive,yieldreducingfactors).Hence,theseexoticmaterials
oftencannotbeuseddirectlyincultivardevelopment.
•Instead,thematerialsaregraduallyintroducedintothecultivar
developmentprogramthroughcrossingandselectingforintermediates
withnewtraits,whilemaintainingagreatamountoftheadaptedtraits.
•Tousewildgermplasm,theunadaptedmaterialisputthrougha
preliminarybreedingprogramtotransferthedesirablegenesinto
adaptedgeneticbackgrounds.
•Theprocessoftheinitialintrogressionofatraitfroman
undomesticatedsource(wild)oragronomicallyinferiorsource,toa
domesticatedoradaptedgenotypeiscalledprebreedingorgermplasm
enhancement.
103Dr. Zekeria Yusuf (PhD)

104. The major uses of germplasmenhancement may be summarized as follows:
1.Preventionsofgeneticuniformityandthe
consequencesofgeneticvulnerability.
2.Potentialcropyieldaugmentation.Historyteachesus
thatsomeofthedramaticyieldincreasesinmajor
worldfoodcrops,suchasrice,wheat,andsorghum,
wereaccomplishedthroughintrogressionof
unadaptedgenes(e.g.,dwarfgenes).
3.Introductionofnewqualitytraits(e.g.,starch,
protein).
4.Introductionofdisease-andinsect-resistancegenes.
5.Introductionofenvironment-resistancegenes(e.g.,
droughtresistance).
Prebreedingcanbeexpensivetoconductandtime
consumingaswell.
104Dr. Zekeria Yusuf (PhD)

105. Genetic vulnerability
•Geneticvulnerabilityisbroughtaboutlargelybythemannerinwhichbreedersgo
aboutdevelopingnewandimprovedcultivarsformodernsociety.
•Geneticvulnerabilityisatermusedtoindicatethegenetichomogeneityand
uniformityofagroupofplantsthatpredisposesittosusceptibilitytoapest,
pathogen,orenvironmentalhazardoflarge-scaleproportions.Acaseinpointisthe
1970epidemicofsouthernleafblight(Helminthosporiummaydis)intheUSAthat
devastatedthecornindustry.Thisgeneticvulnerabilityincornwasattributedto
uniformityinthegeneticbackgroundincornstemmingfromthewidespreaduseof
T-cytoplasmincornhybridseedproduction.
•Geneticuniformityperseisnotnecessarilytheculpritinvulnerabilityofcrops.In
fact,bothproducersandconsumerssometimesdesireandseekuniformityinsome
agronomictraits.Thekeyissueiscommonalityofgeneticfactors.
•Geneticallydissimilarcropscanshareatraitthatissimplyinheritedandthat
predisposesthemtosusceptibilitytoanadversebioticorabioticfactor.
•Acaseinpointisthechestnutblight(Cryphonectriaparasitica)epidemicthat
occurredintheUSAinwhichdifferentspeciesoftheplantwereaffected.
105Dr. Zekeria Yusuf (PhD)

106. Conservation of Plant Genetic Resources
Whyconserveplantgeneticresources?
•Thereareseveralreasonswhyplantgeneticresourcesshould
beconserved:
1.Plantgermplasmisexploitedforfood,fiber,feed,fuel,and
medicinesbyagriculture,industry,andforestry.
2.Asanaturalresource,germplasmisadepletableresource.
3.Withoutgeneticdiversity,plantbreedingcannotbe
conducted.
4.Geneticdiversitydeterminestheboundariesofcrop
productivityandsurvival.
5.Aspreviouslyindicated,variabilityisthelifebloodofplant
breeding.Associetyevolves,itsneedswillkeepchanging.
Similarly,newenvironmentalchallengesmightarise(e.g.,
newdiseases,abioticstresses)forwhichnewvariability
mightbeneededforplantimprovement.
106Dr. Zekeria Yusuf (PhD)

107. Conservation of Plant Genetic Resources…
•Whenagenotypeisunabletorespondfullytotheculturalenvironment,aswellasto
resistunfavorableconditionsthereof,cropproductivitydiminishes.
•Thenaturalpoolsofplantgeneticresourcesareunderattackfromtheactivitiesof
modernsociety–urbanization,indiscriminateburning,andtheclearingofvirginland
forfarming,tonameafew.
•Theseandotheractivitieserodegeneticdiversityinwildpopulations.Consequently,
Theactionsofplantbreedersalsocontributetogeneticerosionaspreviouslyindicated.
High-yielding,narrowgenetic-basedcultivarsarepenetratingcropproductionsystems
allovertheworld,displacingtheindigenoushigh-variabilitylandracecultivars.
Geneticerosion
•Geneticerosioncanbedefinedasthedeclineingeneticvariationincultivatedor
naturalpopulationslargelythroughtheactionofhumans.
•Lossofgeneticvariationmaybecausedbynaturalfactors,andbytheactionsofcrop
producers,plantbreeders,curatorsofgermplasmrepositories,andothersinsocietyat
large.
1.Naturalfactors:
•Geneticdiversitycanbelostthroughnaturaldisasterssuchaslarge-scalefloods,wild
fires,andsevereandprolongeddrought.Theseeventsarebeyondthecontrolof
humans.
107Dr. Zekeria Yusuf (PhD)

108. 2. Action of farmers:
•Rightfromthebeginningsofagriculture,farmershaveengagedinactivities
thatpromotegeneticerosion.
•Theseincludeclearingofvirginlandin,especially,germplasm-richtropical
forests,andthechoiceofplantingmaterial(narrowgenetic-basedcultivars).
•Farmers,especiallyindevelopedeconomies,primarilygrowimprovedseed,
havingreplacedmostoralllandraceswiththesesuperiorcultivars.
•Also,monoculturetendstonarrowgeneticdiversityaslargetractsoflandare
plantedtouniformcultivars.
•Extendinggrazinglandsintowildhabitatsbylivestockfarmers,destroyswild
speciesandwildgermplasmresources.
3.Actionofbreeders:Farmersplantwhatbreedersdevelop.Somemethodsused
forbreeding(e.g.,purelines,singlecross,multilines)promoteuniformityanda
narrowergeneticbase.
Whenbreedersfindsuperiorgermplasm,thetendencyistouseitasmuchas
possibleincultivardevelopment.
Forexample:insoybean,mostofthemoderncultivarsintheUSAcanbetraced
backtoabouthalfadozenparents.Thispracticecausesseverereductionin
geneticdiversity.
108Dr. Zekeria Yusuf (PhD)

109. Problems with germplasmconservation
Inspiteofgoodeffortsbycuratorsofgermplasmrepositoriesto
collectandconservediversity,thereareseveralwaysinwhichdiversity
intheircustodymaybelost.
Themostobviouslossofdiversityisattributedtohumanerrorsinthe
maintenanceprocess(e.g.,improperstorageofmaterialsleadingto
lossofvariability).
Also,whengermplasmisplantedinthefield,naturalselection
pressuremayeliminatesomeunadaptedgenotypes.Also,therecould
bespontaneousmutationsthatcanalterthevariabilityinnatural
populations.
Hybridizationaswellasgeneticdriftincidencesinsmallpopulations
arealsoconsequencesofperiodicmultiplicationofthegermplasm
holdingsbycurators.
109Dr. Zekeria Yusuf (PhD)

110. Germplasmcollection
Plannedcollections(germplasmexplorationsorexpeditions)areconductedby
expertstoregionsofplantorigin.
Thesetripsareoftenmultidisciplinary,comprisingmemberswithexpertisein
botany,ecology,pathology,populationgenetics,andplantbreeding.
Familiaritywiththespeciesofinterestandthecultureoftheregionstobe
exploredareadvantageous.
Mostofthematerialscollectedareseeds,eventhoughwholeplantsand
vegetativeparts(e.g.,bulbs,tubers,cuttings,etc.)andevenpollenmaybe
collected.
Becauseonlyasmallamountofmaterialiscollected,samplingfor
representativenessofthepopulation’snaturalvariabilityiscriticalinthe
collectionprocess,inordertoobtainthemaximumpossibleamountofgenetic
diversity.
Forsomespecieswhoseseedispronetorapiddeterioration,orarebulkyto
transport,invitrotechniquesmaybeavailabletoextractsmallsamplesfrom
theparentsource.
Collectorsshouldbearinmindthatthevalueofthegermplasmmaynotbe
immediatelydiscernible.
Materialsshouldnotbeavoidedforlackofobviousagronomicallydesirable
properties.Ittakestimetodiscoverthefullpotentialofgermplasm.
110Dr. Zekeria Yusuf (PhD)

111. Germplasmcollection…
•Seedmaterialsvaryinviabilitycharacteristics.Thesehaveto
betakenintoaccountduringgermplasmcollection,
transportation,andmaintenanceinrepositories.
•Basedonviability,seedmaybeclassifiedintotwomaingroups
–orthodoxandrecalcitrantseed:
1.Orthodoxseeds:Theseareseedsthatcanprolongtheir
viabilityunderreducedmoisturecontentandlow
temperatureinstorage.Examplesincludecereals,pulses,and
oilseed.Ofthese,somehavesuperior(e.g.,okra)while
othershavepoor(e.g.,soybean)viabilityunderreduced
moisturecoldstorage.
2.Recalcitrantseeds:Lowtemperatureanddecreasedmoisture
contentareintolerabletotheseseeds(e.g.,coconut,coffee,
cocoa).Invitrotechniquesmightbebeneficialtothesespecies
forlong-termmaintenance.
111Dr. Zekeria Yusuf (PhD)

112. Germplasmcollection…
•Theconditionsofstoragedifferdependingonthemode
ofreproductionofthespecies:
1.Seedpropagatedspecies:theseseedsarefirstdriedto
about5%moisturecontentandthenusuallyplacedin
hermeticallysealedmoisture-proofcontainersbefore
storage.
2.Vegetativelypropagatedspecies:thesematerialsmay
bemaintainedasfullplantsforlongperiodsoftimein
fieldgenebanks,naturereserves,orbotanicalgardens.
Alternatively,cuttingsandothervegetativepartsmay
beconservedforashortperiodoftimeunder
moderatelylowtemperatureandhumidity.
•Forlong-termstorage,invitrotechnologyisused.
112Dr. Zekeria Yusuf (PhD)

113. Types of plant germplasmcollections
•Therearefourtypesofplantgeneticresourcesmaintainedbygermplasm
repositories–basecollections,backupcollections,activecollections,and
breeders’orworkingcollections.Thesecategorizationsareonlyapproximate
sinceonegroupcanfulfillmultiplefunctions.
1.Basecollections:
•Thesecollectionsarenotintendedfordistributiontoresearchers,butare
maintainedinlong-termstoragesystems.Theyarethemostcomprehensive
collectionsofthegeneticvariabilityofspecies.Entriesaremaintainedinthe
originalform.Storageconditionsarelowhumidityatsubfreezingtemperatures
(−10to−18°C)orcryogenic(−150to−196°C),dependingonthespecies.
Materialsmaybestoredformanydecadesunderproperconditions.
2.Backupcollections:
•Thepurposeofbackupcollectionsistosupplementthebaseselection.Incase
ofadisasteratacenterresponsibleforabasecollection,aduplicatecollection
isavailableasinsurance.IntheUSA,theNationalSeedStorageLaboratoryat
FortCollins,Colorado,isabackupcollectioncenterforportionsofthe
accessionsoftheCentroInternationaledeMejoramientodeMaizyTrigo
(CIMMYT)andtheInternationalRiceResearchInstitute(IRRI).
113Dr. Zekeria Yusuf (PhD)

114. 3.Activecollections:
•Baseandbackupcollectionsofgermplasmaredesignedforlong-term
unperturbedstorage.Activecollectionsusuallycomprisethesame
materialsasinbasecollections,however,thematerialsinactive
collectionsareavailablefordistributiontoplantbreedersorother
patronsuponrequest.Theyarestoredat0°Candabout8%moisture
content,andremainviableforabout10–15years.Tomeetthis
obligation,curatorsofactivecollectionsatgermplasmbanksmust
increasetheamountofgermplasmavailabletofillrequests
expeditiously.Becausetheaccessionsaremorefrequentlyincreased
throughfieldmultiplication,thegeneticintegrityoftheaccessionmay
bejeopardized.
4.Workingorbreeders’collections:
•Breeders’collectionsareprimarilycomposedofelitegermplasmthatis
adapted.Theyalsoincludeenhancedbreedingstockswithunique
allelesforintrogressionintotheseadaptedmaterials.Inthesetimesof
geneticengineering,breeders’collectionsincludeproductsofrDNA
researchthatcanbeusedasparentsinbreedingprograms.
114Dr. Zekeria Yusuf (PhD)

115. Managing plant genetic resources
•Thekeyactivitiesofcuratorsofgermplasmbanks
include:
1.Regenerationofaccessions,
2.Characterization,
3.Evaluation,
4.Monitoringseedviabilityandgeneticintegrity
duringstorage,&
5.Maintainingredundancyamongcollections.
•Germplasmbanksreceivenewmaterialsona
regularbasis.Thesematerialsmustbeproperly
managedsoastoencourageandfacilitatetheir
usebyplantbreedersandotherresearchers.
115Dr. Zekeria Yusuf (PhD)

116. 1. Periodic Regeneration
•Theregenerationofseeddependsonthelifecycleandbreedingsystemofthespecies
aswellascostoftheactivity.
•Tokeepcoststoaminimumandtoreducelossofgeneticintegrity,itisbesttokeep
regenerationandmultiplicationtoabareminimum.
•Itisagoodstrategytomakethefirstmultiplicationextensivesothatampleoriginal
seedisavailablefordepositinginthebaseandduplicateoractivecollections.
•Amajorthreattogeneticintegrityofaccessionsduringregenerationiscontamination
(fromoutcrossingoraccidentalmigration),whichcanchangethegeneticstructure.
Otherfactorsincludedifferentialsurvivalofallelesorgenotypeswithintheaccession,
andrandomdrift.Theisolationofaccessionsduringregenerationiscritical,especiallyin
cross-pollinatedspecies,tomaintaininggeneticintegrity.
•Thisisachievedthroughproperspacing,caging,coveringwithbags,handpollination,
andothertechniques.
•Regenerationofwildspeciesisproblematicbecauseofhighseeddormancy,seed
shattering,highvariabilityinfloweringtime,andlowseedproduction.
•Somespecieshavespecialenvironmentalrequirements(e.g.,photoperiod,
vernalization)andhenceitisbesttorejuvenateplantsunderconditionssimilartothose
intheplacesoftheirorigin,topreventselectioneffect,whichcaneliminatecertain
alleles.
Dr. Zekeria Yusuf (PhD) 116

117. 2. Characterization:
•Curatorsofgermplasmbankscharacterizetheiraccessions,anactivity
thatentailsasystematicrecordingofselectedtraitsofanaccession.
•Traditionally,thesedataarelimitedtohighlyheritablemorphological
andagronomictraits.
•However,withtheavailabilityofmoleculartechniques,somegermplasm
bankshaveembarkeduponmolecularcharacterizationoftheirholdings.
Forexample,CIMMYThasusedthesimplesequencerepeat(SSR)
markersystemforcharacterizingthemaizegermplasmintheirholding.
•Passportdataareincludedingermplasmcharacterization.Thesedata
includeanaccessionnumber,scientificname,collectionsite(country,
village),source(wild,market),geographyofthelocation,andany
diseaseandinsectpests.
•Tofacilitatedataentryandretrieval,characterizationincludestheuseof
descriptors.Thesearespecificpiecesofinformationonplantor
geographicfactorsthatpertaintotheplantcollection.
•TheInternationalPlantGeneticResourcesInstitute(IPGRI)has
prescribedguidelinesforthecategoriesofthesedescriptors.Descriptors
havebeenstandardizedforsomespeciessuchasrice.
117Dr. Zekeria Yusuf (PhD)

118. 3. Evaluation:
•Geneticdiversityisnotusablewithoutproperevaluation.
•Preliminaryevaluationconsistsofreadilyobservable
traits.Fullevaluationsaremoreinvolvedandmayinclude
obtainingdataoncytogenetics,evolution,physiology,and
agronomy.
•Moredetailedevaluationisoftendoneoutsideofthe
domainofthegermplasmbankbyvariousbreedersand
researchersusingthespecificplanttraitssuchasdisease
resistance,productivity,andqualityofproductare
importantpiecesofinformationforplantbreeders.
•Withoutsomebasicinformationofthevalueofthe
accession,userswillnotbeabletomakeproperrequests
andreceivethemostusefulmaterialsfortheirwork.
118Dr. Zekeria Yusuf (PhD)

119. 4.Monitoringseedviabilityandgeneticintegrity:
•Duringstorage,vigortestsshouldbeconductedatappropriate
intervalstoensurethatseedviabilityremainshigh.During
thesetests,abnormalseedlingsmayindicatethepresenceof
mutations.
5.Exchange:
•Theultimategoalofgermplasmcollection,rejuvenation,
characterization,andevaluationistomakeavailableand
facilitatetheuseofgermplasm.Therearevariouscomputer-
basedgenetic-resourcedocumentationsystemsworldwide,
someofwhicharecrop-specific.
•Thesesystemsallowbreederstorapidlysearchandrequest
germplasminformation.Therearevariouslawsregarding,
especially,internationalexchangeofgermplasm.
•Apartfromquarantinelaws,variousinspectionsandtesting
facilitiesareneededatthecheckpointofgermplasm.
119Dr. Zekeria Yusuf (PhD)

120. 6. Germplasmstorage technologies:
•Oncecollected,germplasmismaintainedinthemostappropriateform
bythegenebankwithstorageresponsibilitiesforthematerials.Plant
germplasmmaybestoredintheformofpollen,seed,orplanttissue.
•Woodyornamentalspeciesmaybemaintainedaslivingplants.Indoor
maintenanceisdoneundercoldstorageconditions,withtemperatures
rangingfrom−18to−196°C.
i.Seedstorage:
•Seedsaredriedtotheappropriatemoisturecontentbeforebeing
placinginseedenvelopes.Theseenvelopesarethenarrangedintrays
thatareplacedonshelvesinthestorageroom.
•Thestorageroomismaintainedat−18°C,atemperaturethatwillkeep
mostseedsviableforupto20yearsormore.
•Thecuratorofthelaboratoryandthestaffperiodicallysampleseedsof
eachaccessiontoconductagerminationtest.
•Whengerminationfallsbelowacertainpredeterminedlevel,the
accessionisregrowntoobtainfreshseed.
120Dr. Zekeria Yusuf (PhD)

121. ii.Fieldgrowing:
•Accessionsareregrowntoobtainfreshseedortoincreaseexisting
supplies(afterfillingordersbyscientistsandotherclients).Tokeep
thegeneticpurity,theaccessionsaregrowninisolation,eachplant
coveredwithacottonbagtokeepforeignsourcesofpollenoutand
alsotoensureself-pollination.
iii.Cryopreservation:
•Cryopreservationorfreeze-preservationisthestorageofmaterials
atextremelylowtemperaturesofbetween−150to−196°Cinliquid
nitrogen.Plantcells,tissue,orothervegetativematerialmaybestored
thiswayforalongtimewithoutloosingregenerativecapacity.
•Whereasseedmayalsobestoredbythismethod,cryopreservationis
reservedespeciallyforvegetativelypropagatedspeciesthatneedtobe
maintainedaslivingplants.
•Shoottipculturesareobtainedfromthematerialtobestoredand
protectedbydippinginacryoprotectant(e.g.,amixtureofsugarand
polyethyleneglycolplusdimethylsulfoxide).
121Dr. Zekeria Yusuf (PhD)

122. iv.Invitrostorage:
•Germplasmofvegetativelypropagatedcropsisnormallystoredanddistributedto
usersinvegetativeformssuchastubers,corms,rhizomes,andcuttings.However,it
islaboriousandexpensivetomaintainplantsintheseforms.
•Invitrogermplasmstorageusuallyinvolvestissueculture.Thereareseveraltypesof
tissueculturesystems(suspensioncells,callus,meristematictissues).
•Tousesuspensioncellsandcallusmaterials,theremustbeanestablishedsystemof
regenerationoffullplantsfromthesesystems,somethingthatisnotavailableforall
plantspeciesyet.Consequently,meristemculturesarefavoredforinvitrostorage
becausetheyaremorestable.
•Thetissueculturematerialmaybestoredusingthemethodofslowgrowth
(chemicalsareappliedtoretardtheculturetemperature)orcryopreservation.
v.Molecularconservation:
•TheadventofbiotechnologyhasmadeitpossibleforresearcherstosequenceDNA
oforganisms.Thesesequencescanbesearchedforgenesatthemolecularlevel.
Specificgenescanbeisolatedbycloningandusedindevelopingtransgenicproducts.
122Dr. Zekeria Yusuf (PhD)

123. What is germplasmconservation?
•Plantgermplasmisthegeneticsourcematerialusedby
theplantbreederstodevelopnewcultivars.
Theymayinclude:
•Seeds
Otherplantpropagulessuchas
•Leaf
•Stem
•Pollen
•Culturedcells
Whichcanbegrownintomatureplant?
•Germplasmprovidetherawmaterial(genes)whichthe
breederusedtodevelopcommercialcropvarieties.
123Dr. Zekeria Yusuf (PhD)

124. Need for Conservation of plant Germplasm
••StorageofEconomicallyimportant,endangered,rarespeciesand
makethemavailablewhenneeded.
•Theconventionalmethodsofstoragefailedtopreventlossescaused
duetovariousreasons.
•Humandependenceonplantspeciesforfoodandmanydifferent
uses.E.g.Basicfoodcrops,buildingmaterials,oils,lubricants,
rubber&otherlatexes,resins,waxes,perfumes,dyesfibresand
medicines.
•Speciesextinctionandmanyothersarethreatenedandendangered
–deforestation.
•Greatdiversityofplantsisneededtokeepthevariousnatural
ecosystemsfunctioningstably–interactionsbetweenspecies.
•Aestheticvalueofnaturalecosystemsandthediversityofplant
species.
124Dr. Zekeria Yusuf (PhD)

125. 125Dr. Zekeria Yusuf (PhD)

126. 126Dr. Zekeria Yusuf (PhD)

127. In-situ Preservation
Preservationofthegermplasmintheirnatural
habitat
Theconservationofdomesticatedandcultivated
speciesinthefarmorinthesurroundings.
However,thereisaheavylossordeclineof
species,populationsandecosystemcomposition,
whichcanleadtoalossofbiodiversity,dueto
habitatdestructionandthetransformationsof
thesenaturalenvironments;therefore,insitu
methodsaloneareinsufficientforsaving
endangeredspecies.
127Dr. Zekeria Yusuf (PhD)

128. 128Dr. Zekeria Yusuf (PhD)

129. Ex-situ preservation
1.Tomaintainthebiologicalmaterialoutsidetheir
naturalhabitats.
2.Storageinseedbanks,fieldgenecollections,in
vitrocollectionsandbotanicalgardens
3.Exsituconservationisaviablewayforsaving
plantsfromextinction,andinsomecases,itisthe
onlypossiblestrategytoconservecertainspecies
4.Invitroconservationisespeciallyimportantfor
vegetativelypropagatedandfornon-orthodox
seedplantspecies
129Dr. Zekeria Yusuf (PhD)

130. 130Dr. Zekeria Yusuf (PhD)

131. 131Dr. Zekeria Yusuf (PhD)

132. 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.
132Dr. Zekeria Yusuf (PhD)

133. In vitro method for germplasmconservation
•Invitromethodemployingshoots,meristemsand
embryosareideallysuitedfortheconservationof
germplasm.Theplantwithrecalcitrantseedsand
geneticallyengineeredcanalsobepreservedbythisin
vitroapproach.
Thereareseveraladvantagesassociatedwithinvitro
germplasmconservation
Largequantitiesofmaterialcanbepreservedinsmall
space
Thegermplasmpreservedcanbemaintainedinan
environmentfreefrompathogens.
Itcanbeprotectedagainstthenature’shazards
Fromthegermplasmstocklargenumberofplantscanbe
obtainedwheneverneeded.
133Dr. Zekeria Yusuf (PhD)

134. 134Dr. Zekeria Yusuf (PhD)

135. CRYOPRESERVATION
•Cryopreservation(Greek,krayos-frost)literallymeaninthefrozen
state.
•Theprincipalinvolvedincryopreservationtobringtheplantcellsand
tissueculturestoazerometabolismornondividingstatebyreducing
thetemperatureinthepresenceofcaryoprotectants.
•Cryopreservationbroadlymeansthestorageofgermplamatverylow
temperature.
1.Oversolidcarbondioxide(at79
o
C)
2.Lowtemperaturedeepfreezers(at-80
o
C)
3.Inliquidnitrogen(at-196
o
C)
•Amongthesethemostcommonlyusedcryopreservationisby
employingliquidnitrogen.Atthetemperatureofliquidnitrogen(-
196
o
C),thecellstayinacompletelyinactivestateandthuscanbe
conservedforlongerperiod.
Infactcryopreservationhasbeensuccessfullyappliedforgermplasm
conservation.Plantspeciese.g.rice,wheat,peanut,sugarcane
,coconut.
135Dr. Zekeria Yusuf (PhD)

136. Principle of Cryopreservation
Cryopreservationistheonlytechniquethatensuresthesafe
andcost-efficientlong-termconservationofvarious
categoriesofplants,includingnon-orthodoxseedspecies,
vegetativelypropagatedplants,rareandendangeredspecies
andbiotechnologyproducts.
StorageofBiomaterialatultralowtemperaturebymeansof
slowfreezing.
Inallcryopreservationprocesses,waterremovalplaysa
centralroleinpreventingfreezinginjuryandinmaintaining
post-thawviabilityofcryopreservedmaterial.
Therearetwotypesofcryopreservationprotocolsthat
basicallydifferintheirphysicalmechanisms:
1. Classical cryopreservation
2. Vitrification
136Dr. Zekeria Yusuf (PhD)

137. Technique of cryopreservation
•Thecryopreservationofplantcellculturefollowed
theregenerationofplantsbroadlyinvolvesthe
followingstages
1. Development of sterile tissue culture.
2. Addition of cryoprotectantand pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival/viability
8. Plant regeneration
137Dr. Zekeria Yusuf (PhD)

138. 138Dr. Zekeria Yusuf (PhD)

139. 139Dr. Zekeria Yusuf (PhD)

140. Classical cryopreservation
•Inthisprocedure,coolingisperformedinthepresenceofice.
•Itinvolvescryoprotectionbyusingdifferentcryoprotectivesolutions
combinedornotwithpregrowthofmaterialandfollowedbyslowcooling
(0.5–2.0°C/min)toadeterminedprefreezingtemperature(usuallyaround
−40°C),rapidimmersionofsamplesinliquidnitrogen,storage,rapidthawing
andrecovery.
•Theyaregenerallyoperationallycomplex,sincetheyrequiretheuseof
sophisticatedandexpensiveprogrammablefreezers.
•Cryopreservationfollowingclassicalprotocolsinducesafreezedehydration
processusingaslowfreezingregime.Astemperaturedecreaseslowly,iceis
initiallyformedintheextracellularsolutionandthisexternalcrystallization
promotestheeffluxofwaterfromthecytoplasmandvacuolestothe
outsideofthecellswhereitfinallyfreezes.
•Therefore,celldehydrationwilldependonthecoolingrateandthe
prefreezingtemperaturesetupbeforeimmersionofsamplestoliquid
nitrogen.
•Classicalcryopreservationtechniqueshavebeensuccessfullyappliedto
undifferentiatedculturesystemsofdifferentplantspecies,suchascell
suspensionsandcalluses.
140Dr. Zekeria Yusuf (PhD)

141. Vitrification
•Inthisprocedure,coolingnormallytakesplace
withouticeformation
•Theprocesswhereformationoficecannottake
placebecauseoftheConcentratedaqueous
solutionwhichpermiticecrystalnucleation.
Instead,watersolidifiesintoanamorphous‘glassy’
state.
•Thevitrification-basedproceduresinvolvecell
dehydrationpriortocoolingbyexposureof
samplestohighlyconcentratedcryoprotective
media(usuallycalledplantvitrificationsolutions,
PVS)and/orbyairdesiccation.
141Dr. Zekeria Yusuf (PhD)

142. Vitrification…
•Coolingratemayberapidorultra-rapid,dependingon
howsamplesareimmersedintoliquidnitrogen.
•Vitrificationperseisaphysicalprocess,definedasthe
transitionoftheliquidphasetoanamorphousglassy
solidattheglasstransition(Tg)temperature.
•Thisglassmaycontributetopreventingtissuecollapse,
soluteconcentrationandpHalterationsduring
dehydration.
•Therefore,thefreeze-induceddehydrationstep
characteristicofclassicalproceduresiseliminatedandthe
slowfreezingregimeisreplacedbyarapidorultra-rapid
coolingprocess,characteristicofthevitrification-based
protocols.
142Dr. Zekeria Yusuf (PhD)

143. 1. Development of sterile tissue culture
•Theselectionofplantspeciesandthetissuewithparticular
referencetothemorphologicalandphysiologicalcharacters
largelyinfluencetheabilityoftheexplantstosurvivein
cryopreservation.
•Anytissuefromaplantcanbeusedforcryopreservation
e.g.meristems,embryos,endosperm,ovules,seeds,
cultureplants.
143Dr. Zekeria Yusuf (PhD)

144. 2. Addition of cryoprotectant
•Cryoprotectantarethecompoundthatcanbepreventthe
damagecausedtocellsbyfreezingorthawing.
•Thereareseveralcryoprotectantwhichinclude(DMSO),
glycerol,ethylene,propylene,sucrose,mannose,glucose,
prolineandacetamide.AmongtheseDMSO,sucrose&glycerol
aremostwidelyused.
144Dr. Zekeria Yusuf (PhD)

145. 3. Freezing
•Thesensitivityofthecelltolowtemperatureisvariable
andlargelydependsontheplantspecies.
•Fourdifferenttypesoffreezingmethodareused:
1.Slowfreezingmethod:thetissueisslowlyfrozenat0.5-
5°C/minfrom0°Cto-100°C,andthentransferredtoliquid
nitrogen.
2.Rapidfreezingmethod:decreaseintemperatureupto-
300to-1000°C.
3.Stepwisefreezingmethod:intermediatetemperaturefor
30min.andrapidlycool.
4.Dryfreezingmethod:reportedthatnon-germinateddry
seedscansurvivefreezingatlowtemperatureincontrast
towaterimbibingseedswhicharesusceptibleto
cryogenicinjuries.
145Dr. Zekeria Yusuf (PhD)

146. 4 : Storage
•Maintenanceofthefrozenculturesatthespecific
temperatureisasimportantasfreezing.
•Ingeneralthefrozencells/tissuesarekeptforstorageat
temperaturesintherangeof-72to-196°C.Storageisideally
doneinliquidnitrogenrefrigerator–at150°Cinthevapour
phase,orat-196°Cintheliquidphase.
•Theultimateobjectiveofstorageistostopallthecellular
metabolicactivitiesandmaintaintheirviability.Forlongterm
storagetemperatureat-196°Cinliquidnitrogenisideal.
146Dr. Zekeria Yusuf (PhD)

147. 5 : Thawing
•Thawingisusuallycarriedoutbyplungingthefrozen
samplesinampoulesintoawarmwater(temp37–45°C)
bathwithvigorousswirling.
•Bythisapproach,rapidthawing(attherateof500-750°C
min¯¹)occurs,andthisprotectsthecellsfromthe
damagingeffectsicecrystalformation.
•Asthethawingoccurs(icecompletelymelts)theampoules
arequicklytransferredtoawaterbathattemperature20-
25°C.Thistransferisnecessarysincethecellsget
damagedifleftforlonginwarm(37-45°C)waterbath.
147Dr. Zekeria Yusuf (PhD)

148. 6. Reculture:
•Ingeneralthawnedgermplasmiswashedseveraltimes
toremovecryoprotectant.Thematerialisthen
reculturedinafreshmedia.
7.Plantregeneration:
•Theultimatepurposeofcryopreservationofgermplasm
istoregeneratethedesiredplant.
•Forappropriateplantgrowthandregeneration,the
cryopreservedcell/tissueshavetobecarefullynursed,
grown.
•Additionofcertaingrowthpromotingsubstances,
besidesmaintenanceofappropriateenvironmental
conditionsisoftennecessaryforsuccessfulplant
regeneration.
148Dr. Zekeria Yusuf (PhD)

149. Applications of germplasmconservation
•Plant materials (cell/tissue) of several species can be cryopreservedand
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 somaclonaland gametoclonalvariation 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 germplasmand other genetic manipulations can be stored.
149Dr. Zekeria Yusuf (PhD)

150. Limitations of germplasmconservation
•Theexpensiveequipmentneededtoprovide
controlledandvariableratesofcooling/warming
temperaturescanhoweverbealimitationinthe
applicationofinvitrotechnologyforlargescale
germplasmconservation.
•Formationoficecrystalinsidethecellshouldbe
preventedastheycauseinjurytothecell.
•Sometimescertainsolutesfromthecellleakout
duringfreezing.
•Cryoprotectantalsoeffecttheviabilityofcells.
150Dr. Zekeria Yusuf (PhD)

151. Concept of gene pools of cultivated crops
HarlananddeWetproposedacategorizationofgenepoolsofcultivatedcrops
accordingtothefeasibilityofgenetransferorgeneflowfromthosespeciesto
thecropspecies.Threecategoriesweredefined,primary,secondary,andtertiary
genepools:
1.Primarygenepool(GP1).GP1consistsofbiologicalspeciesthatcanbe
intercrossedeasily(interfertile)withoutanyproblemswithfertilityofthe
progeny.Thatis,thereisnorestrictiontogeneexchangebetweenmembersof
thegroup.Thisgroupmaycontainbothcultivatedandwildprogenitorsofthe
species.
2.Secondarygenepool(GP2).Membersofthisgenepoolincludebothcultivated
andwildrelativesofthecropspecies.Theyaremoredistantlyrelatedandhave
crossabilityproblems.Nonetheless,crossingproduceshybridsandderivatives
thataresufficientlyfertiletoallowgeneflow.GP2speciescancrosswiththose
inGP1,withsomefertilityoftheF1,butmoredifficultywithsuccess.
3.Tertiarygenepool(GP3).GP3involvestheouterlimitsofpotentialgenetic
resources.GenetransferbyhybridizationbetweenGP1andGP3isvery
problematic,resultinginlethality,sterility,andotherabnormalities.Toexploit
germplasmfromdistantrelatives,toolssuchasembryorescueandbridge
crossingmaybeusedtonurtureanembryofromawidecrosstoafullplantand
toobtainfertileplants.
151Dr. Zekeria Yusuf (PhD)

152. 152Dr. Zekeria Yusuf (PhD)

153. 153Dr. Zekeria Yusuf (PhD)

154. Seed Technology
•Seedtechnologyisthecreationandapplicationoftheknowledgeonseed
foritsbetterusageinagriculture.
•Seedtechnologyreferstomethodsortechniquesusedtomaintainthe
qualityofseedfromharvesttillitisgerminated.
ScopeofSeedTechnology
1.Seedtechnologyencompassesallactivitiescarriedouttoenhance
storability,germinability,vigourandhealthoftheseed.
2.Activitiesincludeharvesting,transporting,handling,storage,testing,
grading,documentation,processingofseedsandgerminationofseeds.
Classes of seed
1.Nucleus or Basic Seed
2.Breeder seed
3.Foundation seed
4.Certified seed
154Haramaya University

155. 155Haramaya University

156. Nucleus or Basic Seed
•Nucleusseed(orbasicseed)istheoriginalorfirstseed(=propagating
material)ofavarietyavailablewiththeproducingbreederoranyother
recognizedbreederofthecrop.
•Thisseedhas100%genetic&physicalpurityalongwithhighstandards
ofallotherseedqualityparameters.
•Whenanewvarietyisreleasedthereisverylittleseed.Theremaybe
onlyahandfulofseedselectedbythebreederfromindividualplants.
ThisseedisthebasisofavarietyandisknownastheNucleusStock.
•Thisnucleusstockmustbemanagedwithgreatcaresothatallseed
producedfromitremainstruetothenewvariety.Thisisamost
importantstepandistheresponsibilityoftheplantbreederwho
developedthevariety.
•Thenucleusstockseedisnotavailabletofarmers.Thenextstepinthe
chainfromplantbreedertofarmeristhattheplantbreederdevelops
BreederSeed.
156Haramaya University

157. BREEDER SEED
•Breederseedistheseedofthehighestpurityofthenewvariety.
•Itisproducedbythebreederandprovidedbythebreeder’sinstitution
toagenciesforfurthermultiplication.
•Ifyouarefromanon-governmentalorganization(NGO)seedbusiness
oraprivatecompanythatisproducingseed,youmayneedtopurchase
breederseedfromaresearchinstitution.Breederseedisthemost
expensiveseedtobuy.
•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.
•Breederseedistheprogenyofthenucleusseedandisthesourcefor
foundationseed.
•Itsproductionisdirectlycontrolledbytheoriginatingplantbreeder
whodevelopedthevariety,oranyotherinstitutionorqualifiedbreeder
recognizedbytheauthorities.
157Haramaya University

158. FOUNDATION SEED
•Foundationseedistheseedproducedfromgrowingbreeder
seed.Itisproducedbytrainedofficersofanagriculturalstationto
nationalstandardsandhandledtomaintainthegeneticpurityof
thevariety.Itmaybeproducedbyagovernmentseedproduction
farmoraprivateorganization–thiswilldependonthe
regulationsofthecountry.Foundationseedislessexpensivethan
breederseed.
•Alsoknowaseliteorbasicseed.Itisthedirectincreaseform
breederseed.Thegeneticidentityandpurityofthevarietyis
carefullymaintainedinfoundationseed.
•Foundationseedisthesourceofcertifiedseed.
158Haramaya University

159. 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.
159Haramaya University

160. CERTIFIED SEED
•Certifiedseedisproducedfromgrowingfoundation,registered
orcertifiedseed.Itisgrownbyselectedfarmerstomaintain
sufficientvarietalpurity.
•Productionissubjecttofieldandseedinspectionspriorto
approvalbythecertifyingagency.Harvestfromthisclassisused
forproducingagain.
•Certifiedseedistheseed,whichiscertifiedbyaSeed
CertificationAgency
•Generally,itisknownastheprogenyoffoundationseedandits
productionissohandledastomaintainspecifiedgenetic
identityandpuritystandardsasprescribedforthecropbeing
certified.
160Haramaya University

161. 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.
161Haramaya University

162. QUALITY DECLARED SEED
•TruthfullylabeledseedorQualityDeclaredSeedisproduced
fromfoundation,registeredorcertifiedseed.
•Itisnotsubjecttoinspectionbyacertifyingagency.Asthis
seedisnotinspected,itsqualityisdependentonthegood
reputationofthefarmerwhohasgrowntheseed.Hisgood
nameinthevillageisimportant.
162Haramaya University

163. MODE OF REPRODUCTION
•Reproductionreferstotheprocessbywhichlivingorganismsproduces
offspringsofsimilarkind(species).Incropplants,themodeofreproductionis
oftwotypes:viz.1)asexualreproduction&2)sexualreproduction.
•Asexualreproductionreferstothemultiplicationofplantswithoutthe
fusionofmaleandfemalegametes.Itcanoccureitherbyvegetativeplant
partsorbyvegetativeembryoswhichdevelopwithoutsexualfusion
(apomixis).Thusasexualreproductionisoftwotypes,viz.,a)vegetative
reproductionand(b)apomixis.
•Vegetativereproductionreferstomultiplicationofplantsbymeansofvarious
vegetativeplantparts.Vegetativereproductionisagainoftwotypes,viz.,a)
naturalvegetativereproductionand(b)artificialvegetativereproduction.
•Naturalvegetativereproductionisthemultiplicationofcertainplantsby
undergroundstems,subaerialstems,rootsandbulbilsnaturally.Insomecrop
species,undergroundstems(amodifiedgroupofstems)giverisetonew
plants.
163Dr. Zekeria Yusuf (PhD)

164. •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
164Dr. Zekeria Yusuf (PhD)

165. •Apomixisreferstothedevelopmentofseedwithoutfertilization.Theembryos
aredevelopedwithoutfertilization.Apomixisisfoundinmanycropspeciesand
isoftwotypesbasedonnature.
•Reproductioninsomespeciesoccursonlybyapomixis.Thisapomixisistermed
asobligateapomixis.Butinsomespeciessexualreproductionalsooccursin
additiontoapomixis.Suchapomixisisknownasfacultativeapomixis.
•Therearefourtypesofapomixisbasedonorigin:viz.1)parthenogenesis,2)
apogamy,3)aposporyand4)adventiveembryony.
•Parthenogenesisreferstodevelopmentofembryofromtheeggcellwithout
fertilization.
•Apogamy–whentheembryooriginatesfromeithersynergidsorantipodalcells
oftheembryosacitiscalledasapogamy.
•Apospory-Inapospory,somediploidcellsofovulelyingoutsidetheembryo
sacdevelopsintoanotherunreducedembryosacthroughaseriesofmitotic
divisionsandwithoutmeiosis.Theembryothendevelopsdirectlyfromthe
diploideggcellofsuchanembryosacwithoutfertilization.
•Adventiveembryony-Thedevelopmentofembryodirectlyfromthediploid
cellsofovulelyingoutsidetheembryosacbelongingtoeithernucellusor
integumentsisreferredtoasadventiveembryony.Itdoesnotinvolvethe
productionofanotherembryosac. 165Dr. Zekeria Yusuf (PhD)

166. Sexualreproduction:
•Reproductionbywhichembryoisdevelopedbythefusionof
maleandfemalegameteisknownassexualreproduction.All
theseedpropagatingspeciesbelongtothisgroup.
MODEOFPOLLINATION
•Theprocessbywhichpollengrainsaretransferredfromanthers
tostigmaisreferredtoaspollination.
•Pollinationisoftwotypes,viz.,1)Autogamyorself
pollinationand2)Allogamyorcrosspollination.
A.Autogamy
•Transferofpollengrainsfromtheanthertothestigmaofsame
flowerisknownasautogamyorselfpollination.Autogamyis
theclosestformofinbreeding.
•Autogamyleadstohomozygosity.Suchspeciesdevelop
homozygousbalance&donotexhibitsignificantinbreeding
depression.
166Dr. Zekeria Yusuf (PhD)

167. Thereareseveralmechanismswhichpromoteautogamy.
1.Bisexualityisthepresenceofmaleandfemaleorgansinthesame
flower.Thepresenceofbisexualflowersisamustforselfpollination.
Alltheselfpollinatedplantshavehermaphroditeflowers.Eg.Rice
2. HomogamyMaturation of anthers and stigma of a flower at the same
time is called homogamywhich is essential for self-pollination Eg.
Bhindi
3. Cleistogamyis when pollination and fertilization occur in an unopened
flower bud. It ensures self pollination and prevents cross pollination.
Eg. cowpea
4. Chasmogamyis 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.
167Dr. Zekeria Yusuf (PhD)

168. B.Allogamy
•Thetransferofpollengrainsfromtheantherofoneplantto
thestigmaofanotherplant(crosspollination).
•Allogamyisthecommonformofout-breedingandleadsto
heterozygosity.Suchspeciesdevelopheterozygousbalance
andexhibitsignificantinbreedingdepressiononselfing.
Theconditionswhichpromoteallogamyareasfollows:
1.Diclinyreferstounisexualflowers.Thisisoftwotypes:viz.
i)monoecyandii)dioecy.
•Whenmaleandfemaleflowersareseparatebutpresentinthe
sameplant,itisknownasmonoecy.
•Insomecrops,themale&femaleflowersarepresentinthe
sameinflorescencesuchasincoconut,mango,castor&
banana.Insomecases,theyareonseparateinflorescenceas
inmaize,cucurbits,cassavaandrubber.Whenstaminateand
pistillateflowersarepresentondifferentplants,itiscalled
dioecyasinpapaya,nutmeganddatepalm
168Dr. Zekeria Yusuf (PhD)

