Horticulture, Environment, and Biotechnology

, Volume 60, Issue 1, pp 69–80 | Cite as

Genotyping of octoploid strawberry inbred lines by SNP discovery using genotyping-by-sequencing

  • Jinhee KimEmail author
  • Sun Yi Lee
  • Dosun Kim
  • Eun-Su Lee
  • Hye-Eun Lee
  • Koen Han
  • Byoung-Cheorl Kang
Research Report


The ‘common’ strawberry (Fragaria × ananassa) is a popular fruit in Korea and is often consumed fresh. Polyploidy is common in horticultural crops, and Fragaria × ananassa is octoploid, having 8 sets of each chromosome; however, the molecular genetic information of octoploid strawberry is still insufficient to develop effective markers for agronomic traits. In this study, a set of high-quality single nucleotide polymorphisms (SNPs) was collected via the genotyping-by-sequencing (GBS) method to genotype the strawberry inbred lines developed in Korea. We used a population of 89 strawberry inbred lines, which were derived from nine strawberry cultivars. The FANhybrid_r1.2 octoploid assembly was used as a reference genome. The average length of reads was 87,458,702 bp, and a total of 437,058 SNPs were collected. Several filtering criteria (SNP quality, read depth, and genotype quality) were used to identify 20,923 potential SNPs. Finally, 2863 high-quality SNPs were selected for the study. The genetic diversity of the inbred lines was analyzed by phylogenetic tree and structure analysis using the SNP genotype data. This is the first report of genotyping strawberry inbred lines using the GBS technique, and can be used to accelerate strawberry molecular breeding.


Fragaria × ananassa Molecular breeding Selfing generation Cluster analysis 



This work was supported by grants from the Export Promotion Technology Development Research Programs (315047-3) funded by the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

Supplementary material

13580_2018_100_MOESM1_ESM.docx (734 kb)
Supplementary material 1 (DOCX 734 kb)


  1. