Genetic Resources and Crop Evolution

, Volume 57, Issue 6, pp 841–851 | Cite as

Exploitation of Malus EST-SSRs and the utility in evaluation of genetic diversity in Malus and Pyrus

  • Lihua Yao
  • Xiaoyan Zheng
  • Danying Cai
  • Yuan Gao
  • Kun Wang
  • Yufen Cao
  • Yuanwen Teng
Research Article


A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES96) to 0.83 (MES138), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.


Expressed sequence tags (ESTs) Genetic diversity Malus Pyrus Simple sequence repeat (SSR) 



We gratefully acknowledge Prof. David Spooner of Department of Horticulture, University of Wisconsin, for critical review of the manuscript. This research work was funded by the project (30871690) of the National Natural Science Foundation of China, project (R307605) from Natural Science Foundation of Zhejiang Province, China, the grant (2008C32011) from Science & Technology Department of Zhejiang Province and the earmarked fund for Modern Agro-industry Technology Research System of China.

Supplementary material

10722_2009_9524_MOESM1_ESM.pdf (81 kb)
Supplementary material 1 (PDF 82 kb)


  1. Bao L, Chen KS, Zhang D, Cao YF, Yamamoto T, Teng Y (2007) Genetic diversity and similarity of pear (Pyrus L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers. Genet Resour Crop Evol 54:959–971CrossRefGoogle Scholar
  2. Bao L, Chen KS, Zhang D, Li XG, Teng Y (2008) An assessment of genetic variability and relationships within Asian pears based on AFLP (amplified fragment length polymorphism) markers. Sci Hortic 116:374–380CrossRefGoogle Scholar
  3. Barrett B, Griffiths A, Schreiber M, Ellison N, Mercer C, Bouton J, Ong B, Forster J, Sawbridge T, Spangenberg G, Bryan G, Woodfield D (2004) A microsatellite map of white clover. Theor Appl Genet 109:596–608PubMedGoogle Scholar
  4. Bassil N, Postman JD (2010) Identification of European and Asian pears using EST-SSRs from Pyrus. Genet Resour Crop Evol. doi: 10.1007/s10722-009-9474-7
  5. Bell RL, Quamme HA, Layne REC, Skirvin RM (1996) Pears. In: Janick J, Moore JN (eds) Fruit breeding, tree and tropical fruits, vol 1. John Wiley, London, pp 441–514Google Scholar
  6. Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331PubMedGoogle Scholar
  7. Celton JM, Tustin DS, Chagné D, Gardiner SE (2008) Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences. Tree Genet Genomes 2:86–97. doi: 10.1007/s11295-008-0171-z Google Scholar
  8. Chagné D, Carlisle CM, Blond C, Volz RK, Whitworth CJ, Oraguzie NC, Crowhurst RN, Allan AC, Espley RV, Hellens RP, Gardiner SE (2007) Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple. BMC Genomics 8:212. doi: 10.1186/1471-2164-8-212 CrossRefPubMedGoogle Scholar
  9. Chen C, Zhou P, Choi YA, Huang S, Gmitter FG Jr (2006) Mining and characterizing microsatellites from Citrus ESTs. Theor Appl Genet 112:1248–1257CrossRefPubMedGoogle Scholar
  10. DeCandolle AP (1825) Prodromus Systematis Naturalis Regni Vegetabilis. Paris:Sumptibus Sociorum Treuttel et Würtz. Rece de Pourbon No.17. Venitque in Eotumdem Bibliopolüs Argentorati et Londini 2:633–636Google Scholar
  11. Decroocq V, Fave MG, Hagen L, Bordenave L, Decroocq S (2003) Development and transferability of apricot and grape EST microsatellite markers across taxa. Theor Appl Genet 106:912–922PubMedGoogle Scholar
  12. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21CrossRefGoogle Scholar
  13. Dirlewanger E, Cosson P, Tavaud M, Aranzana J, Poizat C, Zanetto A, Arus P, Laigret F (2002) Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theor Appl Genet 105:127–138CrossRefPubMedGoogle Scholar
  14. Ellegren H, Moore S, Robinson N, Byrne K, Ward W, Sheldon BC (1997) Microsatellites evolution—a reciprocal study of repeat lengths at homologous loci in cattle and sheep. Mol Biol Evol 14:854–860PubMedGoogle Scholar
  15. Gonzalez-Martinez SC, Robledo-Arnuncio JJ, Collada C, Diaz A, Williams CG, Alia R, Cervera MT (2004) Cross-amplification and sequence variation of microsatellite loci in Eurasian hard pines. Theor Appl Genet 109:103–111CrossRefPubMedGoogle Scholar