169. 2.Dichogamyreferstothematurationofanthersand
stigmaofthesameflowersatdifferenttimes.
•Dichogamypromotescrosspollinationeveninthe
hermaphroditespecies.
•Dichogamyisoftwotypes:viz.i)protogynyandii)
protandry.
•Whenpistilmaturesbeforeanthers,itiscalled
protogynysuchasinblackpepperandpearlmillet.
•Whenanthersmaturebeforepistil,itisknownas
protandryasincoconutandseveralotherspecies.
169Dr. Zekeria Yusuf (PhD)

170. 3.Heterostyly:Whenstylesandfilamentsinaflowerareof
differentlengths,itiscalledheterostyly.Itpromotescross
pollination,suchaslinseed.
4.Herkogamy:
Hinderancetoself-pollinationduetosomephysicalbarrierssuch
aspresenceofhylinemembranearoundtheantherisknownas
herkogamy.Suchmembranedoesnotallowthedehiscenceof
pollenandpreventsself-pollinationsuchasinalfalfa.
5.Selfincompatibilityistheinabilityoffertilepollenstofertilize
thesameflower.
Itpreventsself-pollinationandpromotescrosspollination.Self
incompatibilityisfoundinseveralcropspecieslikeBrassica,
Radish,Nicotiana,andmanygrassspecies.Itisoftwotypes
sporophyticandgametophytic.
170Dr. Zekeria Yusuf (PhD)

171. 6.Malesterility:insomespecies,thepollengrainsarenon
functional.Suchconditionisknownasmalesterility.It
preventsself-pollination&promotescrosspollination.It
isofthreetypes:viz.genetic,cytoplasmicand
cytoplasmicgenetic.Itisausefultoolinhybridseed
production.
•Selfincompatibilityandmalesterilityarethetwogenetic
mechanismsfavouringcrosspollination.
•Themodeofpollinationplaysanimportantroleinplant
breeding.Ithasimpactonfiveimportantaspects:
1)geneaction
2)geneticconstitution
3)adaptability
4)geneticpurity
5)transferofgenes.
171Dr. Zekeria Yusuf (PhD)

172. Estimation of Genetic variability parameters
172Dr. Zekeria Yusuf (PhD)

173. 173Dr. Zekeria Yusuf (PhD)

174. VARIANCES AND COVARIANCES
•Thevariabilitypresentinapopulationisofpolygenic
natureandthispolygenicvariationisofthreetypes
•1)Phenotypic
•2)Genotypic
•3)Environmental
•Thestatisticalprocedurewhichseparates(or)splitsthe
totalvariationintodifferentcomponentsiscalled
analysisofvariance(or)ANOVA.
•ANOVAisusefulinestimatingthedifferent
componentsofvariance.Itprovidesbasisforthetestof
significanceanditiscarriedoutonlywithreplicated
dataobtainedfromstandardstatisticalexperimental
results.
174Dr. Zekeria Yusuf (PhD)

175. Dr. Zekeria Yusuf (PhD) 175
Genetic variability at a single location

176. 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:
176Dr. Zekeria Yusuf (PhD)

177. 177Dr. Zekeria Yusuf (PhD)
Genetic variability at multilocations

178. Dr. Zekeria Yusuf (PhD) 178

179. Dr. Zekeria Yusuf (PhD) 179

180. Dr. Zekeria Yusuf (PhD) 180

181. 181Dr. Zekeria Yusuf (PhD)

182. Heritabilityandgeneticadvanceareimportantselectionparametersand
heritabilityestimatealongwithgeneticadvanceareinterpretedas
follows
1)Highheritabilityaccompaniedwithhighgeneticadvanceindicates
heritabilityisduetoadditive(or)fixablevariationandselectionmay
beeffective.
2)Highheritabilityaccompaniedwithlowgeneticadvanceindicatesnon
additivegeneactionandselectionforsuchcharactersmaynotbe
rewarding.
3)Lowheritabilityaccompaniedwithhighgeneticadvancerevealsthat
charactersaregovernedbyfixablegeneeffectsandlowheritabilityis
duetohighenvironmentalinfluenceandselectionmaybeeffective.
4)Lowheritabilityaccompaniedwithlowgeneticadvanceindicatesthat
characterishighlyinfluencedbyenvironmentandselectionis
ineffective.
182Dr. Zekeria Yusuf (PhD)

183. 183Dr. Zekeria Yusuf (PhD)

184. ESTIMATION OF HETEROSIS AND INBREEDING DEPRESSION
•referstothesuperiorityofF1inoneormorecharactersoverits
parents.Itisalsodefinedasincreaseinfitnessandyieldoverits
parentalvalues.
•Heterosis,orhybridvigor,oroutbreedingenhancement,isthe
improvedorincreasedfunctionofanybiologicalqualityina
hybridoffspring.Itistheoccurrenceofageneticallysuperior
offspringfrommixingthegenesofitsparents.
•Itisalsocalledashybridvigour.Thethreemaincausesof
heterosisareoverdominance,dominanceandepistasis,ofthis
dominanceisthewidelyacceptedone.
•Incropplantstherearethreemainwaysforfixationofheterosis
i.e.asexualreproduction,polyploidyandapomixis
184Dr. Zekeria Yusuf (PhD)

185. 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.
185Dr. Zekeria Yusuf (PhD)

186. . Four different methods are used to estimate heterosis.
186Dr. Zekeria Yusuf (PhD)

187. •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 vigouris 38.46%
2. Hbp= (F –bp)/bp= (90 –80)/80 =0.125
•Herobeltiosisis 12.5%
•The better parent heterosisis more significant as far as breeding is
concerned because individual progenies are more superior to the
better parent.
187Dr. Zekeria Yusuf (PhD)

188. Gene action
•Allelesmayinteractwithoneanotherinanumberofwaystoproduce
variabilityintheirphenotypicexpression.Thefollowingmodelsmayhelpus
understandvariousmodesofgeneaction.
•Additivegeneaction:absenceofDOMINANCEincaseofsinglelocus.
•Therefore,thebreedingprocedurechosenforacropgenotypewilldependon
theprevalenceofgeneactione.gadditivegeneactionwillbeeffectivein
accumulatingfavourableallelesinbreedingmaterialsespeciallyinself-
pollinatingcrops.
•Heritabilityinthisnarrowersenseistheratiooftheadditivegeneticvariance
tothephenotypicvariance:
188Dr. Zekeria Yusuf (PhD)

189. 189Dr. Zekeria Yusuf (PhD)

190. Additive gene action
•Whenbothallelescontributeforthephenotype.
•Thus,offspringphenotyperesembletheirparents
•Itcanbedirectlyinheritedfromparentstotheiroffspringsas
phenotypeiseffectofeachallelesfrombothparentsbeingadded
together.
•Itcanbefixable
Dominancegeneaction:
✓Inwhichoneallelecontributesmoreorlesstothefinalphenotype.
✓Dominancevarianceisnotdirectlyinheritedfromparentstotheir
offsprings.Sinceitisduetointeractionofgenesfrombothparents
withinindividuals,andofcourseonlyonealleleispassedfrom
eachparenttooffsprings.
190Dr. Zekeria Yusuf (PhD)

191. •Dominancevariancehastwocomponents:variancedueto
homozygousalleles(w/cisadditive)andvariancedueto
heterozygousgenotypicvalues.
•Dominanceeffectsaredeviationsfromadditivitythatmake
heterozygoteresembleoneparentmorethantheother.
•Whendominanceiscompleteheterozygoteisequalto
homozygotesineffects(i.e.Aa=AA).
•Breedercan’tdistinguishbetweenheterozygousandhomozygous
phenotypesasaresultbothAaandAAwillbeselected.
•Thusfixingsuperiourgeneswillbelesseffectivewithdominance
geneactionsinceAawillsegregateinnextgeneration.
191Dr. Zekeria Yusuf (PhD)

192. 192Dr. Zekeria Yusuf (PhD)

193. 193Dr. Zekeria Yusuf (PhD)

194. Dr. Zekeria Yusuf (PhD) 194

195. Dr. Zekeria Yusuf (PhD) 195

196. Overdominancegene action
•Thehetrozygoteismorevaluablethaneitherhomozygotes.
•Existswheneachalleleatalocusproducesaseparateeffecton
thephenotypeandeithercombinedeffectexceedsindependent
effectofthealleles.
•breedercanfixoverdominanceeffectsonlyinF1generation
throughapomixisorthroughchromosomedoulblingofthe
productofwidecross.
Epistasisgeneaction(nonalleleicinteraction)
-interactionbetweenallelesatdifferentloci.
VG=VA+VD+VI
Vp=VG+VE
Forinbreedingpopulation,Vp=Ve,sinceVg=0
Vp=VA+VD+VI+VE+Vgxe
Narrowsenseheritability=
196Dr. Zekeria Yusuf (PhD)

197. Dr. Zekeria Yusuf (PhD) 197

198. Dr. Zekeria Yusuf (PhD) 198

199. Path interpretation
•Inthepathanalysisforgeneticcorrelations,thedirecteffectofNBP
traitonO/LratiowassmallerthantheindirecteffectofAGBP,
NSPODandoleicacidtraitsonO/Lratio;inthiscase,thesignificant
correlationvaluebetweenNBPandO/Lratiowasattributedtothe
indirecteffectsofAGBP,NSPODandoleicacidtraits.Thatis
breedingforhighoilqualitycanbeeffectivethroughindirect
selectionforhighAGBP.InthepathanalysisofAGBPonO/Lratio,
thedirecteffectofAGBPonO/Lratiowassmallerthantheindirect
effectsofNSPODandoleicacidonO/Lratio;inthiscase,the
significantandpositivecorrelationvaluebetweenAGBPandO/L
ratioisattributedtotheindirecteffectsofNSPODandoleicacid
traits.ThepathanalysishasshownthatbreedingforhighO/Lratio
canbeconductedthroughselectionforAGBPtrait.Thecoefficient
ofdetermination()inthepathanalysisforgenotypiccorrelation
indicatesthat92%oftheO/Lratiovariabilitywasexplainedbythe
variableswhichisagoodfitforthemodelandshowsthe
importanceoftheexplainingvariablesintheO/Lratio.
Dr. Zekeria Yusuf (PhD) 199

200. Dr. Zekeria Yusuf (PhD) 200

201. Dr. Zekeria Yusuf (PhD) 201

202. Dr. Zekeria Yusuf (PhD) 202

203. Dr. Zekeria Yusuf (PhD) 203

204. Dr. Zekeria Yusuf (PhD) 204

205. Dr. Zekeria Yusuf (PhD) 205

206. Detection of G×Einteraction
Dr. Zekeria Yusuf (PhD) 206

207. Interpretation of Gene Action
•Ifgcavariancesarehigherthanscavariances,itmeansthat
thereispreponderanceofadditivegeneactionandprogeny
selectionwillbeeffectiveforthegeneticimprovementofsuch
traits.
•Ifscavariancesarehigherthangcavariances,itindicatesthat
thereispreponderanceofnonadditivegeneaction(dominance
&epistasis)andtherefore,heterosisbreedingmaybe
rewarding.
•Ifbothgca&scavariancesareofequalmagnitude,itshowsthat
additiveandnon-additivegenesareequallyimportantinthe
expressionofcharacterthenweusereciprocalrecurrent
selection.
Dr. Zekeria Yusuf (PhD) 207

208. Dr. Zekeria Yusuf (PhD) 208

209. Combining ability analysis
Dr. Zekeria Yusuf (PhD) 209

210. Combining ability analysis…
Dr. Zekeria Yusuf (PhD) 210

211. Combining ability analysis…
Dr. Zekeria Yusuf (PhD) 211

212. Combining ability analysis…
Dr. Zekeria Yusuf (PhD) 212

213. •Parentsshowingahighaveragecombiningabilityin
crossesareconsideredtohavegoodGCAwhileiftheir
potentialtocombinewellisboundedtoaparticularcross,
theyareconsideredtohavegoodSCA.
•Fromastatisticalpointofview,theGCAisamaineffect
andtheSCAisaninteractioneffect[6].BasedonSprague
andTatum[5],GCAisowingtotheactivityofgeneswhich
arelargelyadditiveintheireffectsaswellasadditive×
additiveinteractions.Specificcombiningabilityisregarded
asanindicationoflociwithdominancevariance(non-
additiveeffects)andallthethreetypesofepistatic
interactioncomponentsifepistasiswerepresent.They
includeadditive×dominanceanddominance×dominance
interactions.
Dr. Zekeria Yusuf (PhD) 213

214. 214
Breeding
Self Pollinated Crops

215. 215
➢Cultivar
Isagroupofgeneticallysimilarplants,which
maybeidentified(bysomemeans)fromother
groupsofgeneticallysimilarplants
➢EssentialCharacteristics:
•Identity:cultivarmustbedistinguishable
fromothercultivars
•Reproducibility: the distinguishing
characteristic(s)needtobereproducedinthe
progenyfaithfully
Cultivars

216. 216
Types of Cultivars
Open-Pollinatedcultivars
➢O.P.seedsarearesultofeithernaturalor
humanselectionforspecifictraitswhichare
thenreselectedineverycrop.
➢Theseediskepttruetotypethrough
selectionandisolation;theflowersofopen-
pollinatedorO.P.seedvarietiesare
pollinatedbybeesorwind.

217. 217
Types of Cultivars
Syntheticcultivars
➢Apopulationdevelopedbyinter-crossingaset
ofgoodcombinerinbredlineswith
subsequentmaintenancethroughopen-
pollination.
➢Thecomponentsofsyntheticsareinbredsor
clonessothecultivarcanbeperiodically
reconstituted.

218. 218
➢Multi-linecultivars
Amixtureofisolineseachofwhichisdifferent
forasinglegenecontrollingdifferentformsof
thesamecharacter(e.g.,fordifferentracesof
pathogens)
➢F1cultivars
Thefirstgenerationofoffspringfromacrossof
geneticallydifferentplants
➢Pure-linecultivars
Theprogenyofasinglehomozygousindividual
producedthroughself-pollination
Types of Cultivars

219. 219
Cultivars and Self-pollinated Crops
Inself-pollinatedspecies:
➢Homozygouslociwillremainhomozygous
followingself-pollination
➢Heterozygouslociwillsegregateproducing
halfhomozygous progenyandhalf
heterozygousprogeny
➢Plantsselectedfrommixedpopulationsafter5-
8selfgenerationswillnormallyhavereacheda
practicallevelofhomozygosity

220. 220
➢Ingeneral,amixedpopulationofself-pollinated
plantsiscomposedofplantswithdifferent
homozygousgenotypes(i.e.,aheterogeneous
populationofhomozygotes
➢Ifsingleplantsareselectedfromthispopulation
andseedincreased,eachplantwillproducea
‘pure’population,buteachpopulationwillbe
different,basedontheparentalselection
Cultivars and Self-pollinated Crops

221. Genetic Basis of Self pollinated crops
•Inself-pollinatedspecies:
•Homozygouslociwillremainhomozygousfollowingself-
pollination
•Heterozygouslociwillsegregateproducinghalfhomozygous
progenyandhalfheterozygousprogeny
•Plantsselectedfrommixedpopulationsafter5-8self
generationswillnormallyhavereachedapracticallevelof
homozygosity.
•Ingeneral,amixedpopulationofself-pollinatedplantsis
composedofplantswithdifferenthomozygousgenotypes
•Ifsingleplantsareselectedfromthispopulationandseed
increased,eachplantwillproducea‘pure’population,but
eachpopulationwillbedifferent,basedontheparental
selection
Dr. Zekeria Yusuf (PhD) 221

222. Dr. Zekeria Yusuf (PhD) 222

223. Johanson’sPure line theory
Dr. Zekeria Yusuf (PhD) 223

224. 224
➢SelectioninvolvestheIDandpropagationof
individualgenotypesfromalandracepopulation,
orfollowingdesignedhybridizations
➢Geneticvariationmustbeidentifiedand
distinguishedfromenvironment-basedvariation
➢Selectionprocedurespracticedinmixed
populationsofself-pollinatedcropscanbe
dividedintotwoselectionprocedures
Breeding Self-pollinated Crops

225. 225
Breeding Methods of Self
Pollinated Crops
1. Pure line
2. Mass
3. Bulk
4. Pedigree
5. Single Seed Descent (modified pedigree)
6. Backcross

226. 226
➢Apurelineconsistsofprogenydescended
solelybyself-pollinationfromasingle
homozygousplant
➢Purelineselectionisthereforeaprocedurefor
isolatingpureline(s)fromamixedpopulation
Pure-line Selection

227. 227
➢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:
1.Rapid improvement in land-race or mixed cultivars
2.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

228. 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.
228

229. •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 selfedfamily selection.
•i) Slself family selection
•ii) S2self family selection.
229

230. 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.
230

231. Inbred line development:
c)Inbredorselfedfamilyselection
Familiesproducedbyselfing.
S1familyselection
Familiesproducedbyonegenerationofselfing.Theseareusedforevaluationand
superiorfamiliesareintermated(Simplerecurrentselection).
S2familyselection
Familiesobtainedbytwogenerationsofselfingandusedforevaluation.Superior
familiesareintermated.
Meritsofprogenytestingandselection
1.Selectionbasedonprogenytestandnotonphenotypeofindividualplants.
2.Inbreedingcanbeavoidedifcareistakenraisingalargerpopulationfor
selection.
3.Selectionschemeissimple.
Demerits
1.Nocontroloverpollensource.Selectionisbasedonlyonmaternalparentonly.
2.Comparedtomassselection,thecyclerequires2-3yearswhichistime
consuming.
231

232. Recurrent
parent
Donor
parent
aa AA
Aa F
1
aaAa BC
1F
1
BC
2F
1
aaAa
BC
3F
1
BC
2F
1
aa
AA
aa
Aa
Aa aa
BC
4F
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
232

233. Donor parent Recurrent parent
Backcross for a recessive allele
AA aa
F
1-Aa
BC
1F
1
S**S= selfing BC
1F
2
AAremoved
Aaremoved
aamaintained
BC
2F
1-Aa
BC
3F
1SBC
3F
2
AAremoved
Aaremoved
aamaintained
BC
4F
1-Aa
BC
5F
1-Aa SBC
5F
2
AAremoved
Aaremoved
aamaintained
233

234. 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 propogatedby seeds or by vegetative parts. The
vegetative propogationis 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
234

235. MethodsofBreedingAsexuallyPropagatedSpecies
Importantbreedingmethodsapplicabletoasexuallypropagated
speciesare
(1)PlantIntroduction
(2)Clonalselection
(3)Massselection
(4)Heterosisbreeding
(5)Mutationbreeding
(6)Polyploidybreeding
(7)Distanthybridization
(8)Transgenicbreeding.
•Massselectionisrarelyusedinasexuallypropagatedspecies.
235

236. 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
236

237. Characteristicsofaclones:
1.Alltheindividualbelongingtoasinglecloneareidenticalingenetype
2.Thephenotypicvariationwithinacloneinduetoenvironmentonly
3.Thephenotypeofacloneisduetotheeffectsofgenotype(g),theenvironment(e)
andthegenotypexenvironmentinteraction(GxE),overthepop.mean(M)
4.Theoraticallyclonesareimmortal.Theydeteriorateduetoviral/bacterialinfection
andmutations.
5.Clonesarehighlyheterozygousandstable
6.Theycanbepropagatedgenerationaftergenerationwithoutanychange.
Importanceofaclone
1.Owingtoheterozygosityandsterilityinmanycropsclonesaretheonlymeansof
propagation.
2.Clonesareusedtoproducenewvarieties.
3.Clonesareveryusefultoolstopreservetheheterozygosityonceobtained.Inmany
cropsthesuperiorplantsaremaintained.(Mango,orange,apple,sugarcane)
Sourcesofclonalselection:1.Localvarieties2.Introducedmaterial3.Hybrids
and4.Segregatingpopulations
237

238. 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.
238

239. •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.
239

240. 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
240

241. Plant breeding for Biotic stresses
Dr. Zekeria Yusuf (PhD) 241

242. 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:
1.Non preference,
2.Antibiosis,
3.Tolerance,
4.Avoidance.

243. •Non preference: Non acceptance or AntixenosisUn
attractive or unsuitable for colonization, Ovipositionor 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.

244. Nature of Insect Resistance
1. Hairiness: Hairiness of leaves is associated with resistance.
Eg. Jassidresistance –cotton and cereal leaf beetle.
2. Colourof Plant: Induces non-preference for oviposition. Red cabbage -
Lepidopteran. Red colourCotton -Boll worms.
3. Thickness of plant Tissue: Cotton -Jassidresistance. 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 exudatersetc.
Solanumsp -Gum exudate-Aphids are trapped in it.
Genetics of Insect resistance :
1. Oligogenic-Monogene3 : 1. e.g. Jassidresistance , Cotton Wheat rust
resistance Green bug resistance.
2. Polygenic More durable Wheat cereal leaf beetle resistance.
3. CytoplasmicPlasmogenes: European corn borer in maize.

245. Sources of Resistance
1. Cultivated variety
2. GermplasmCollection.
3. Related Wild species -S.nitidum-shoot fly resistance –
Sorghum, G.anamalum–Jassidresistance -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.

246. II. Breeding for Disease Resistance
•NeedforDiseaseResistanceBreeding
i)Topreventyieldloss.
ii)Highcostofreduction.
iii)Preventionofenvironmentalpollution.
KindsofDiseaseReaction
i)Susceptiblereaction:Diseasereactionisprofuse,if
uncheckeditmayleadtototalyieldloss.
ii)Immunereaction:Hostdoesnotshowthesymptomsofa
disease.
iii)Resistancereaction:Infectionandestablishmenttakesplace
butgrowthofthepathogeninthehostisrestricted.
iv)Tolerance:Hostisattackedbythepathogeninthesame
mannerasthesusceptiblevarietybuttheremaynotbeyield
loss.

247. MechanismofDiseaseResistance
a)Mechanical:Certainmechanicaloranatomicalfeaturesofhostmay
preventinfectione.g.Closedfloweringhabitofwheatandbarley
preventsinfectionbysporesofovaryinfectingfungi.
b)Hypersensitivity:Immediatelyafterinfectionseveralhostcells
surroundingthepointofinfectiondie.Thisleadstodeathof
pathogenalso.Phytoalexinspresentinplantbodyisresponsiblefor
hypersensitivityreaction.
c)Antibiosis:Presenceofsometoxicsubstance.Thisismorecorrect
forinsectresistance.e.g.Gossypolcontentincotton.
d)Nutritionalfactors:Thereductioningrowthandsporeformation
maybeduetonutritionalfactorsofthehost.
GeneticsofDiseaseResistance
a)OligogenicResistance:Resistanceisgovernedbyoneorfewmajor
genesandresistanceisgenerallydominant.Theactionofmajor
genesmaybealteredbymodifiers.

248. MethodsofDiseaseResistanceBreeding
1.Plantintroduction:Resistantvarietiesfromothercanbedirectly
introducedforcultivation.e.g.IR20riceresistanttoblast.
2.Selection:Thismaybefromlocallandracesorfromintroduced
cultivars.
3.HybridisationandSelection:dependingongeneactiontheselection
proceduremayvary.Iftheresistanceisgovernedbypolygenes,then
pedigreemethodofselectionistobefollowed.
•Iftheresistanceisgovernedbymajorgeneslinkedwithother
undesirablecharacterswehavetogoforbackcrossmethodof
breeding.
3.MutationBreeding
4. Polyploidy Breeding:
•Nicotianacrosses 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

249. ScreeningTechniquesforDiseaseResistance
•Dependingonmodeofspreadofdiseasethescreeningtechnique
maydiffer.Thescreeningcanbedonebothatscreenorglasshouse
levelandfieldlevel.Thedifferentscreeningtechniquesareas
follows.
1.SoilBorneDiseases
•Wilt,rootrotareproducedbysoilbornefungi.Inthiscasesickplot
techniqueisfollowed.
•Susceptiblevarietiescanbegrownandinfectedplantscanbe
ploughedinsitutomaintainoptimumconditionforinfection.
2.AirBorneDiseasese.g.Rust,Smut,mildews,blights.
•Forgroundnutrust,infestorrowscanbesown15daysearlieras
borderrowsandthediseasewillinfestthesusceptibleinfestor
rows.After15daysthevarietiestestedtobearetobesown.
•Sprayingthesporesuspensionfromaffectedleaveswillalso
increasetheload.

250. SeedBorneDisease
•Smut,buntetc.Artificialinoculationcanbedone
bysoakingtheseedsinsolutionofpathogen
undervaccumcondition.
InsecttransmittedDiseasese.g.VirusDiseases,
Redgramsterilitymosaicvirus.Saptransmitted.
Herethestaplingtechniqueisused.Leavesfrom
affectedplantscanbestapledtotheentriestobe
tested.Theinsectfeedinginsusceptibleleafwill
transmitvirustotestentries.

251. Breeding for AbioticStress Resistance
(Drought, Cold, Salinity & alkalinity)
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252. 1.TemperatureStress
a.Coldresistance/Tolerance
b.HighTemperature:Duetohightemperatureseedset
maybeaffected.Incaseofmalesterilelines,the
sterilitymaybebrokendown.Inthiscasealsotesting
singleplantsforhightemperatureresistanceistime
consumingandskillisrequired.Testslikeheattestwith
leafdiscsanddesiccationtolerancetestarefollowed.
2.WaterStress
a.Lowwateri.e.,Droughtresistance:Thisismore
importantforallthedrylandcrops.

253. 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 AbioticStress Breeding
i. Screening techniques require high skill and they are time
consuming.
ii. Creation of artificalconditions 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. Abioticstress breeding depends mostly on physiological traits
which are often not stable.

254. Droughtresistanceincropplantscanbedividedinto
threecategories.
i.Droughtescape-abilityofaplanttocompleteits
lifecyclebeforeserioussoilandplantwater
deficitoccurs.
ii.Droughttolerancewithhightissuewaterpotential.
iii.Droughttolerancewithlowtissuewaterpotential.
•Droughtresistanceincropplantsaremoredueto
physiologicalconditionsofplantlikestomatal
aperture& photosyntheticrates,root
characteristics.

255. 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 PhotosynthatesVegetative Parts
•Total Dry matter should be taken as a criterion for selection.
•Drought Resistance
•Drought avoidance & Drought tolerance
1. Xeromorphictraits
2. Root Growth
3. Stomalcontrol,
4. Cuticularresistance(water permeability of leaf cuticle)
4. Stomatalnumber (transpiration low, low stomatalfrequency &
high photosynthetic rate)
5. Cell turgor(Inhibit plant growth) (root water absorption &
stomatalwater loss)

256. Breeding for Drought resistance variety
-High yield x High cuticularwax 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 cuticularwax &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.

257. GeneticDiversity:thedifferencesthatdistinguishoneplantfrom
anotherareencodedintheplant’sgeneticmaterial,theDNA.DNAis
packagedinchromosomepairs,onecomingfromeachparent.Thegenes,
whichcontrolaplant’scharacteristics,arelocatedonspecificsegmentsof
eachchromosome.
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Measurement of Genetic diversity

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259. Why genetic diversity is important in populations...
1.genetic diversity required to evolve or to adapt to new environment or
environmental modifications.
2.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 (Bistonbetularia) 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 sulphurpollution
• light form: visible / dark form: camouflaged
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261. 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 bis 0.4, under linkage
equilibrium:
frequency of ab= 0.2*0.4 = 0.08
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262. Variation from recombination
•linkagedisequilibrium-originalnonrandom
associationbetweenallelesofdifferentgenes
onsamechromosome
•linkagedisequilibriumchangesslowlythrough
time,atrateproportionaltoamountof
recombinationbetweengenes
•geneticvariationthroughrecombinationcan
bemuchfasterthanthroughmutation
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Methods used to measure genetic variation at molecular level:

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275. Genetic Mapping
•Geneticmappingisbasedontheprinciplethatgenes(markersor
loci)segregateviachromosomerecombinationduringmeiosis
(i.e.sexualreproduction),thusallowingtheiranalysisinthe
progeny(Paterson,1996).
Whatismeangeneticmarkers?
•Geneticmarkersrepresentgeneticdifferencesbetweenindividual
organismsorspecies
•Theydonotrepresentthetargetgenesthemselvesbutactas
‘signs’or‘flags’
•Geneticmarkersthatarelocatedincloseproximitytogenesmay
bereferredtoas‘genetags’
•Allgeneticmarkersoccupyspecificgenomicpositionswithin
chromosomescalled‘loci’
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277. Genetic Markers
AgeneticmarkerisageneorDNAsequencewithaknown
locationonachromosomeandassociatedwithaparticular
geneortrait.
Itcanbedescribedasavariation,whichmayarisedueto
mutationoralterationinthegenomicloci,thatcanbe
observed.
AgeneticmarkermaybeashortDNAsequence,suchasa
sequencesurroundingasinglebase-pairchange(single
nucleotidepolymorphism,SNP),oralongone,like
minisatellites.
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278. Genetic Markers
Geneticmarkersarethebiologicalfeaturesthataredeterminedby
allelicformsofgenesorgeneticlociandcanbetransmittedfromone
generationtoanother,andthustheycanbeusedasexperimentalprobes
ortagstokeeptrackofanindividual,atissue,acell,anucleus,a
chromosomeoragene.
Geneticmarkersusedingeneticsandplantbreedingcanbeclassified
intotwocategories:classicalmarkersandDNAmarkers.
Classicalmarkersincludemorphologicalmarkers,cytologicalmarkers
andbiochemicalmarkers.
DNAmarkershavedevelopedintomanysystemsbasedondifferent
polymorphism-detectingtechniquesormethods(southernblotting–
nuclearacidhybridization,PCR–polymerasechainreaction,&DNA
sequencing)suchasRFLP,AFLP,RAPD,SSR,SNP,etc.
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279. Morphological markers
•Duringtheearlyhistoryofplantbreeding,themarkersused
mainlyincludedvisibletraits,suchasleafshape,flowercolor,
pubescencecolor,podcolor,seedcolor,seedshape,hilumcolor,
awntype&length,fruitshape,rind(exocarp)colorandstripe,
fleshcolor,stemlength,etc.
•Thesemorphologicalmarkersgenerallyrepresentgenetic
polymorphismswhichareeasilyidentified&manipulated.
Therefore,theyareusuallyusedinconstructionoflinkagemaps
byclassicaltwo-and/orthreepointtests.
•Someofthesemarkersarelinkedwithotheragronomictraitsand
thuscanbeusedasindirectselectioncriteriainpractical
breeding.
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280. Cytological markers
Incytology,thestructuralfeaturesofchromosomescanbeshownbychromosome
karyotypeandbands.
Thebandingpatterns,displayedincolor,width,orderandposition,
revealthedifferenceindistributionsofeuchromatinand
heterochromatin.Forinstance,Qbandsareproducedbyquinacrine
hydrochloride,GbandsareproducedbyGiemsastain,andRbandsare
thereversedGbands.
Thesechromosomelandmarksareusednotonlyforcharacterizationof
normalchromosomesanddetectionofchromosomemutation,butalso
widelyusedinphysicalmappingandlinkagegroupidentification.
Thephysicalmapsbasedonmorphologicalandcytologicalmarkerslaya
foundationforgeneticlinkagemappingwiththeaidofmolecular
techniques.
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281. Biochemical/protein markers-Allozymes& Isozyme
•Biochemical/proteinmarkers:Proteinmarkersmayalsobecategorizedinto
molecularmarkers.
•Isozymesarealternativeformsorstructuralvariantsofanenzymethat
havedifferentmolecularweightsandelectrophoreticmobilitybuthave
thesamecatalyticactivityorfunction.
•Isozymesreflecttheproductsofdifferentallelesratherthandifferent
genesbecausethedifferenceinelectrophoreticmobilityiscausedby
pointmutationasaresultofaminoacidsubstitution.Therefore,
iso‐zymemarkerscanbegeneticallymappedontochromosomesand
thenusedasgeneticmarkerstomapothergenes.
•Theyarealsousedinseedpuritytestandoccasionallyinplant
breeding.Thereareonlyasmallnumberofisozymesinmostcrop
speciesandsomeofthemcanbeidentifiedonlywithaspecificstain.
Therefore,theuseofenzymemarkersislimited.
•Anotherexampleofbiochemicalmarkersusedinplantbreedingishigh
molecularweightgluteninsubunit(HMW-GS)inwheat.
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282. Biochemical Marker -Allozymes(Isozyme)….
•Allozymesare allelic variants of enzymes encoded by structural
genes.
•Becauseofchangesinelectricchargeandconformationcanaffectthe
migrationrateofproteinsinanelectricfield,allelicvariationcanbe
detectedbygelelectrophoresisandsubsequentenzyme-specificstainsthat
containsubstratefortheenzyme,cofactorsandanoxidizedsalt(e.g.
nitro-bluetetrazolium).
•Usuallytwo,orsometimesevenmorelocicanbedistinguishedforan
enzymeandthesearetermedisoloci.Therefore,allozymevariationis
oftenalsoreferredtoasisozymevariation.
•Althoughproteinmarkerscircumventtheeffectsofenvironment,they
havethedrawbacksofalimitationinthenumberofdetectableisozymesas
wellastissueanddevelopmentstagespecificity.
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283. Biochemical Marker -Allozymes(Isozyme)….
Advantages:
Thestrengthofallozymesissimplicity.
BecauseallozymeanalysisdoesnotrequireDNAextractionorthe
availabilityofsequenceinformation,primersorprobes,theyarequick
andeasytouse.
Simpleanalyticalprocedures,allowsomeallozymestobeappliedat
relativelylowcosts,dependingontheenzymestainingreagentsused.
Allozymesarecodominantmarkersthathavehighreproducibility.
Zymograms(thebandingpatternofisozymes)canbereadilyinterpreted
intermsoflociandalleles,ortheymayrequiresegregationanalysisof
progenyofknownparentalcrossesforinterpretation.
Sometimes,however,zymogramspresentcomplexbandingprofiles
arisingfrompolyploidyorduplicatedgenesandtheformationof
intergenicheterodimers,whichmaycomplicateinterpretation.
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284. Biochemical Marker -Allozymes(Isozyme)….
•Disadvantages:
•relativelylowabundanceandlowlevelofpolymorphism.
•Moreover,proteinswithidenticalelectrophoreticmobility(co-migration)may
notbehomologousfordistantlyrelatedgermplasm.Inaddition,theirselective
neutralitymaybeinquestion.
•Lastly,oftenallozymesareconsideredmolecularmarkerssincetheyrepresent
enzymevariants,andenzymesaremolecules.However,allozymesareinfact
phenotypicmarkers,andassuchtheymaybeaffectedbyenvironmental
conditions.
•Forexample,thebandingprofileobtainedforaparticularallozymemarkermay
changedependingonthetypeoftissueusedfortheanalysis(e.g.rootvs.leaf).
Thisisbecauseagenethatisbeingexpressedinonetissuemightnotbe
expressedinothertissues.
•Onthecontrary,molecularmarkers,becausetheyarebasedondifferencesin
theDNAsequence,arenotenvironmentallyinfluenced,whichmeansthatthe
samebandingprofilescanbeexpectedatalltimesforthesamegenotype.
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285. DNA markers
•Mostwidelyusedtypeofmarker
predominantlyduetotheirabundance
•Selectivelyneutralbecausetheyarenon-
codingsequences
•Notaffectedbyenvironmentalfactors
and/orthedevelopmentalstageoftheplant
•Numerousapplicationsinplantbreedingsuch
asassessingthelevelofgeneticdiversity
withingermplasmandcultivaridentity
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286. DNA Makers/ Molecular markers
•DNAmarkersaredefinedasafragmentofDNArevealingmutations/variations,which
canbeusedtodetectpolymorphismbetweendifferentgenotypesorallelesofagene
foraparticularsequenceofDNAinapopulationorgenepool.
•AmolecularmarkerissegmentofDNAwhosecharacteristicscanbe
measuredandmakeinferencetotheecologyandevolutionofindividuals,
populations,andspecies
Therearethreemethodstodetectthepolymorphism:
1.Southernblotting,anuclearacidhybridizationtechnique(Southern1975),
2.PCR,apolymerasechainreactiontechnique(Mullis,1990),aswellas
3.microarraychiptechniquesuseDNAhybridizationcombinedwithlabeled
nucleotides,andnewsequencingtechniquesdetectpolymorphismbysequencing.
•UsingPCRand/ormolecularhybridizationfollowedbyelectrophoresis(e.g.PAGE–
polyacrylamidegelelectrophoresis,AGE–agarosegelelectrophoresis,CE–capillary
electrophoresis),thevariationinDNAsamplesorpolymorphismforaspecificregion
ofDNAsequencecanbeidentifiedbasedontheproductfeatures,suchasbandsizeand
mobility.InadditiontoSothernblottingandPCR,moredetectionsystemshavebeen
alsodeveloped.
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287. Applications of molecular markers
•DNAmarkersystems,whichwereintroducedtogeneticanalysisinthe1980s,havemanyadvantagesoverthe
traditionalmorphological&proteinmarkersthatareusedingenetic&ecologicalanalysesofplantpopulations:
1.anunlimitednumberofDNAmarkerscanbegenerated;
2.DNAmarkerprofilesarenotaffectedbytheenvironment,&
3.DNAmarkers,unlikeisozymemarkers,arenotconstrainedbytissueordevelopmentalstagespecificity.
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. QTLMapping
•-We will discuss today
8. Marker-Assisted Selection
•-Backcrossing in a transgene
•-Maintaining or crossing in a QTL
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288. Properties of ideal molecular markers
An ideal molecular marker must have some desirable properties which are enlisted as follows:
1. Highly polymorphic/hypervariablenature: It must be polymorphic as it is
polymorphism that is measured for genetic diversity studies.
2. Codominantinheritance: 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
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289. Properties of ideal molecular markers…
12.Freedomfromenvironmentalandpleiotropiceffects.Molecularmarkersdo
notexhibitphenotypicplasticity,whilemorphologicalandbiochemicalmarkers
canvaryindifferentenvironments.DNAcharactershaveamuchbetterchance
ofprovidinghomologoustraits.Mostmorphologicalorbiochemicalmarkers,in
contrast,areunderpolygeniccontrol,andsubjecttoepistaticcontrol&
environmentalmodification(plasticity);
13.Potentiallyunlimitednumberofindependentmarkersareavailable,unlike
morphologicalorbiochemicaldata;
14.DNAcharacterscanbemoreeasilyscoredasdiscretestatesofallelesorDNA
basepairs,whilesomemorphological,biochemicalandfieldevaluationdata
mustbescoredascontinuouslyvariablecharactersthatarelessamenableto
robustanalyticalmethods;
Theseadvantagesdonotimplythatothermoretraditionaldatausedtocharacterize
biodiversityarenotvaluable.Onthecontrary,morphological,ecologicaland
other“traditional”datawillcontinuetoprovidepracticalandoftencritical
informationneededtocharacterizegeneticresources.
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290. Properties of ideal molecular markers…
Itisextremelydifficulttofindamolecularmarker,whichwouldmeetalltheabove
criteria.
Awiderangeofmoleculartechniquesisavailablethatdetectspolymorphismatthe
DNAlevel.Dependingonthetypeofstudytobeundertaken,amarkersystemcanbe
identifiedthatwouldfulfillatleastafewoftheabovecharacteristics.
VarioustypesofmolecularmarkersareutilizedtoevaluateDNApolymorphismandare
generallyclassifiedashybridization-basedmarkersandpolymerasechainreaction
(PCR)-basedmarkers.Intheformer,DNAprofilesarevisualizedbyhybridizingthe
restrictionenzyme-digestedDNA,toalabeledprobe(whichisaDNAfragmentof
knownoriginorsequence).
PCRbasedmarkersinvolveinvitroamplificationofparticularDNAsequencesorloci,
withthehelpofspecificallyorarbitrarilychosenoligonucleotidesequences(primers)
andathermostableDNApolymeraseenzyme.
Theamplifiedfragmentsareseparatedelectrophoreticallyandbandingpatternsare
detectedbydifferentmethodssuchasstainingandautoradiography.
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291. Genetic markers: dominance / co-dominance principle
dominance:whenheterozygotesarenot
distinguishablefromhomozygotes
‣AAwithPCRproduct,aawithoutPCRproduct,Aa
withPCRproduct
co-dominance:whenheterozygotesare
distinguishablefromhomozygotes
‣AAwithlowmobility,aawithhighmobility,Aa
withamediummobility
→difficultiesintheanalyses
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295. Applications of molecular markers
1.Assessmentofgeneticdiversityandcharacterizationof
germplasm
2.Identification&fingerprintingofgenotypes
3.Estimationofgeneticdistancebetweenpopulations,inbreds&
breedingmaterials
4.Detectionofmonogenic/qualitative&quantitativetrait
loci(QTL)
5.Markerassistedbreeding
6.Identificationofsequencesofusefulcandidategenesetc.
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296. CLASSIFICATION OF DNA MARKERS
•Basedonthemethodoftheirdetectionbroadlydivided
into
1.Hybridization-based
2.PCRbased
3.DNAsequence-based
•Visualisesthegeneticdifferencesbygelelectrophoresis
•Stainingwithchemicals(ethidiumbromideorsilver)
•Detectionwithradioactiveorcolourimetricprobes
PolymorphicDNAmarkers:helpsinrevealingthe
differencesbetweenindividualsofthesameordifferent
species.
MonomorphicDNAmarkers:Markersthatdonot
differentiatebetweengenotypesarecalledmonomorphic
markers
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299. First generation molecular markers
•ThefirstgenerationofDNAmarkersystemsemployedSouthernblot
basedmarkers.
•RFLPs(restrictionfragmentlengthpolymorphisms):resultfrompoint
mutationsinrestrictionenzymerecognitionsites.Chromosomal
mutationssuchasinsertions,deletions,inversions,andtranslocations
canalsocauserestrictionfragmentsizepolymorphisms.TheRFLP
techniqueemploysmolecularhybridizationofcDNAorgenomicDNA
probeswithgenomicDNAfragmentedbyrestrictionenzymes.
•AnotherSouthernblotbasedmarkersystemreliedonminisatellite
probesfor‘fingerprinting’individualspecifichumanDNA.
MinisatelliteDNAsareshortstretchesofDNAthatarepresentin
tandemrepeatsineukaryotes.Theyarehighlyabundant,and
individualsoftencarrydifferentnumbersoftandemrepeatswhichcan
bedetectedasVNTRs(variablenumberoftandemrepeat)byPCR
amplification.
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300. Minisatellites, Variable Number of Tandem Repeats (VNTR)
•ThetermDNAfingerprintingwasintroducedforminisatellites,though
DNAfingerprintingisnowusedinamoregeneralwaytorefertoa
DNA-basedassaytouniquelyidentifyindividuals.
•Minisatellitesareparticularlyusefulinstudiesinvolvinggeneticidentity,
parentage,clonalgrowthandstructure,andidentificationofvarieties
andcultivars,andforpopulation-levelstudies.Minisatellitesareof
reducedvaluefortaxonomicstudiesbecauseofhypervariability.
DNA fingerprints (minisatellites)
•‣core repeat sequences of 10-100 bp
•highly variable, high quantity of DNA, difficult to set-up / old
method
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305. Second Generation DNA Markers
•ThesecondgenerationDNAmarkersforgeneticanalysiswerethose
derivedfromPCRpolymerasechainreaction.
•PCRrevolutionizedgeneticandecologicalanalysesofpopulationsin
severalwaysbecauseithadtwomajoradvantagesoverSouthernblot
basedmarkers:
1.itrequiresonlysmallDNAamountstoallowanalysisatveryearly
stages,thusreducingtheneedforplantnurseries.
2.itisinexpensive,andsimpleenoughthatlargescaleexperimentscanbe
carriedoutrapidlyonalargescale.OfthemanyPCR-marker
techniquesthathavebeendeveloped,RAPD,AFLPISSR,SSRandSNP
arethemajorsystems,withtheothersystemsbeingmodificationsof
thesethree.
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306. Inter Simple Sequence Repeats (ISSR)
•ISSRinvolvesamplificationofDNAsegmentspresentatanamplifiabledistancein
betweentwoidenticalmicrosatelliterepeatregionsorientedinoppositedirection.
•ThetechniqueusesmicrosatellitesasprimersinasingleprimerPCRreactiontargeting
multiplegenomiclocitoamplifymainlyintersimplesequencerepeatsofdifferentsizes.
•ThemicrosatelliterepeatsusedasprimersforISSRscanbedi-nucleotide,trinucleotide,
tetranucleotideorpenta-nucleotide.
•ISSRsuselongerprimers(15–30mers)ascomparedtoRAPDprimers(10mers),
whichpermitthesubsequentuseofhighannealingtemperatureleadingtohigher
stringency.
•IncontrasttotheSSRmarkertechniquethatamplifieswithprimerslocatedonthe
flankingsinglecopyDNA,microsatellitesanchoredprimersthatannealtoanSSRregion
canamplifyregionsbetweenadjacentSSRs.
•TheISSRtechniqueusesprimersthatarecomplimentarytoasingleSSRandanchoredat
eitherthe5'or3'endwithaone-tothree-baseextension.Theampliconsgenerated
consistofregionsbetweenneighbouringandinvertedSSRs.Asaresult,thehighcomplex
bandingpatternobtainedwilloftendiffergreatlybetweengenotypesofthesamespecies.
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307. Microsatellites or Simple sequence Repeat (SSR)
SSRs,alsocalledmicrosatellites,SimpleSequenceRepeats(SSRs),short
tandemrepeats(STRs)orsequence-taggedmicrosatellitesites(STMS),are
sectionsofDNA,consistingoftandemrepeatsofshortnucleotidemotifs(1-6
bp/nucleotideslong),mono-,di-,tri-,tetra-orpenta-nucleotideunitsthatare
randomlyarrangedthroughoutthegenomesofmosteukaryoticspecies.e.g.
(GT)n,(AAT)nand(GATA)n.
Microsatellitemarkers,developedfromgenomiclibraries,canbelongtoeither
thetranscribedregionorthenontranscribedregionofthegenome,andrarelyis
thereinformationavailableregardingtheirfunctions.
BecausetheDNAsequencesflankingmicrosatelliteregionsareusually
conserved,primersspecificfortheseregionsaredesignedforuseinthePCR
reaction.SSRlociareindividuallyamplifiedbyPCRusingpairsof
oligonucleotideprimersspecifictounique/conservedDNAsequencesflanking
theSSRsequence.
Inaddition,primersmaybeusedthathavealreadybeendesignedforclosely
relatedspecies.
307Haramaya University

