Eva ZyprianJulius Kühn-Institut, Institut für Rebenzüchtung Geilweilerhof

www.jki.bund.de

COST Action FA1003, Sofia workshop, May 7, 2012

SNP genotyping for genetic mapping and association genetics for grapevine breeding

Wanted:

Molecular markers in genetic linkage to desired traits for grapevine breeding to enable Marker-assisted selection applying combinatorial approaches

Examples for desired traits:

Disease resistances

Downy mildewPowdery mildewBotrytisBlack rotPhylloxera

Quality traits

Berry size and berry skin colourMust quality Enrich positive flavor compoundsAvoid negative aroma compounds

Phenology

Optimize sprouting and ripening time

Trait-linked markers can be identified through

•Genetic mapping followed by QTL analysis

•Identification of genetic diversity in candidate genes potentially determining a trait

•Association genetics in comprehensive populations

•Genetic mapping

You need•Controlled crosses•Large progeny•Genotyping of many individuals•At many loci

To develop genetic maps

•ordering the markers by linkage/recombination analysis (meiotic recombination frequencies)

Use the genetic maps to look for QTL

quantitative trait loci

(genomic regions with factors affecting the trait of interest)

•Following QTL analysis

`Regent´ and `Lemberger´

Gf.Ga-47-42 (`Bacchus´ x `Seyval´) and `Villard blanc´

2

Amplicon-Sequencing of alleles of resistance gene analogs of RGA137 identified sequence polymorphisms

Welter and Zyprian, in prep.

SNP markers (Single nucleotide polymorphisms)

Heterozygous SNPs found in a sequence alignment of the coding region of a candidate gene for Erysiphe necator resistance)

S = G or C

R = A or G

K = G or T

SNP markers (Single nucleotide polymorphisms)

(Bonow-Rex and Zyprian, in prep.

SNP markers (Single nucleotide polymorphisms)

References:Troggio et al. 2007, doi:10.1534/genetics.106.067462Cabezas et al. 2011, doi:10.1186/1471-2229-11-153 Myles et al. 2011, doi: 10.1073/pnas.1009363108 Emanuelli et al. 2011, doi:10.1186/1471-2229-10-241

•Collaborative work with

Stella GrandoJose Miguel Martinez ZapaterSean Myles

•JKI Geilweilerhof work with

Iris FechterLudger Hausmann

Eva Zyprian Leocir WelterMartina Bonow-RexTina Moser

The SNPs we use currently (384 + 48) come from

•Literature

SNP Genotyping

for1

for2

rev1

rev2

•Allele-specific PCR

XXXY YY

SNP Genotyping

•Taq Man technology qRT-PCR

Allelic discrimination of 160 Individuals for their genotype at a SNP of a candidate gene for Erysiphe necator resistance

SNP Genotyping

Using nested PCR over the target region followed by allele-specific PCR in duplex amplifications with fluorophore labeling

Nanofluidic FluiDigm PCR System 48 x 48

SNP Genotyping

SNP Genotyping

48 Genotypes at a SNP in the Erysiphe necator inducible Promotor of PR10

48 Genotypes at a SNP in the Erysiphe necator inducible Transcription factor gene

Genetic mapping can be facilitated by SNP markers

SNP marker applications

Genetic mapping: Conversion of SNP genotypes

Genetic mapping study of Gf.Ga-47-42 x `Villard blanc´

Segregating population:152 individuals

In total about 700 markers of different types

178 out of 384 SNP markers were informative and could be mapped:

54 double heterozygous hk x hk76 heterozygous in female lm x ll48 heterozygous in male nn x np

