Genetic structure of European apple germplasm

Molecular markers as tools to manage practical issues in germplasm collections

C.E. DURELINRA, Angers

Major questions when curating a germplasm collection :

• Is my accession corresponding to the true genotype ? (TTT = « True To Type »)

• Is my accession unique or redundant within my collection or with other collections ?

• Are these 2 accessions related ? • Are these 2 accessions genetically close or distant ?• How representative of the genetic diversity is my

collection ?• Is my collection structured in subgroups ?

Markers available in apple(isozymes, RAPD, AFLP)

SSR :(Simple Sequence Repeats)> 660 publishedhttp://www.hidras.unimi.it/

SNP :(Single Nucleotide Polymorphism)8k / 18k / … 420k

GBS :(Genotyping by Sequencing)

(Elshire et al., 2011)

Fingerprinting SSRCvrs

SSR1 SSR2 SSR3 SSR4 …

Cvr A 90-100 120-126 164-168 205-209

Cvr B 96-100 126-132 164-170 205-211

Cvr C 98-102 122-130 162-170 207-213

Cvr D 90-100 120-126 164-168 205-209

…

Genetic fingerprinting

SNPCvrs

SNP1 SNP2 SNP3 SNP4 …

Cvr A AC AG CC GT

Cvr B AA AG CT GG

Cvr C CC AA TT TT

Cvr D AC AG CC GT

…

The more markers, the better …

i.e., the more specific the fingerprint is.

Genetic distances SSRCvrs

SSR1 SSR2 SSR3 SSR4

Cvr A 90-100 120-126 164-168 205-209

Cvr B 96-100 126-132 164-170 205-211

Cvr C 98-102 122-130 162-170 207-213

Cvr D 90-100 120-126 164-168 205-209

Genetic distances (dissimilarities) according to common/distinct marker profiles

CvrsCvrs

Cvr A Cvr B Cvr C Cvr D

Cvr A 0Cvr B 0.5 0Cvr C 1 0.87 0Cvr D 0 0.5 1 0

Is my accession True To Type ?

SSRCvrs

SSR1 SSR2 SSR3 SSR4

Cvr A 90-100 120-126 164-168 205-209

Cvr B 96-100 126-132 164-170 205-211

Cvr C 98-102 122-130 162-170 207-213

Cvr D 90-100 120-126 164-168 205-209

A and D : Clones

(+/- mutations)

Samefingerprints

dij = 0

Disctinctness

SSRCvrs

SSR1 SSR2 SSR3 SSR4

Cvr A 90-100 120-126 164-168 205-209

Cvr B 96-100 126-132 164-170 205-211

Cvr C 98-102 122-130 162-170 207-213

Cvr D 90-100 120-126 164-168 205-209

A (or B) and C ~ Unrelated

~ Nomatching

alleles

dij ~ 0.9 - 1

Triploidy checkingN°

echantillonDiploid / Triploid CH01f03b CH01h01 CH02c06 CH02d08 CH04e05 CH05f06 NZ05g08

0001 Bédange de Nantes Diploid 174/186 121/127 254/256 215/258 205/205 171/185 127/163

0002 Belle de fumée Triploid 141/174 119/123/127 232/242/254 215/229 178/213 179/189 127/127

0003 Belle fille du Penthièvre Triploid 141/162/186 119/121/127 - 229/258 178/205/213 179/187/189 133/149

0004 Blanc d'été Diploid 141/141 125/127 254/254 211/231 205/205 185/189 127/142

0005 Bercelien Triploid 162/174/186 125/137 232/254 215/258 178/213/219 171/181/187 127/129

0006 Boblin Diploid 141/174 105/137 232/262 217/260 178/219 177/189 121/121

0009 Charles pitrel Diploid 174/186 133/137 254/254 258/258 178/219 179/185 129/142

0010 Coaquin Triploid 141/174 125/133 234/238/252 215/229/258 178/205/219 171/183/185 144/151/163

0011 Cul d'oie Diploid 141/141 127/137 232/246 209/229 178/205 177/181 127/127

0012 Cul na Diploid 141/174 119/133 220/232 215/256 178/222 179/181 127/127

0013 Cyriac Diploid 141/186 137/137 232/232 215/229 178/219 181/187 127/127

…

Accessions with 3 alleles per marker triploid apple varieties

Is my accession unique or redundant within my collection or with other collections ?

1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 1030

1

2

3

4

5

6

7

8

Duplicates - Belgium

Belgium Czech_Republic FranceItaly Sweden United_Kingdom

unique 2n 190 genotypes (209 accessions)duplicate 2n

69 genotypes(88 accessions)

unique 3n59 genotypes

(79 accessions)

duplicate 3n10 genotypes

(16 accessions)

Collection 1(408 DNA samples received)

Are these 2 accessions related ?

Large data set Relatedness coefficient (ML-Relate software ; Kalinowski et al., 2006)

Parent-Offspring (0.5) / Full-sibs (0.25) / Half-sibs (0.125) / …

http://en.wikipedia.org/wiki/File:Pedigree_marker_information.jpg

Parentage analysis

Parentage inference conditionally to the available marker data:

Software CERVUS (Kalinowsky et al., 2005)

Known variety

Female parent ? Male parent ?

Correct marker allele inheritance ?

Parentage analysis

Rose de Berne Rose d’Ajoie Blaser

Pomme raisin Calville rouge d’hiver

Inference of unknown parents:

Full-Sibs:

Lassois et al. (in prep.)

Requirements:- very large data set- >= 20 SSR

Caution !:- Are the accessions TTT ??- The true cross could derive from mutants of the identified parents

(2 Swiss cvrs)

Global relatednessPutative relatedness tree in grape:IBD computed on >5300 SNPs

(Myles et al., 2010)

Is my collection structured in subgroups ?

Full-Sibs

Dessert - New

Dessert - Old

Cider

Lassois et al. (in prep.)

FRB-funded project

Is my collection structured in subgroups ?

Full-Sibs

Wild applesDomesticated apple

Is my collection structured in subgroups ?

Full-Sibs

FruitBreedomics – WP4 :2750 accessions fingerprinted with 16 SSR

Is my collection structured in subgroups ?

Full-Sibs

Faint structure in 3 European regions

North-East

West

South

(1550 diploid genotypeswith known geographic origin)

How representative of the genetic diversity is my collection ?

Full-Sibs

Collection 1

Full-Sibs

Collection 2

How representative of the genetic diversity is my collection ?

Can I get a subset of cvrs representative of my (neutral) genetic diversity ?

0

20

40

60

80

100

0 100 200 300 400 500 600

N° of accessions of the core

Cap

ture

d a

llel

ic r

ich

nes

s (%

)

97%

Lassois et al. (in prep.)

Core collection

Technical issue for SSR : ajusting allele sizes choice of genotypes covering almost all SSR alleles

96 accessions covering almostall SSR alleles

Facilitating the alignment of SSR alleles

Limits - Marker versus Phenotype mutation

Phenotypic mutations :

Colour Gala, Red DeliciousArchitecture McIntosh vs WijickAcidity Usterapfel LA vs HAVigor M9 rootstock…

Genetic mutationRegulation modification

Cell layers L1/L2/L3

Marker mutation rate = 10-4 for SSR10-6 for SNP

SSR marker mutation

1 single allelic difference over 24 markers

Allelic mutation

Marker CH03d12

Des moissons

Pomme Livre

Belle Louronnaise

Demie sûre

Markers can help germplasm curators

• Quality insurance :- Grafting/labelling error- Erroneous pomological identification • Information about duplicates within and between collections (but not mutants !)• Possible inferences of parentage (when enough markers and large data set)

looking backwards how selection has been done empirically by farmers and gardeners

• Representativeness/specificity of the collection

To be combined with phenotypic data• Optimal choice for core collection• Genome-Wide Association Studies

Technical and financial issues

Multiplexing Analysis Cost Information

SSR : Low Long Low High (multi-allelic)

SNP : High Short High Low / marker 8k ~ 50 € but numerous SNP 18k ~ 65 € 420k ~ 165 €

GBS : High Long Low Missing data (bioinformatic) (imputation)

Major goal : ~500-1000 SNP at 5-10 € ???

Thank you

• M. Lateur, P. Houben (CRA-W)• S. Tartarini, L. Dondini (UNIBO)• F. Paprstein, J. Sedlak (RBIPH)• M. Ordidge (Reading Univ.)• F. Fernandez, K.M. Evans (EMR)• H. Nybom, L. Garkava-Gustavsson (SLU)• C. Miranda, J. Urrestarazu (Un. Navarra)• J. Gassmann (Agroscope)• K. Antonius (MTT)• I. Suprun (SKZNIISIV, Krasnodar)• A. Pikunova (VNIISPK, Orel)• C. Denancé, E. Ravon, L. Feugey, A. Guyader, R. Guisnel, L. Lassois (INRA)

Thank you for your attention!