Tree Genetics & Genomes

, 13:14 | Cite as

Unravelling genetic diversity and cultivar parentage in the Danish apple gene bank collection

  • Bjarne Larsen
  • Torben Bo Toldam-Andersen
  • Carsten Pedersen
  • Marian Ørgaard
Original Article
Part of the following topical collections:
  1. Germplasm Diversity


Characterization of apple germplasm is important for conservation management and breeding strategies. A set of 448 Malus domestica accessions, primarily of local Danish origin, were genotyped using 15 microsatellite markers. Ploidy levels were determined by flow cytometry. Special emphasis was given to pedigree reconstruction, cultivar fingerprinting and genetic clustering. A reference set of cultivars, mostly from other European countries, together with a private nursery collection and a small set of Malus sieversii, Malus sylvestris and small-fruited, ornamental Malus cultivars, was also included. The microsatellite markers amplified 17–30 alleles per loci with an average degree of heterozygosity at 0.78. We identified 104 (23%) duplicate genotypes including colour sports. We could infer first-degree relationships for many cultivars with previously unknown parentages. STRUCTURE analysis provided no evidence for a genetic structure but allowed us to present a putative genetic assembly that was consistent with both PCA analysis and parental affiliation. The Danish cultivar collection contains 10% duplicate genotypes including colour sports and 22% triploids. Many unique accessions and considerable genetic diversity make the collection a valuable resource within the European apple germplasm. The findings presented shed new light on the origin of Danish apple cultivars. The fingerprints can be used for cultivar identification and future management of apple genetic resources. In addition, future genome-wide association studies and breeding programmes may benefit from the findings concerning genetic clustering and diversity of cultivars.


Malus domestica cultivars Germplasm Pedigree Genetic structure Microsatellites Ploidy 

Supplementary material

11295_2016_1087_MOESM1_ESM.pdf (75 kb)
Online Resource 1List of accessions and accession numbers. Accessions originate from the gene bank collection, “Pometet” (Taastrup, Copenhagen Region), University of Copenhagen, unless otherwise specified. (PDF 75 kb)
11295_2016_1087_MOESM2_ESM.pdf (10 kb)
Online Resource 2STRUCTURE analysis for 344 M. domestica cultivars. The graph shows delta K vs. K for K = 2–12, tested over 20 runs. (PDF 10 kb)
11295_2016_1087_MOESM3_ESM.pdf (60 kb)
Online Resource 3List of ploidy level for Malus domestica cultivars and M. sieversii, determined by flow cytometry. (PDF 60 kb)
11295_2016_1087_MOESM4_ESM.pdf (37 kb)
Online Resource 4Each horizontal row contains accessions with identical genotype profile of 15 SSR-loci. The duplicate genotypes are divided into four categories: previously (beige) and not previously reported synonyms (blue), subclones such as colour sports from an original genotype (red) and accessions from the private nursery collection Assens (green). (PDF 36 kb)
11295_2016_1087_MOESM5_ESM.pdf (57 kb)
Online Resource 5Network of first degree relationships. Arrows point from parent to offspring. Information given in each box: cultivar name, accession number, ploidy level, S-RNase alleles and approximate geographical origin and year of origin. (PDF 57 kb)