308. SSR…
ThePCR-amplifiedproductscanbeseparatedinhigh-resolutionelectrophoresis
systems(e.g.AGEandPAGE)andthebandscanbevisuallyrecordedby
fluorescentlabelingorsilver-staining.
Microsatellites,likeminisatellites,representtandemrepeats,buttheirrepeat
motifsareshorter.
PolymeraseslippageduringDNAreplication,orslippedstrandmispairing,is
consideredtobethemaincauseofvariationinthenumberofrepeatunitsofa
microsatellite,resultinginlengthpolymorphismsthatcanbedetectedbygel
electrophoresis.
Microsatellitesequencesareespeciallysuitedtodistinguishcloselyrelated
genotypes;becauseoftheirhighdegreeofvariability/polymorphism,theyare,
therefore,favouredinpopulationstudies&fortheidentificationofclosely
relatedcultivars.
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309. Haramaya University 309

310. The third generation of molecular markers
•Thethirdgenerationofmolecularmarkersisthesystemthatutilizes
SNPs(singlenucleotidepolymorphisms)andmicroarrays.
•Comparedtothegel-basedmolecularmarkersystems,SNPdetection
andanalysiscanbecarriedoutwithnon-gelbasedassays.
•Thepolymorphismofasinglebasedifferencecanbeassessedby
through-putanalysis:hybridizationwithallele-specificoligonucleotides
(ASO),primerextension,oligonucleotideligationassay(OLA),&
invasivecleavage.
•ThefrequencyoftheSNPscanrangefromapproximatelyoneper30bp
tooneper500bpinotherplantspecies.
•Althoughthesenewgenerationmarkersystemsarepowerfultoolsin
linkagedisequilibriumanalysis,germplasmassaybyhaplotyping,QTL
(quantitativetraitloci)analysisandafewothers,theyareonly
amenabletouseinthosespeciesforwhichextensivenucleotide
sequenceinformationisavailableinmajorcrops.
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311. SNPmarkers
AnSNPisasinglenucleotidebasedifferencebetweentwoDNAsequencesor
individuals.
SNPscanbecategorizedaccordingtonucleotidesubstitutionseitheras
transitions(C/TorG/A)ortransversions(C/G,A/T,C/AorT/G).
Inpractice,singlebasevariantsincDNA(mRNA)areconsideredtobeSNPs
asaresinglebaseinsertionsanddeletions(indels)inthegenome.
Thefactthatinmanyorganismsmostpolymorphismsresultfromchangesina
singlenucleotideposition(pointmutations),hasledtothedevelopmentof
techniquestostudysinglenucleotidepolymorphisms(SNPs).
SNPsprovidetheultimate/simplestformofmolecularmarkersasasingle
nucleotidebaseisthesmallestunitofinheritance,andthustheycanprovide
maximummarkers.SNPsoccurverycommonlyinanimalsandplants.
Typically,SNPfrequenciesareinarangeofoneSNPevery100-300bpin
plants.
SNPsmaypresentwithincodingsequencesofgenes,non-codingregionsof
genesorintheintergenicregionsbetweengenesatdifferentfrequenciesin
differentchromosomeregions. 311Haramaya University

312. Gene microarrays
–Genemicroarraysenableresearcherstostudyallofthe
genesexpressedinatissueveryfast
–Microarray(genechip)iscreatedwithuseofsmallglass
microscopeslide
•SinglestrandedDNAmoleculesarespottedontheslideusingan
arrayer(computercontrolledroboticarm)whichfixesDNA
(multiplecopiesofcDNA)atdifferentspotsontheslidewhichis
recordedbyacomputer.
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336. Haramaya University 336

337. 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)
337Haramaya University

338. Some important findings from HapMap project (and earlier studies using other genetic
markers)
(1)About 10-15% of total human genetic variation is among populations; rest is within
populations
(2)Africa harbours substantially higher levels of human genetic variation than other
regions
(3)Patterns of natural selection ‘for’ given alleles (positive selection) vary substantially
among populations
-> local adaptation
338Haramaya University

339. Molecular Breeding
Molecularbreeding(MB)maybedefinedinabroad-senseasthe
useofgeneticmanipulationperformedatDNAmolecular
levelstoimprovecharactersofinterestinplantsandanimals
(MAB+GMO).
Marker-assistedbreeding(MAB)isdefinedastheapplicationof
molecularbiotechnologies,specificallymolecularmarkers,in
combinationwithlinkagemapsandgenomics,toimprove
plantoranimaltraitsonthebasisofgenotypicassays.
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340. 340Haramaya University

341. 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
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343. 343Haramaya University

344. 344Haramaya University

345. 345Haramaya University

346. Prerequisites for an efficient marker-assisted selection program
1.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
346Haramaya University

347. Prerequisites for an efficient marker-assisted selection program…
3.Geneticmaps:
•Linkagemapsprovideaframeworkfordetectingmarker-traitassociationsand
forchoosingmarkerstoemployinMAS.
•Onceamarkerisfoundtobeassociatedwithatraitinagivenpopulation,a
densemolecularmarkermapinastandardreferencepopulationwillhelp
identifymarkersthatarecloserto,orthatflank,thetarget.
4.ThemostcrucialingredientforMASisknowledgeofmarkersthatare
associatedwithtraitsimportanttoabreedingprogram.
5.Datamanagementsystem
•LargenumbersofsamplesarehandledinaMASprogram,witheachsample
potentiallyevaluatedformultiplemarkers.
•Thissituationrequiresanefficientsystemforlabeling,storing,retrieving,and
analyzinglargedatasets,andproducingreportsusefultothebreeder.
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348. 348Haramaya University

349. 349Haramaya University

350. REQUIREMENTS FOR GENETIC MAPPING
•Geneticlinkagemapconstructionrequiresthatthe
researcher;
1)Developappropriatemappingpopulationanddecidethe
samplesize.
2)Decidethetypeofmolecularmarker(s)forgenotyping
themappingpopulation.
3)Screenparentsformarkerpolymorphism,andthen
genotypethemappingpopulation(parentsplusall
progenies).
4)Performlinkageanalyses(calculatepairwise
recombinationfrequenciesbetweenmarkers,establish
linkagegroups,estimatemapdistances,anddetermine
maporder)usingstatisticalprograms.
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351. Gene Mapping principle
•Genesandmarkerssegregateviachromosomerecombination(called
crossing-over)duringmeiosis
•Genesormarkersthatareclosetogetherortightly-linkedwillbe
transmittedtogetherfromparenttoprogenymorefrequentlythan
genesormarkersthatarelocatedfurtherapart
•Inasegregatingpopulation,thereisamixtureofparentaland
recombinantgenotypes
•Thefrequencyofrecombinantgenotypes:inferthegeneticdistance
betweenmarkers
•Thelowerthefrequencyofrecombinationbetweentwomarkers,the
closertheyaresituatedonachromosomeandthehigherthe
frequencyofrecombinationbetweentwomarkers,thefurtheraway
theyaresituatedonachromosome).
•Markersthathavearecombinationfrequencyof50%aredescribedas
‘unlinked’andassumedtobelocatedfarapartonthesame
chromosomeorondifferentchromosomes
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352. 352Haramaya University

353. Gene Mapping principle….
•Inasegregatingpopulation,thereisamixtureof
parentalandrecombinantgenotypes
•Thefrequencyofrecombinantgenotypes:inferthe
geneticdistancebetweenmarkers
•Thelowerthefrequencyofrecombinationbetween
twomarkers,theclosertheyaresituatedona
chromosomeandthehigherthefrequencyof
recombinationbetweentwomarkers,thefurtheraway
theyaresituatedonachromosome).
•Markersthathavearecombinationfrequencyof50%are
describedas‘unlinked’andassumedtobelocatedfar
apartonthesamechromosomeorondifferent
chromosomes.
353Haramaya University

354. QTLMapping
•Theprocessofconstructinglinkagemapsandconducting
QTLanalysistoidentifygenomicregionsassociatedwith
traitswiththehelpofmolecularmarkersisknownasQTL
mapping;Alsocalledasgenetic,geneorgenome
mapping;
•Manyagriculturallyimportanttraitssuchasyield,quality
andsomeformsofdiseaseresistancearecontrolledby
manygenesandareknownas“quantitativetraitsor
polygenicormultifactorialorcomplextraits”.
•Thesetraitsshowcontinuousvariationinapopulation.
•Thesetraitsdonotfallintodiscreteclasses.
•Theyaremeasurable.
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355. 355Haramaya University

356. Quantitative Trait Loci (QTL)
•ThelocicontrollingquantitativetraitsarecalledquantitativetraitlociorQTL.
•TermfirstcoinedbyGeldermanin1975.
•Itistheregionofthegenomethatisassociatedwithaneffectona
quantitativetrait.
•Itcanbeasinglegeneorclusteroflinkedgenesthataffectthetrait.
•QTLsarecontrolledbymultiplegenes,eachsegregatingaccordingtoMendel's
laws.
•Thesetraitscanalsobeaffectedbytheenvironmenttovaryingdegrees.
Individualgeneeffectsissmall&Thegenesinvolvedcanbedominant,or
codominant.Thegenesinvolvedcanbesubjecttoepistasisorpleiotrophic
effect.
•QuantitativeTraitLocus(QTL):astatisticallysignificantlocus(notnecessarily
agene)thatquantitativelyaffectsaphenotypeofinterestwithphysical
boundariesdefinedbylinkedmolecularmarkers.
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357. 357Haramaya University

358. 358Haramaya University

359. 359Haramaya University

360. 360Haramaya University

361. Objectives of QTLMapping
•ThebasicobjectiveistodetectQTL,whileminimizing
theoccurrenceoffalsepositives(TypeIerrors,thatis
declaringanassociationbetweenamarkerandQTL
wheninfactonedoesnotexist).
•Toidentifytheregionsofthegenomethataffectsthe
traitofinterest.
•ToanalyzetheeffectoftheQTLonthetrait.
•Howmuchofthevariationforthetraitiscausedbya
specificregion?
•WhatisthegeneactionassociatedwiththeQTL–
additiveeffect?Dominanteffect?
•Whichalleleisassociatedwiththefavorableeffect?
361Haramaya University

362. Prerequisites for QTLmapping
1.Availabilityofagoodlinkagemap(thiscanbe
doneatthesametimetheQTLmapping)
2.Asegregatingpopulationderivedfromparents
thatdifferforthetrait(s)ofinterest,&which
allowforreplicationofeachsegregant,sothat
phenotypecanbemeasuredwithprecision(such
asRILsorDHs)
3.Agoodassayforthetrait(s)ofinterest
4.Softwareavailableforanalyses
5.MolecularMarkers
6.SophisticatedLaboratory
362Haramaya University

363. Prerequisites for QTLmapping…
•Selection of parental lines
•Sufficient polymorphism
•Parentallinesarehighlycontrasting
phenotypicallyGeneticallydivergent
•Selectionof molecular markers
(dominant/codominant)
•Making crosses
•Creation of mapping population
363Haramaya University

364. Principle of QTLanalysis
•Genesandmarkerssegregateviachromosomerecombinationduring
meiosis,thusallowingtheiranalysisintheprogeny.
•QTLanalysisdependsonthelinkagedisequilibrium.
•QTLanalysisisusuallyundertakeninsegregatingmappingpopulations.
•Asignificantdifferencebetweenphenotypicmeansofthegroupsindicates
thattheparticularmarkerlocusislinkedtoaQTLcontrollingthetrait.
•Itisbasedontheprincipleofdetectinganassociationbetweenphenotype
andthegenotypeofthemarkers.
•Markersareusedtopartitionthemappingpopulationintodifferent
genotypicgroupsbasedonthepresenceorabsenceofaparticularmarker
locusandtodeterminewhethersignificantdifferencesexistbetweengroups
withrespecttothetraitbeingmeasured.
•Asignificantdifferencebetweenphenotypicmeansofthegroups,
dependingonthemarkersystemandtypeofmappingpopulation,
•Itisnoteasytodothisanalysismanuallyandsowiththehelpofacomputer
andasoftwareitisdone.
364Haramaya University

365. Principle of QTLanalysis contd…
•If,QTL&markeriscloselylinked,chanceof
recombinationwillbeless.
•SoQTLandmarkerwillbeinheritedtogetherand
meanofthegroupwillhavesignificant
difference,
•Iflooselylinkedorunlinked,thereisindependent
segregationofthemarkerandQTL.Thus,there
willbenosignificantdifferencebetweenmeans
ofthegenotypegroups.
365Haramaya University

