Accelerated Grape BreedingAccelerated Grape Breeding

Daryl J. SomersDaryl J. Somers

Director of Applied GenomicsDirector of Applied GenomicsDirector of Applied GenomicsDirector of Applied GenomicsVineland Research and Innovation CentreVineland Research and Innovation Centre

CCOVI 2010

•• Complex traits and molecular breedingComplex traits and molecular breeding

•• Lessons learned from wheat molecular breedingLessons learned from wheat molecular breeding

•• Strategies and tools for grape breedingStrategies and tools for grape breeding

C l t itC l t itComplex traits:Complex traits:

•• Typically controlled by multiple genesTypically controlled by multiple genesoo various modes of inheritancevarious modes of inheritance

•• Often influenced by the environmentOften influenced by the environmentOften influenced by the environmentOften influenced by the environmentoo temperaturetemperatureoo nutritionnutrition

moist remoist reoo moisturemoisture

•• Testing in multiple environments if essential to Testing in multiple environments if essential to quantify the genetics component of trait expression.quantify the genetics component of trait expression.

Tools to characterize complex traitsTools to characterize complex traits

•• Genetic map Genetic map –– underpins most of the researchunderpins most of the research

•• High throughput DNA diagnostic methodsHigh throughput DNA diagnostic methodsoo DNA extractionDNA extractionoo SSRsSSRs DaRTDaRT SNPsSNPsoo SSRs, SSRs, DaRTDaRT, SNPs, SNPsoo next Gen next Gen SeqSeq

SS l bl b titi t t l h tt t l h t•• Space, Space, labourlabour, time , time –– to accurately phenotypeto accurately phenotype

•• Statistical analysis softwareStatistical analysis softwareoo merges genotypes with phenotypesmerges genotypes with phenotypesoo maps QTLs.maps QTLs.

Cereal Quality and Segregation• Seed quality testing will begin at F4 (micro tests on 50-100g)• Continues through F6, F8

y g g

• Quadramat mill – flour yield• NIR – protein content• mixograph – dough strength

CWADKyle

CPSW

KVD

•“A” test – F9 Advanced testing begins• Buhler mill – flour yield

CWRSCDC Teal

CPSWAC Karma

y• NIR – protein content• Chemical/physiological tests• Dough strength (multiple methods)

CWSWSFielder

CWRWg g ( p )• Baking (multiple methods)

•“B” test – F10•“C” test – yr 1-3 – F11-13

CWESGlenlea

CDC Clair

C test yr 1 3 F11 13• Registration CPSRAC Crystal

Canada

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Canada

HT – MAS techniques and costs

DNA extraction96 well format, freeze dried leaf or crushed seedNon organic ammonim acetate ethanol pptNon-organic, ammonim acetate, ethanol ppt.

DNA quantification with Hoechst 3325896 well fomat96 well fomatdilute DNA to 6ng/uL

PCR tPCR set up384 well format on Tecan Robot10 uL rxn with 24 ng gDNAM13 t ili th d f i d iM13 tailing method for primer design

Canada

Microsatellite Allele Database and Image Archive

Many wheat microsatellites are not locus specific.

Knowledge of allele size and image quality is important to apply markers.

Allele database records allele sizeAllele database records allele size of 330 accessions at 125 loci/genome.

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Canada

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FUSARIUM HEAD BLIGHT

19941994

10 d f FHB h i i10 d f FHB h i i10 years spread of FHB on the prairies.10 years spread of FHB on the prairies.

19961996

PrecipitationPrecipitation

19991999

TemperatureTemperature

20032003

TemperatureTemperature

Fusarium Head Blight

Fusarium graminearum

Polygenic resistance from Asian sources

G x E interaction impacts disease reaction and impairs selection

Affects all level of grain industry from production – marketing - consumer

Good candidate for MAS!

Canada

Canada

Version 2.0 - April/2003Accelerated development of FHB resistant bread wheat using Marker-Assisted Selection

Phase 1: Parental Genotyping 6 seeds of each parent lines are screened with 3 AFLP primercombinations and 12 Msats to detect off types and select a single“normal” plant of each accession.

The list of parents for the project includes:

98B69-L4798B69-R28ProdigyBW301AC ElsaPT205BW264

Additional genotype checks include:

WuhanMaringaOpataSyntheticChinese SpringBW252BW278

The selected parent plants and genotype checks are genotypedat 700 Msat loci with ~550 Msats.

During parental genotyping, the elite lines and pest resistancedonars will be intercrossed in multiple combinations, usingelite cytoplasm to generate F1 seed. The genotyping informa-tion will be used to select 4 streams beginning with a singlecross.

Investigators:

Daryl Somers Molecular breedingJulian Thomas GeneticsStephen Fox BreedingG i h di

STREAM 4

Gavin Humphreyscross = 38% polymorphism

STREAM 3

Ron DePauwcross = 70% polymorphism

STREAM 2

George Fedakcross = 76% polymorphism

STREAM 1

Stephen Foxcross = 79% polymorphism

Phase 2: Marker-assisted selection of BCF1

TIME LINE:Milestones and ProjectionsSTART APRIL 1/2002

BW264BW263HC374HC736HC81498B08-A11198B08-A41

BW278DH181AC ForemostSumai 3

Gavin Humphreys BreedingRon DePauw BreedingGeorge Fedak BreedingJeannie Gilbert FHB PathologyBrent McCallum Lr PathologyBob Lamb Entomology

HC374 [3BSm, 3BMm, 4Bw](Wuhan/Maringa)

98B69-L47(midge)

X

F1 98B69L47XA few F1 backcrossed to generate>1,200 BC1F1 seed.

F1 BW301X

HC736 [3BSs, 4Bs, 6Bs](AC Foremost//Biggar/Sumai3)

98B69-R2898B69-L47

(midge)

X

F1 ProdigyX

98B08-A111 [3BMs, 5As](BW252//AC Domain*2/Sumai3)

BW263X

F1 BW263X

HC374 [2Dw, 5Am, 3BSm](Wuhan/Maringa)

BW301(Lr21)

X

Self elite andretain forfuture BC

A few F1 backcrossed to generate>1,200 BC1F1 seed. A few F1 backcrossed to generate>1,200 BC1F1 seed.

July /02

Self elite andretain forfuture BCs.

A few F1 backcrossed to generate>1,600 BC1F1 seed.

Self elite andretain forfuture BCs.

Self elite andretain forfuture BCs.

Completed July 5/02, all F1 seeddistributed to collaborators.

Nov. /02

Mar. /03

BC1F1 98B69L47X- 1076 BC1F1 plants screened for 3 markers in QTLregions- 154 plants selected and further screened for QTLintervals (5 additional markers)- 88 plants selected for genome scan with 76markers- 5 BC1F1 backcrossed to generate >1,200 BC2F1seed

BC2F1

BC1F1 BW301X

BC2F1

BC1F1 ProdigyX

BC2F1

BC1F1 BW263X

BC2F1>800 BC2F1 plants screened for 3 genes; ~100 sel. >800 BC2F1 plants screened for 3 genes; ~100 sel.

>1,600 BC1F1 plants screened for 4 genes; ~100 sel.plants screened for unfixed, elite alleles.

A few BC1F1 backcrossed to generate>1,600 BC2F1 seed.

>1 536 BC2F1 plants screened for 4 genes; ~100 >800 BC2F1 plants screened for 2 genes + second

Planted selected Stream 1 BC1F1.

Dec. /02 Planted selected Stream 4 BC1F1.Planted selected Stream 2 BC1F1.

- 1151 BC1F1 plants screened for 3 markers in QTLregions- 134 plants selected and further screened for QTLintervals (3 additional markers)- 64 plants selected for genome scan with 71markers- 5 BC1F1 backcrossed to generate >1,200 BC2F1seed

- 968 BC1F1 plants screened for 2 markers in QTLregions- 220 plants selected and further screened for QTLintervals (4 additional markers)- 96 plants selected for genome scan with VVmarkers- 5 BC1F1 backcrossed to generate >1,200 BC2F1seed

Apr. 2003, BC2F1 stream 1,2,4and BC1F1 steam 3 arrives to

l l b di VC i d t

July /03

Phase 3: Stream convergence

BC2F2Screen 1152 BC2F2 plants to select homozygous for3 genes (1 in 64). Stream should be fixed for midgeby this point.

Self best 3 plants to make 500 seeds.

Elite fixed for midge, 3BSm, 3BMm, 4Bw

BC2F2

Elite fixed for Lr21, 2Dw, 5Am, 3BSm

BC2F2

Elite fixed for midge, 3BSs, 4Bs, 6Bs

BC2F2

Elite fixed for 3BMs, 5As, white seed?

C p g ;plants screened for unfixed, elite alleles. BSA usedto make genotyping efficient.

Screen 1152 BC2F2 plants to select homozygous for3 genes (1 in 64). Stream should be fixed for Lr21 bythis point.

Self best 3 plants to make 500 seeds.

p g ;plants screened for unfixed, elite alleles. BSA usedto make genotyping efficient.

Screen 1,536 BC2F2 plants to select homozygous for4 genes (1 in 256).

Self best plants to make 1,600 seeds.

>1,536 BC2F1 plants screened for 4 genes; 100sel. plants screened for unfixed, elite alleles.

Screen 1,152 BC2F2 plants to select homozygous for2 genes + last colour loci (1 in 32). Stream may befixed for 3 colour loci at this point.

Self best 3 plants to make 500 seeds.

>800 BC2F1 plants screened for 2 genes + secondcolour locus; ~100 sel. plants screened for unfixed,elite alleles. BSA used to make genotyping effi-cient.

molecular breeding. VC is used tomodify workplan.

Sept/Oct 2003, BC2F2 plantsbeing selected, to plant for inter-cross first week of Nov.

Nov. /03

Mar. /04

and DH production

BC2F1 (str1, str2) BC2F1 (str3, str4)

BC2F1 (str1, str2, str3, str4)

DH production

DH PopulationsDH production

Note:A variety or 2 and 3 way crosses can be made forDH production in addition to the 4 way cross.

Oct. /04DH Populations

1st Cdn field trials - Summer 2005

NZ or glass house increaseusing DH S0 seed source.

p(3 centres?)

Mar. /05

Canada

QTL introgression – the details

BC1F1

S l hi “h h h” h i lSelect everything “h‐h‐h” across the intervalCheck 70‐80 background markers, select for homozygosity

BC2F1

S l hi “h h h” h i lSelect everything “h‐h‐h” across the intervalCheck unfixed background markers, select for homozygosity

Canada

QTL introgression – the details

BC1F1

BC2F1

Self the elite, heterozygous plants

BC2F2

Select plants homozygous donor across the interval

Canada

Fixation of recurrent parent using molecular breeding.

25 BC1F1BC1F1BC2F1BC2F1

20

BC1F1BC1F1

No. ofLinesNo. ofLines

10

15FinalFinal

5

% Fixed Elite Alleles% Fixed Elite Alleles

030 40 50 60 70 80 90 10020

Canada

120 BC2F3 entries x 3 reps – Ottawa ON – 2004

Canada

BC2F3 (homozygous rows)BC2F3 (homozygous rows)

STREAM 2 - FHB INDEX

50.0

60.0

20.0

30.0

40.0

0.0

10.0N

one

2DL

3BS

5AS

L 3B

S

L 5A

S

S 5A

S

S 5A

S

HB

37

Mor

se

Vist

a

Cor

a

D27

10

C37

4

HY6

44

Als

en

enzi

e

uper

b

2DL

2DL

3BS

2DL

3BS FH

AC

M

AC

V

AC

ND H H A

McK

e

Su

2 years of replicated data ‐ Ottawa ON – FedakClear effect of single genes.2 years of replicated data ‐ Ottawa ON – FedakClear effect of single genes.

Canada

Complex traits + diverseFHB

Complex traits   diverse genetics require haplotype informaton.

D l d h l tDeveloped haplotypes images for major FHB loci.

Routinely used to screen:yParents, F1, … advanced.

Grape BreedingGrape Breeding••Grape is a hybrid cropGrape is a hybrid crop

oo high degree of high degree of heterozygosityheterozygosity

••Perennial, Perennial, vegetativelyvegetatively propagatedpropagated

••Buds and Seeds require a cold / dormant period for Buds and Seeds require a cold / dormant period for growth/germinationgrowth/germination

oo stratificationstratificationoo stratificationstratification

Freezing Freezing temperturestempertures ((--20C to 20C to --25C)25C)t d t d d b dt d t d d b doo stress dormant wood and budsstress dormant wood and buds

oo kill buds, no renewed growthkill buds, no renewed growthoo vines lose productivity for yearsvines lose productivity for yearsp y yp y y

Freezing toleranceFreezing tolerance• Some elements of freezing tolerance can be controlledSome elements of freezing tolerance can be controlled

oo vineyard managementvineyard managementoo rootstocksrootstocksoo rootstocksrootstocksoo varietyvariety

Bud freeze tolerance can be measured after acclimationBud freeze tolerance can be measured after acclimation•• Bud freeze tolerance can be measured after acclimationBud freeze tolerance can be measured after acclimationoo low low temperturetemperture exothermexotherm –– ice formationice formationoo TennyTenny DTA freezer systemDTA freezer systemoo Good sample throughputGood sample throughput

•• Niagara varieties show LT50 atNiagara varieties show LT50 at --20C to20C to --25C25CNiagara varieties show LT50 at Niagara varieties show LT50 at 20C to 20C to 25C25C

VitisVitis ripariariparia will survive to will survive to --40C.40C.strategy is to BCstrategy is to BC VV ripariariparia intointo VV viniferaviniferastrategy is to BC strategy is to BC V. V. ripariariparia into into V. V. viniferavinifera

Genetic markers and Genetic mapsGenetic markers and Genetic mapspp

Microsatellite markers (simple sequence repeats)

TGGTGGTGGTGG

Var. 1 Var. 2

TGGTGGTGGTGGACCACCACCACC

TGGTGGTGGACCACCACC +

•International community uses 6 markers to distinguish varieties.••Vineland is set up to generate and deliver this informationVineland is set up to generate and deliver this information.

Grape GenotypingGrape Genotyping

•• ABI3130 XLABI3130 XL genotypergenotyper

Grape GenotypingGrape Genotyping

ABI3130 XL ABI3130 XL genotypergenotyper

•• Established HT DNA Established HT DNA extractionextraction

•• Screened >800 SSRs on 24 Screened >800 SSRs on 24 NiagaraNiagaraVitisVitis cvscvs..

•• Adapting DNA melt point Adapting DNA melt point tech for SNP detection.tech for SNP detection.

•• DaRTDaRT marker technology marker technology from from TriticarteTriticarte (Australia).(Australia).

Genetic AnalysisGenetic Analysis Aux Zweig Chard96

Genetic AnalysisGenetic Analysis

Chard96 Chard PinotGris GamNoir PinotBlanc Pixie

MW

PinotNoir Gew Dorn Reisling239 Reisling49 Limb

Syrah/Pinot Noir – 994 loci – 1245 cMLimb

CabSauv ChenBlanc SauvBlanc Sem CabFranc

0 09 0 31 0 54 0 77 1 00

Merlot Chamb Baco MarFoch Vidal256

Coefficient0.09 0.31 0.54 0.77 1.00

Grape GenotypingGrape Genotyping

•• DiscoverDiscover chimerismchimerism

Grape GenotypingGrape Genotyping

Discover Discover chimerismchimerismwithin leaf tissue.within leaf tissue.

•• May be derived from L1,May be derived from L1,May be derived from L1, May be derived from L1, L2 cell layers.L2 cell layers.

•• Gives rise toGives rise to clonalclonal

Limberger

Gives rise to Gives rise to clonalclonalvariation.variation.

Pinot noir

Periclinal chimeraPericlinal chimera

Cell layers generally do not mix.

Plant meristems are generally composed of 3 cell layers.

L1

L2

L3

y g y

Configuration was discovered by observing mutations in each cell layerL3 observing mutations in each cell layer.

Derive plants from individual cellSomatic embryogenesis

Derive plants from individual cell layers

G i ll di i ll lGenetically distinct cell layers (mutants) derive phenotypically distinct plants

Dwarf Grape – rapid cyclingDwarf Grape – rapid cycling

Wildtype

Dwarf Grape  rapid cyclingDwarf Grape  rapid cycling

Vegetative growthTendrils

GA

VvGAI VvGAIGA

GA Inhibits flowering in grape.

Boss & Thomas (2002) Nature 416:847‐850

DwarfContinual flowering

Vvgai

DELLA mutation Few, no tendrils

GAI/gai detection with PCRGAI/gai detection with PCR

Wildtype

Mutant

Accelerated Grape BreedingAccelerated Grape BreedingAccelerated Grape BreedingAccelerated Grape BreedingVitis sp.* Trait *

V. ViniferaPinot MeunierX

Can use SSR or Can use SSR or DaRTDaRT Trait GAI ‐WT

Pinot Meuniergai – Pixie

F1 x V. vinifera

Xmarkers to track progress.markers to track progress.

A l t t ti fA l t t ti f

BCF1 x V. vinifera

Accelerates restoration of Accelerates restoration of elite genetic background.elite genetic background.

BCF2 x V. vinifera

BCF3

Risky aspects of researchRisky aspects of researchRisky aspects of researchRisky aspects of researchFreeze tolerance testingFreeze tolerance testing

C ld li dli 4CC ld li dli 4C 22 3 k3 k•• Cold acclimate seedlings at 4C Cold acclimate seedlings at 4C –– 22‐‐3 weeks3 weeks•• Determine bud hardiness in programmable freezer.Determine bud hardiness in programmable freezer.

Fruit ripening and seed germinationFruit ripening and seed germination•• Plant maintenancePlant maintenance•• Stratification and GA applicationsStratification and GA applicationsStratification and GA applicationsStratification and GA applications•• Embryo rescueEmbryo rescue

P f f tP f f tPerformance of new grape genotypesPerformance of new grape genotypes•• What can be selected in the GH? What can be selected in the GH? 

Future PlanningFuture PlanningVitis sp.* Trait *G

V. ViniferaPinot Meunieri i i

X Create dwarf versions of all current and f i l b d d i hGAI ‐WT gai – Pixie

F1 x V. vinifera

future varietals to be produced in the region

BCF1 x V. viniferaThis creates a resource to cross future traits into multiple varietals within 1 or 2 generations

BCF2 x V. vinifera

2 generations.

Collect pollen from important 

BCF3accessions worldwide that may contribute traits to Canadian vines.