  1. Al-Atiyat RM (2015) The power of 28 microsatellite markers for parentage testing in sheep. Electron J Biotechnol 18:116–121. doi:10.1016/j.ejbt.2015.01.001 CrossRefGoogle Scholar
  2. Bergström I (1938) Tetraploid apple seedlings obtained from the progeny of triploid varieties. Hereditas 24:210–215CrossRefGoogle Scholar
  3. Bianco L et al (2014) Development and validation of a 20K single nucleotide polymorphism (SNP) whole genome genotyping array for apple (Malus × domestica Borkh). PLoS One 9:e110377. doi:10.1371/journal.pone.0110377 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bianco L et al (2016) Development and validation of the Axiom®Apple480K SNP genotyping array. Plant J 86:62–74. doi:10.1111/tpj.13145 CrossRefPubMedGoogle Scholar
  5. Bredsted HC (1893) Haandbog i dansk Pomologi, 2. æbler. vol Book, Whole. Hempelske Bog- og Papirhandels Forlag, OdenseGoogle Scholar
  6. Chagné D et al (2012) Genome-wide SNP detection, validation, and development of an 8K SNP array for apple. PLoS One 7:e31745. doi:10.1371/journal.pone.0031745 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chagné D et al (2015) Polyploid and aneuploid detection in apple using a single nucleotide polymorphism array. Tree Genet Genomes 11:1–6CrossRefGoogle Scholar
  8. Clark LV, Jasieniuk M (2011) Polysat: an R package for polyploid microsatellite analysis. Mol Ecol Resour 11:562–566. doi:10.1111/j.1755-0998.2011.02985.x CrossRefPubMedGoogle Scholar
  9. Coart E, Van Glabeke S, De Loose M, Larsen AS, RoldÁN-Ruiz I (2006) Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh). Mol Ecol 15:2171–2182. doi:10.1111/j.1365-294X.2006.02924.x CrossRefPubMedGoogle Scholar
  10. Cornille A et al (2012) New insight into the history of domesticated apple: secondary contribution of the European wild apple to the genome of cultivated varieties. PLoS Genet 8:e1002703. doi:10.1371/journal.pgen.1002703 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cornille A, Gladieux P, Giraud T (2013) Crop-to-wild gene flow and spatial genetic structure in the closest wild relatives of the cultivated apple. Evol Appl 6:737–748. doi:10.1111/eva.12059 CrossRefGoogle Scholar
  12. Crane MB, Lawrence WJC (1930) Fertility and vigour of apples in relation to chromosome number. J Genet 22:153–163. doi:10.1007/BF02983844 CrossRefGoogle Scholar
  13. Cuenca J, Aleza P, Juárez J, García-Lor A, Froelicher Y, Navarro L, Ollitrault P (2015). Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus. Sci Rep 5:9897. doi:10.1038/srep09897
  14. Druart P (2000) Aneuploids and variants of apple (Malus domestica Borkh.) through in vitro culture techniques. In: XXV International Horticultural Congress, Part 10: Application of Biotechnology and Molecular Biology and Breeding-In Vitro 520, pp 301–310Google Scholar
  15. Earl DA, vonHoldt BM (2012) Structure harvester: a website and program for visualizing structure output and implementing the Evanno method. Conserv Genet Resour 4(2):359-361. doi: 10.1007/s12686-011-9548-7
  16. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14:2611–2620. doi:10.1111/j.1365-294X.2005.02553.x CrossRefPubMedGoogle Scholar
  17. Evans KM, Fernándes F, Laurens F, Feugey L, Van de Weg E (2007) Harmonizing fingerprinting protocols to allow comparisons between germplasm collections. Eucarpia. XII Fruit Selection Symposium. Zaragoza, Spain, pp57–58Google Scholar
  18. Evans KM et al (2011) Genotyping of pedigreed apple breeding material with a genome-covering set of SSRs: trueness-to-type of cultivars and their parentages. Mol Breed 28:535–547CrossRefGoogle Scholar
  19. Fernández-Fernández F (2010) Fingerprinting the National Apple and Pear Collections Final report of DEFRA research project GC0140 1–18