366. What are linkage maps?
•Itisthe‘roadmap’ofthechromosomes
derivedfromtwodifferentparents
•Indicatethepositionandrelativegenetic
distancesbetweenmarkersalongchromosomes
(signsorlandmarksalongahighway)
•Helpstoidentifychromosomallocations
containinggenesandQTLsassociatedwith
traitsofinterest;suchmapsmaythenbe
referredtoas‘QTL’/or‘genetic’maps
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367. 367Haramaya University

368. 368Haramaya University

369. How linkage map constructed?
•Thethreemainstepsoflinkagemap
constructionare:
(1)Productionofamappingpopulation
(2)Identificationofpolymorphism
(3)Linkageanalysisofmarkers
369Haramaya University

370. 1. Production of a mapping population
•Requiresasegregatingplantpopulation(i.e.apopulation
derivedfromsexualreproduction)
•Theparentsselectedwilldifferforoneormoretraitsofinterest
Populationsizes:generallyrangefrom50to250individuals
•IfmapisusedforQTLstudiesthenthemappingpopulation
mustbephenotypicallyevaluated(i.e.traitdatamustbe
collected)beforesubsequentQTLmapping
•Inselfpollinatedspecies,parentsthatarebothhighly
homozygous(inbred)
•Incrosspollinatingspecies,thesituationismorecomplicated
beingheterozygous
370Haramaya University

371. Mapping population
•Thefirststepinproducingamappingpopulationis
selectingtwogeneticallydivergentparents,whichshow
cleargeneticdifferencesforoneormoretraitsofinterest
(e.g.,therecipientorrecurrentparentcanbeahighly
productiveandcommerciallysuccessfulcultivarbutlacks
diseaseresistance,whichispresentinanotherdonor
parent).
•Theparentsshouldbegeneticallydivergentenoughto
exhibitsufficientpolymorphismandatthesametimethey
shouldnotbetoogeneticallydistantsoasto:
a)Causesterilityoftheprogeniesand/or,
b)Showveryhighlevelsofsegregationdistortionduring
linkageanalysis.
371Haramaya University

372. Mapping populations developed for self pollinating species.
•Inself-pollinatingspecies,mappingpopulationsoriginatefromparentsthat
arebothhighlyhomozygous(inbred).
•AsshowninFigure2,progeniesfromthesecondfilialgeneration(F2),
backcross(BC),recombinantinbredlines(RILs),doublehaploids(DHs),and
nearisogeniclines(NILs)canbeusedforgeneticmappinginselfpollinating
species.
•Theirmainadvantagesarethattheyareeasytoconstructandrequireonlya
shorttimetoproduce.InbreedingfromindividualF2plantsallowsthe
constructionofrecombinantinbred(RI)lines,whichconsistofaseriesof
homozygouslines,eachcontainingauniquecombinationofchromosomal
segmentsfromtheoriginalparents.
•ThelengthoftimeneededforproducingRIpopulationsisthemajor
disadvantage,becauseusuallysixtoeightgenerationsarerequired.Doubled
haploid(DH)populationsmaybeproducedbyregeneratingplantsbythe
inductionofchromosomedoublingfrompollengrains,however,the
productionofDHpopulationsisonlypossibleinspeciesthatareamenableto
tissueculture(e.g.cerealspeciessuchasrice,barleyandwheat).
•ThemajoradvantagesofRIandDHpopulationsarethattheyproduce
homozygousor‘true-breeding’linesthatcanbemultipliedandreproduced
withoutgeneticchangeOccurring.
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373. Haramaya University 373

374. Mapping populations developed for cross pollinating species
•Two-waypseudo-testcross,half-sibandfull-sibfamiliesderivedfromcontrolledcrosses
havebeenproposedformappinginoutcrossingspecies.
•F2populationsaredevelopedbyselfingF1hybridsderivedbycrossingthetwoparents
whileBCpopulationisproducedbycrossingF1backintooneoftheparents(the
recipientorrecurrentparent).
•RILsaredevelopedbysingle-seedselectionsfromindividualplantsofanF2population;
suchselectionscontinueforsixtoeightgenerations.
•Ifbackcrossselectionisrepeatedatleastforsixgenerations,morethan99%ofthe
genomefromBC6andabovewillbederivedfromrecurrentparent(Babuetal.,2004).
•SelfingofselectedindividualsfromBC7F1willproduceBC7F2linesthatare
homozygousforthetargetgene,whichissaidtobenearlyisogenicwiththerecipient
parent(NILs).
•NILsarefrequentlygeneratedbyplantbreedersastheytransfermajorgenesbetween
varietiesbybackcrossbreeding(Tanksleyetal.,1995).
•ADHpopulationisproducedbydoublingthegametesofF1orF2population.Plants
willberegeneratedusingtissueculturetechniquesafterinductionofchromosome
doublingfrompollengrainsorhaploidembryosresultingfromspeciescrosses.
•Incrosspollinating(outcrossing)species,thesituationismorecomplicatedsincemost
ofthesespeciesdonottolerateinbreeding.
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375. 2. Identification of polymorphism
•Selectanappropriatemarkerthatshowsdifferences
betweenparents(polymorphicmarkers)
•Itiscriticalthatsufficientpolymorphismexistsbetween
parents
•InCrosspollinatingspecies,higherDNApolymorphism
exitscomparedtoinbreedingspecies,
•Soininbreedingspecies,parentsselectedshouldbe
distantlyrelated,
•Thenmarkershouldbescreenedacrosstheentire
mappingpopulation,includingtheparents:marker
genotyping.
375Haramaya University

376. Identification of polymorphism contd..
•Generally,markerswillsegregateinaMendelian
fashionalthoughdistortedsegregationratios
maybeencountered
•Significantdeviationsfromexpectedratioscanbe
analysedusingchisquaretests
•Inpolyploidspecies,identifyingpolymorphic
markersismorecomplicated
•Somappingofdiploidrelativesofpolyploid
speciesisdone
•However,diploidrelativesdonotexistforall
polyploidspecies
376Haramaya University

377. 3. Linkage analysis of markers
•CodedataforeachDNAmarkeroneachindividual
ofapopulationandconductlinkageanalysisusing
computerprograms
•Linkagebetweenmarkersisusuallycalculatedusing
Oddsratios:theratiooflinkageversusnolinkage
•LODvalue/LODscore:Logarithmoftheoddratio
•Manualanalysisisnotfeasible,socomputer
programmesneeded
•Commonlyusedsoftwareprograms
•Mapmaker/EXP,MapManagerQTX,JoinMapetc
377Haramaya University

378. Linkage map…
•Linkedmarkersaregroupedtogetherinto‘linkage
groups,’whichrepresentchromosomalsegments
orentirechromosomes
•Polymorphicmarkersdetectedarenotnecessarily
evenlydistributedoverthechromosome,but
clusteredinsomeregionsandabsentinothers
•Theaccuracyofmeasuringthegeneticdistance
anddeterminingmarkerorderisdirectlyrelated
tothenumberofindividualsstudiedinthe
mappingpopulation,min50individuals
378Haramaya University

379. Dr. Zekeria Yusuf (PhD) 379

380. Haramaya University 380

381. Genetic distance and mapping functions
•Greaterthedistancebetweenmarkers,the
greaterthechanceofrecombinationoccurring
duringmeiosis
•Distancealongalinkagemapismeasuredin
termsofthefrequencyofrecombination
betweengeneticmarkers
•Mappingfunctionsarerequiredtoconvert
recombinationfractionsintocentiMorgans(cM)
•Whenmapdistancesaresmall(<10cM),themap
distanceequalstherecombinationfrequency
•However,thisrelationshipdoesnotapplyformap
distancesthataregreaterthan10cM
Haramaya University 381

382. QTLMapping
Five primary types of QTLmapping with increasing complexity
and (theoretically) power
1.Single marker analysis
2.Interval mapping (IM)
3.Composite interval mapping (CIM)
4.Multiple interval mapping (MIM)
5.Bayesian ( Hidden Markov Model)
•Others that are more rare.
382Haramaya University

383. 1. Single-Marker Analysis (SMA)
Alsoknownassingle-point
analysis.
Itisthesimplestmethodfor
detectingQTLsassociatedwith
singlemarkers.
•Thismethoddoesnotrequirea
completelinkagemapandcanbe
performedwithbasicstatistical
softwareprograms.
•Thestatisticalmethodsusedfor
single-markeranalysisincludet-
tests,analysisofvariance
(ANOVA)andlinearregression.
•Linearregressionismost
commonlyusedbecausethe
coefficientofdetermination(R2)
fromthemarkerexplainsthe
phenotypicvariationarisingfrom
theQTLlinkedtothemarker.
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384. 384Haramaya University

385. 385Haramaya University

386. 386Haramaya University

387. 2. Simple Interval Mapping (SIM)
•ItwasfirstproposedbyLanderandBolstein.
•Ittakesfulladvantagesofthelinkagemap.
•Thismethodevaluatesthetargetassociationbetweenthetrait
valuesandthegenotypeofahypotheticalQTL(targetQTL)at
multipleanalysispointsbetweenpairofadjacentmarkerloci(target
interval).
•PresenceofaputativeQTLisestimatedifthelogofoddsratio
exceedsacriticalthreshold.
•Theuseoflinkedmarkersforanalysiscompensatesfor
recombinationbetweenthemarkersandtheQTL,andisconsidered
statisticallymorepowerfulcomparedtosingle-pointanalysis.
•MapMaker/QTLandQGeneareusedtoconductSIM.
•Theprinciplebehindintervalmappingistotestamodelforthe
presenceofaQTLatmanypositionsbetweentwomappedloci.
387Haramaya University

388. Statistical methods used for SIM
i.MaximumLikelihoodApproach
•ItisassumedthataQTLislocatedbetweentwomarkers,
thetwolocimarkergenotypes(i.e.AABB,AAbb,aaBB,
aabbforDHprogeny)eachcontainmixturesofQTL
genotypes.
•MaximumlikelihoodinvolvessearchingforQTLparameters
thatgivethebestapproximationforquantitativetrait
distributionthatareobservedforeachmarkerclass.
•Modelsareevaluatedbycomparingthelikelihoodofthe
observeddistributionswithandwithoutfindingQTLeffect
•ThemappositionofaQTLisdeterminedasthemaximum
likelihoodfromthedistributionoflikelihoodvalues.
388Haramaya University

389. ii. Logarithm of the odds ratio (LODscore):
•Linkagebetweenmarkersisusuallycalculatedusingoddsratio.
•Thisratioismoreconvenientlyexpressedasthelogarithmofthe
ratio,andiscalledalogarithmofodds(LOD)valueorLODscore.
•LODvaluesof>3aretypicallyusedtoconstructlinkagemaps.
•LODof2meansthatitis100timesmorelikelythataQTLexists
intheintervalthanthatthereisnoQTL.
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390. ii. Logarithm of the odds ratio (LODscore):
•LODof3betweentwomarkersindicatesthat
linkageis1000timesmorelikely(i.e.1000:1)
thannolinkage.
•LODvaluesmaybeloweredinordertodetecta
greaterleveloflinkageortoplaceadditional
markerswithinmapsconstructedathigherLOD
values.
•TheLODscoreisameasureofthestrengthof
evidenceforthepresenceofaQTLata
particularlocation.
390Haramaya University

391. 3. Composite Interval Mapping (CIM)
•DevelopedbyJansenandStamin1994
•ItcombinesintervalmappingforasingleQTLin
agivenintervalwithmultipleregression
analysisonmarkerassociatedwithotherQTL.
•Itismorepreciseandeffectivewhenlinked
QTLsareinvolved.
•Itconsidersmarkerintervalplusafewother
wellchosensinglemarkersineachanalysis,so
thatn-1testsforinterval–QTLassociationsare
performedonachromosomewithnmarkers.
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392. Dr. Zekeria Yusuf (PhD) 392

393. Understanding interval mapping results
•SIMandCIMproducea
profileofthelikelysites
foraQTLbetween
adjacentlinkedmarkers.
•Thestatisticalresultsare
typicallypresentedusing
alogarithmicofodds
(LOD)scoreorlikelihood
ratiostatistic(LRS)
•LRS=4.6×LOD
•PositionforaQTL:position
wherethehighestLOD
valueisobtained.
Haramaya University 393

394. Understanding interval mapping results contd…
•RealQTL:Thepeakmustalsoexceedaspecified
significanceandisdeterminedusingpermutationtests.
•Thephenotypicvaluesofthepopulationare‘shuffled’
whilstthemarkergenotypicvaluesareheldconstantand
QTLanalysisisperformedsome500-1000timestoassess
theleveloffalsepositivemarker-traitassociationsand
significantlevelsaredetermined.
•Previously,LODscoreofbetween2.0to3.0(most
commonly3.0)wasusuallychosenasthesignificance
threshold.
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395. Haramaya University 395

396. Haramaya University 396

397. 4. Multiple Interval Mapping (MIM)
•Itisalsoamodificationofsimpleintervalmapping.
•Itutilizesmultiplemarkerintervalssimultaneously
tofitmultipleputativeQTLdirectlyinthemodel
formappingQTL.
•Itprovidesinformationaboutnumberandposition
ofQTLinthegenome.
•ItalsodeterminesinteractionofsignificantQTLs
andtheircontributiontothegeneticvariance.
•ItisbasedonCockerham’smodelforinterpreting
geneticparameters.
397Haramaya University

398. 5. Bayesian Interval Mapping (BIM)
DevelopedbySatagopanetal.in1996.
•Itprovidesinformationaboutnumberandpositionof
QTLandtheireffects
•TheBIMestimatesshouldagreewithMIMestimates
andshouldbesimilartoCIMestimates.
•Itprovidesinformationposteriorestimatesofmultiple
QTLintheintervals.
•ItcanestimateQTLeffectandpositionseparately.
398Haramaya University

399. Minor and major QTL
•QTLcanbemajororminorbasedontheproportionofthe
phenotypicvariationi.e.R2value:
•MajorQTLs:>10%),stableQTL
•MinorQTLs:<10%.environmentallysensitiveespecially(disease
Resistance):
(1)Suggestive;
(2)Significant;
(3)Highlysignificantto“avoidafloodoffalsepositiveclaims”and
toensurethat“truehintsoflinkage”werenotmissed
Significantandhighly-significant:significancelevelsof5and
0.1%,RespectivelySuggestive:Expectedtooccuronceat
randominaQTLmappingstudy.
399Haramaya University

400. Genomic Selection
•Genomicselection(GS)isanewapproachfor
improvingquantitativetraitsinlargeplant
breedingpopulationsthatuseswholegenome
molecularmarkersandcombinesmarkerdata
withphenotypicdatainanattempttoincrease
theaccuracyofthepredictionofbreedingand
genotypicvalues.
400Haramaya University

401. Genomic Selection…
401Haramaya University

402. Dr. Zekeria Yusuf (PhD) 402