16 apple germplasm strcture and tools for germplasm curators durel charles eric

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16 apple germplasm strcture and tools for germplasm curators durel charles eric

  1. 1. Genetic structure of European apple germplasm Molecular markers as tools to manage practical issues in germplasm collections C.E. DUREL INRA, Angers
  2. 2. 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 ?
  3. 3. Markers available in apple (isozymes, RAPD, AFLP) SSR : (Simple Sequence Repeats) > 660 published http://www.hidras.unimi.it/ SNP : (Single Nucleotide Polymorphism) 8k / 18k / … 420k GBS : (Genotyping by Sequencing) (Elshire et al., 2011)
  4. 4. Fingerprinting SSR Cvrs 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 SNP vrs SNP1 SNP2 SNP3 SNP4 … vr A AC AG CC GT vr B AA AG CT GG vr C CC AA TT TT vr D AC AG CC GT The more markers, the better … i.e., the more specific the fingerprint is.
  5. 5. Genetic distances SSR Cvrs 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 Cvrs Cvrs Cvr A Cvr B Cvr C Cvr D Cvr A 0 Cvr B 0.5 0 Cvr C 1 0.87 0 Cvr D 0 0.5 1 0
  6. 6. Is my accession True To Type ? SSR Cvrs 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) Same fingerprints dij = 0
  7. 7. Disctinctness SSR Cvrs 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 ~ No matching alleles dij ~ 0.9 - 1
  8. 8. Triploidy checking N° echantillon Diploid / 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
  9. 9. Is my accession unique or redundant within my collection or with other collections ? 0 1 2 3 4 5 6 7 8 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 Duplicates - Belgium Belgium Czech_Republic France Italy Sweden United_Kingdom unique 2n 190 genotypes (209 accessions) duplicate 2n 69 genotypes (88 accessions) unique 3n 59 genotypes (79 accessions) duplicate 3n 10 genotypes (16 accessions) Collection 1 (408 DNA samples received)
  10. 10. 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
  11. 11. 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 ?
  12. 12. 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)
  13. 13. Global relatedness Putative relatedness tree in grape: IBD computed on >5300 SNPs (Myles et al., 2010)
  14. 14. Is my collection structured in subgroups ? Dessert - New Dessert - Old Cider Lassois et al. (in prep.) FRB-funded project
  15. 15. Is my collection structured in subgroups ? Wild apples Domesticated apple
  16. 16. Is my collection structured in subgroups ? FruitBreedomics – WP4 : 2750 accessions fingerprinted with 16 SSR
  17. 17. Is my collection structured in subgroups ? Faint structure in 3 European regions North-East West South (1550 diploid genotypes with known geographic origin)
  18. 18. How representative of the genetic diversity is my collection ? Collection 1
  19. 19. Collection 2 How representative of the genetic diversity is my collection ?
  20. 20. 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 Capturedallelicrichness(%) 97% Lassois et al. (in prep.) Core collection
  21. 21. Technical issue for SSR : ajusting allele sizes  choice of genotypes covering almost all SSR alleles 96 accessions covering almost all SSR alleles  Facilitating the alignment of SSR alleles
  22. 22. Limits - Marker versus Phenotype mutation Phenotypic mutations : Colour  Gala, Red Delicious Architecture  McIntosh vs Wijick Acidity  Usterapfel LA vs HA Vigor  M9 rootstock … Genetic mutation Regulation modification Cell layers L1/L2/L3 Marker mutation rate = 10-4 for SSR 10-6 for SNP
  23. 23. SSR marker mutation 1 single allelic difference over 24 markers Allelic mutation Marker CH03d12 Des moissons Pomme Livre Belle Louronnaise Demie sûre
  24. 24. 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
  25. 25. 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 € ???
  26. 26. 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)
  27. 27. Thank you for your attention!

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