  20. Florin R (1926) Pollen production and incompatibilities in apples and pears Mem Hort Soc New York 3Google Scholar
  21. Gardner KM et al (2014) Fast and cost-effective genetic mapping in apple using next-generation sequencing. G3: Genes|Genomes|Genetics 4:1681–1687CrossRefPubMedPubMedCentralGoogle Scholar
  22. Garkava-Gustavson L, Nybom H (2003) DNA-analyser afslöjar våra äpplesorter Fakta Trädgård Fritid 94Google Scholar
  23. Garkava-Gustavsson L, Kolodinska Brantestam A, Sehic J, Nybom H (2008) Molecular characterisation of indigenous Swedish apple cultivars based on SSR and S-allele analysis. Hereditas 145:99–112CrossRefPubMedGoogle Scholar
  24. Garkava-Gustavsson L, Mujaju C, Sehic J, Zborowska A, Backes GM, Hietaranta T, Antonius K (2013) Genetic diversity in Swedish and Finnish heirloom apple cultivars revealed with SSR markers. Sci Hortic 162:43–48. doi:10.1016/j.scienta.2013.07.040 CrossRefGoogle Scholar
  25. Gasi F, Simon S, Pojskic N, Kurtovic M, Pejic I (2010) Genetic assessment of apple germplasm in Bosnia and Herzegovina using microsatellite and morphologic markers. Sci Hortic 126:164–171CrossRefGoogle Scholar
  26. Gianfranceschi L, Seglias N, Tarchini R, Komjanc M, Gessler C (1998) Simple sequence repeats for the genetic analysis of apple. Theor Appl Genet 96:1069–1076. doi:10.1007/s001220050841 CrossRefGoogle Scholar
  27. Gross BL, Volk GM, Richards CM, Forsline PL, Fazio G, Chao CT (2012) Identification of “duplicate” accessions within the USDA-ARS National Plant Germplasm System Malus Collection. J Am Soc Hortic Sci 137:333–342Google Scholar
  28. Guarino C, Santoro S, De Simone L, Lain O, Cipriani G, Testolin R (2006) Genetic diversity in a collection of ancient cultivars of apple (Malus × domestica Borkh.) as revealed by SSR-based fingerprinting. Journal of horticultural science & biotechnology 81:39–44CrossRefGoogle Scholar
  29. Hardy OJ, Vekemans X (2002) Spagedi: a versatile computer program to analyse spatial genetic structure at the individual or population levels. Mol Ecol Notes 2:618–620. doi:10.1046/j.1471-8286.2002.00305.x CrossRefGoogle Scholar
  30. Ingvarsson PK, Street NR (2011) Association genetics of complex traits in plants. The New phytologist 189:909–922. doi:10.1111/j.1469-8137.2010.03593.x CrossRefPubMedGoogle Scholar
  31. Jones OR, Wang J (2010) COLONY: a program for parentage and sibship inference from multilocus genotype data. Mol Ecol Resour 10:551–555. doi:10.1111/j.1755-0998.2009.02787.x CrossRefPubMedGoogle Scholar
  32. Juniper BE, Mabberley DJ (2006) The story of the apple, vol 20. Timber Press Portland, OregonGoogle Scholar
  33. Kalinowski ST, Wagner AP, Taper ML (2006) Ml-relate: a computer program for maximum likelihood estimation of relatedness and relationship. Mol Ecol Notes 6:576–579. doi:10.1111/j.1471-8286.2006.01256.x CrossRefGoogle Scholar
  34. Kobel F (1927) Zytologische Untersuchungen an Prunoideen und Pomoideen Archiv der julius klaus-stiftung für vererbungsforschung sozialanthropologie und rassenhygiene 3:69–77Google Scholar
  35. Kvaale E (1926) Abortive and sterile apple pollen Mem Hort Soc New York 3Google Scholar
  36. Larsen A, Asmussen C, Coart E, Olrik D, Kjær E (2006) Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris). Tree Genet Genomes 2:86–97. doi:10.1007/s11295-005-0030-0 CrossRefGoogle Scholar
  37. Larsen B, Ørgaard M, Toldam-Andersen TB, Pedersen C (2016a) A high-throughput method for genotyping S-RNase alleles in apple. Mol Breed 36:1–10. doi:10.1007/s11032-016-0448-0 CrossRefGoogle Scholar
  38. Larsen B, Pedersen C, Ørgaard M, Toldam-Andersen TB (2016b) Danish apple cultivars: genetic diversity, parentage and breeding potential Acta HorticulturaeGoogle Scholar
  39. Lassois L et al (2016) Genetic diversity, population structure, parentage analysis, and construction of core collections in the French apple germplasm based on SSR markers. Plant Mol Biol Report 34:827–844. doi:10.1007/s11105-015-0966-7 CrossRefGoogle Scholar
  40. Liang W, Dondini L, De Franceschi P, Paris R, Sansavini S, Tartarini S (2015) Genetic diversity, population structure and construction of a core collection of apple cultivars from Italian germplasm. Plant Mol Biol Report 33:458–473. doi:10.1007/s11105-014-0754-9 CrossRefGoogle Scholar
  41. Matthiessen C (1913) Dansk Frugt. Hagerup, CopenhagenGoogle Scholar
  42. Patzak J, Paprštein F, Henychová A, Sedlák J, Somers D (2012) Comparison of genetic diversity structure analyses of SSR molecular marker data within apple (Malus × domestica) genetic resources. Genome 55:647–665CrossRefPubMedGoogle Scholar
  43. Pedersen A (1925) Danmarks Frugtavl, Beretning fra Fællesudvalget for lokale Iagttagelsesplantninger og Frugtsortundersøgelser. CopenhagenGoogle Scholar
  44. Pedersen A (1950) Danmarks Frugtsorter, 1. del. æbler vol 2. vol Book, Whole. Alm. Dansk Gartnerforening, Copenhagen,Google Scholar
  45. Potts SM, Han Y, Khan MA, Kushad MM, Rayburn AL, Korban SS (2012) Genetic diversity and characterization of a core collection of Malus germplasm using simple sequence repeats (SSRs). Plant Mol Biol Report 30:827–837CrossRefGoogle Scholar
  46. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedPubMedCentralGoogle Scholar
  47. Riester M, Stadler PF, Klemm K (2009) FRANz: reconstruction of wild multi-generation pedigrees. Bioinformatics 25:2134–2139. doi:10.1093/bioinformatics/btp064 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Rodzen JA, Famula TR, May B (2004) Estimation of parentage and relatedness in the polyploid white sturgeon (Acipenser transmontanus) using a dominant marker approach for duplicated microsatellite loci. Aquaculture 232:165–182. doi:10.1016/S0044-8486(03)00450-2 CrossRefGoogle Scholar
  49. Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotech 18:233–234CrossRefGoogle Scholar
  50. Urrestarazu J, Miranda C, Santesteban LG, Royo JB (2012) Genetic diversity and structure of local apple cultivars from northeastern Spain assessed by microsatellite markers. Tree Genet Genomes 8:1163–1180CrossRefGoogle Scholar
  51. Urrestarazu J et al (2016) Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level. BMC Plant Biol 16(130). doi:10.1186/s12870-016-0818-0
  52. van Treuren R, Kemp H, Ernsting G, Jongejans B, Houtman H, Visser L (2010) Microsatellite genotyping of apple (Malus domestica Borkh.) genetic resources in the Netherlands: application in collection management and variety identification. Genet Resour Crop Evol 57:853–865CrossRefGoogle Scholar
  53. Varming C, Amigo JM, Petersen MA, Toldam-Andersen T (2013) Aroma analysis and data handling in the evaluation of niche apple juices from 160 local Danish apple cultivars. In: Flavour Science: Proceedings from XIII Weurman Flavour Research Symposium, Academic Press, p 277Google Scholar
  54. Velasco R et al (2010) The genome of the domesticated apple (Malus [times] domestica Borkh). Nat Genet 42:833–839 CrossRefPubMedGoogle Scholar
  55. Vinatzer BA, Patocchi A, Tartarini S, Gianfranceschi L, Sansavini S, Gessler C (2004) Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab-resistant accessions in Malus germplasm. Plant Breed 123:321–326. doi:10.1111/j.1439-0523.2004.00973.x CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Bjarne Larsen
    • 1
  • Torben Bo Toldam-Andersen
    • 1
  • Carsten Pedersen
    • 1
  • Marian Ørgaard
    • 1
  1. 1.Department of Plant and Environmental SciencesUniversity of CopenhagenFrederiksberg CDenmark

Personalised recommendations