The genetic map was significantly improved

SNP marker applications

Ra108_Vb10.0

S_IN036211.0UDV122_23411.9Ra108_Ga118.0VVMD1723.4Ra137_Ga325.7Ra137_GaVb227.8A_E4450_M00531.2UDV122_22731.5Ra137_Ga235.8Ra137_Ga137.1VVMD17_22141.9UDV122_22346.9VMC2D2_10250.4A_E4451_M00352.8A_E3850_M03054.9UDV13456.1R_A15_100057.2A_E4450_M03457.7VMC2D2_10460.3UDV11764.5S_VV_18_1220757567.0S_SNP883_16067.4S_DFR S_VV_18_1123873973.3S_VV_18_958280575.0A_E3850_M01978.2R_A19_177681.0S_SNP453_37585.2VMCNG1B986.4S_VV_18_662452089.0A_E3348_M01589.5A_E3650_M02490.0A_E4451_M01991.9S_SNP355_154 S_SNP219_17292.7A_E4450_M02294.8A_E3850_M01895.0SCU1095.3S_VV_18_259134799.1S_1082L02F99.3VMC8B599.8VMC2A3108.7A_E3348_M008110.2

LG18

Zyprian et al., unpublished

Rpv3

LOD max 7.322% var. expl.

SNP marker applications

PlasmoparaR QTL

SNP marker applications

SNP markers in association studies

SNP markers in a contigous piece of DNA form a hapolotype. They are genetically linked to each other (they are in Linkage Disequilibrium) and may be also linked to factors determining traits of interest or be residing in causative or candidate genes!

Recombination results in break down of LD !

SNP marker applications

In comprehensive unrelated populations of individuals the frequent recombination events from the past result in the breakdown of Linkage Disequilibrium

… unless there is a selection to keep a specific haplotypic combination!

… so a trait of interest may be linked to a specific haplotype of SNP markers!

…we can find these trait-linked SNPs in association studies

… but we have to control the level of relatedness

SNP marker applications

357 diverse accessions from the Geilweilerhof germplasm collection were selected due to different levels of expression of resistance traits to

•Erysiphe necator (powdery mildew)•Plasmopara viticola (downy mildew)

Primer pairs: Bowers et al., 1996, 1999b, Merdinoglu et al., 2005Vitis Microsatellite Consortium

SSR1SSR3

SSR4

SSR6SSR8

SSR9

SSR10 SSR12

SSR13

SSR14

SSR15

SSR17SSR 18

SSR19SSR20

SSR2SSR5

SSR16

SSR11

SSR7

IGGP1 IGGP4IGGP3IGGP2 IGGP5 IGGP6 IGGP7 IGGP8

IGGP11IGGP10 IGGP12 IGGP13 IGGP14 IGGP15 IGGP17 IGGP18 IGGP19IGGP16

IGGP9

Controlling the levels of relatedness: Determination of general genetic diversity with SSR markers

Trilateral project CoreGrapeGene

GABI-BMBF

Genetic structure of 357 genotypes

K = 4

K = 7

K = 6

K = 5

There are at least four genetic groups in this set of accessions !

V3125 2,0

Börner 8,0

V002 6,9

V008 6,7

V020 6,4

V024 5,5

V031 5,5

+site allele p_marker significance

SNP1 1 0.7364 n.s.

2 0.0181 *

SNP2 1 0.7364 n.s.

2 - -n.s.= not significant, * significant at p<0.05

SNP1 SNP2

allele 1 allele 2 allele 1 allele 2

V3125 A A A G

Börner T T G G

V002 A T A G

V008 A T A G

V020 A T A G

V024 A T A G

V031 A T A G

=

Association-Analysis (TASSEL software V2.1)

5´ 3´

coding region (633bp) of candidate gene

SNP1 SNP2 Bonow-Rex and Zyprian, in prep.

Use this set for association analysis with SNPs from candidate genes for disease resistance (Powdery mildew resistance of leaves)

PM leaf resistance scores

SNP marker applications

SNP marker applications

... Now to be confirmed in the complete genotype set

Tina Moser Deutsche Forschungsgemeinschaft

Martina Bonow-Rex GRASP Project BMBF EraNet

Leocir Welter DAAD/CAPES

All colleagues from Geilweilerhof

Reinhard Töpfer

Thanks to...