  16. Gupta PK, Rustgi S, Sharma S, Singh R, Kumar N, Balyan HS (2003) Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat. Mol Genet Genomics 270:315–323CrossRefPubMedGoogle Scholar
  17. Hackauf B, Wehling P (2002) Identification of microsatellite polymorphisms in an expressed portion of the rye genome. Plant Breed 121:17–25CrossRefGoogle Scholar
  18. Huckins CA (1972) A revision of the sections of the genus Malus Miller. Ph.D. Thesis, Cornell University, Ithaca, NYGoogle Scholar
  19. Jang JT, Tanabe K, Tamura F, Banno K (1992) Identification of Pyrus species by leaf peroxidase isozyme phenotypes (in Japanese with English summary). J Jpn Soc Hortic Sci 61:273–286CrossRefGoogle Scholar
  20. Jia XP, Shi YS, Song YC, Wang GY, Wang TY, Li Y (2007) Development of EST-SSR in foxtail millet (Setaria italica). Genet Resour Crop Evol 54:233–236CrossRefGoogle Scholar
  21. Jiang D, Zhong GY, Hong QB (2006) Analysis of microsatellites in Citrus Unigenes. Acta Genetica Sinica 33:345–353 (in Chinese)CrossRefPubMedGoogle Scholar
  22. Kajiura I, Sato Y (1990) Recent progress in Japanese pear (Pyrus pyrifolia Nakai) breeding and descriptions of cultivars based on literature review (in Japanese with English summary). Bull Fruit Tree Res Stn Extra 1:1–329Google Scholar
  23. Kantety RV, Rota ML, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48:501–510CrossRefPubMedGoogle Scholar
  24. Kikuchi A (1948) Horticulture of fruit trees, vol 1. Yokendo, Tokyo (in Japanese)Google Scholar
  25. Langenfeld WT (1991) Apple trees. Morphological evolution, phylogeny, geography and systematics. Riga (Zinatne) 232 (in Russian)Google Scholar
  26. Lewin B (1994) Genes V. Oxford University Press, New YorkGoogle Scholar
  27. Li Y (1996) A critical review of the species and the classification of the genus Malus Mill. in the world. Journal of Fruit Science, Vol 10 (Suppl) Zhengzhou Fruit Research Institute, pp 63–81 (in Chinese)Google Scholar
  28. Li Y (1999) An investigation and studies on the origin and evolution of Malus domestica Borkh. in the world. Acta Hortic Sinica 26:213–220Google Scholar
  29. Li Y (2001) Researches of germplasm resources of Malus Mill. Agriculture Press, Beijing (in Chinese)Google Scholar
  30. Linnaeus C (1753) Species plantarum. Bernard Quaritch, LondonGoogle Scholar
  31. Miyao A, Zhong HS, Monna L, Yano M, Yamamoto K, Havukkala I, Minobe Y, Sasaki T (1996) Characterization and genetic mapping of simple sequence repeats in the rice genome. DNA Res 3:233–238CrossRefPubMedGoogle Scholar
  32. Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273CrossRefPubMedGoogle Scholar
  33. Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EHA, Allan AC, Beuning LL, Bowen JH, Gera E, Jamieson KR, Janssen BJ, Laing WA, McArtney S, Nain B, Ross GS, Snowden KC, Souleyre EJF, Walton EF, Yauk YK (2006) Analysis of expressed sequence tags from apple. Plant Physiol 141:147–166CrossRefPubMedGoogle Scholar
  34. Pierantoni L, Cho KH, Shin IS, Chiodini R, Tartarini S, Dondini L, Kang SJ, Sansavini S (2004) Characterisation and transferability of apple SSRs to two European pear F1 populations. Theor Appl Genet 109:1519–1524CrossRefPubMedGoogle Scholar
  35. Pillen K, Binder A, Kreuzkam B, Ramsay L, Waugh R, Förster J, Léon J (2000) Mapping new EMBL-derived barley microsatellites and their use in differentiating German barley cultivars. Theor Appl Genet 101:652–660CrossRefGoogle Scholar
  36. Poncet V, Hamon P, Minier J, Carasco C, Hamon S, Noirot M (2004) SSR cross-amplification and variation within coffee trees (Coffea spp.). Genome 47:1071–1081CrossRefPubMedGoogle Scholar
  37. Powell W, Machray GC, Provan J (1996) Polymorphism revealed by simple sequence repeats. Trends Plant Sci 1:215–222Google Scholar
  38. Pu F, Wang Y (1963) Pomology of China. Pears, vol 3. Shanghai Science and Technology Press, Shanghai (in Chinese)Google Scholar
  39. Pu F, Huang L, Sun B, Li S (1989) Pear cultivars. Agriculture Press, Beijing (in Chinese)Google Scholar
  40. Rallo P, Tenzer I, Gessler C, Baldoni L, Dorado G, Martin A (2003) Transferability of olive microsatellite loci across the genus Olea. Theor Appl Genet 107:940–946CrossRefPubMedGoogle Scholar
  41. Rehder A (1940) Manual of cultivated trees and shrubs, 2nd edn. Macmillan, New YorkGoogle Scholar
  42. Robinson JP, Harris SA, Juniper BE (2001) Taxonomy of the genus Malus Mill. (Rosaceae) with emphasis on the cultivated apple, M. domestica Borkh. Plant Syst Evol 226:35–58CrossRefGoogle Scholar
  43. Roder MS, Korzun V, Wendehake K, Plaschke J, Tixier MH, Leroy P, Ganal MW (1998) A microsatellite map of wheat. Genetics 149:2007–2023PubMedGoogle Scholar
  44. Rohlf FJ (1998) Numerical taxonomy and multivariate analysis system. Version 2.0. Exeter Software, SetauketGoogle Scholar
  45. Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  46. Scott KD, Eggler P, Seaton G, Rossetto M, Ablett EM, Lee LS, Henry RJ (2000) Analysis of SSRs derived from grape ESTs. Theor Appl Genet 100:723–726CrossRefGoogle Scholar
  47. Silfverberg-Dilworth E, Matasci CL, Van DE, Weg WE, Kaauwen MPW, Walser M, Kodde LP, Soglio V, Gianfranceschi L, Durel CE, Costa F, Yamamoto T, Koller B, Gessler C, Patocchi A (2006) Microsatellite markers spanning the apple (Malus × domestica Borkh.) genome. Tree Genet Genomes 2:202–224CrossRefGoogle Scholar
  48. Teng Y, Tanabe K (2004) Reconsideration on the origin of cultivated pears native to East Asia. Acta Hortic 634:175–182Google Scholar
  49. Teng Y, Tanabe K, Tamura F, Itai A (2001) Genetic relationships of pear cultivars in Xinjiang, China as measured by RAPD markers. J Hortic Sci Biotechnol 76:771–779Google Scholar
  50. Teng Y, Tanabe K, Tamura F, Itai A (2002) Genetic relationships of Pyrus species and cultivars native to East Asia revealed by randomly amplified polymorphic DNA markers. J Am Soc Hortic Sci 127:262–270Google Scholar
  51. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106:411–422PubMedGoogle Scholar
  52. Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23:48–55CrossRefPubMedGoogle Scholar
  53. Vendramin E, Dettori MT, Giovinazzi J, Micali S, Quarta R, Verde I (2007) A set of EST-SSRs isolated from peach fruit transcriptome and their transportability across Prunus species. Mol Ecol Notes 7:307–310CrossRefGoogle Scholar
  54. Watkins R (1981) The main species of Malus. In: Hora B (ed) The Oxford encyclopedia of trees of the world. Oxford University Press, Oxford, pp 188–192Google Scholar
  55. Way RD, Aldwinckle HS, Lamb RC, Rejman A, Sansavini S, Shen T, Watkins R, Westwood MN, Yoshida Y (1991) Apples (Malus). In: Moore JN, Ballington JR (eds) Genetic resources of temperate fruit and nut crops 1. International Society of Horticultural Science, Wageningen, pp 3–62Google Scholar
  56. Yamamoto T, Kimura T, Sawamura Y, Kotobuki K, Ban Y, Hayashi T, Matsuta N (2001) SSRs isolated from apple can identify polymorphism and genetic diversity in pear. Theor Appl Genet 102:865–870CrossRefGoogle Scholar
  57. Yamamoto T, Kimura T, Terakami S, Nishitani C, Sawamura Y, Saito T, Kotabuki K, Hayashi T (2007) Integrated reference genetic linkage maps of pear based on SSR and AFLP markers. Breed Sci 57:321–329CrossRefGoogle Scholar
  58. Yang RC, Yeh FC (1993) Multilocus structure in Pinus contorta Dougl. Theor Appl Genet 87:568–576CrossRefGoogle Scholar
  59. Yu T (1979) Taxonomy of the fruit tree in China. Agriculture Press, Beijing (in Chinese)Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Lihua Yao
    • 1
    • 2
  • Xiaoyan Zheng
    • 1
    • 2
  • Danying Cai
    • 1
    • 2
  • Yuan Gao
    • 3
  • Kun Wang
    • 3
  • Yufen Cao
    • 3
  • Yuanwen Teng
    • 1
    • 2
  1. 1.Department of HorticultureZhejiang UniversityHangzhouChina
  2. 2.The State Agricultural Ministry Laboratory of Horticultural Plant Growth, Development & Quality ImprovementHangzhouChina
  3. 3.Research Institute of PomologyChinese Academy of Agricultural SciencesXingchengChina

Personalised recommendations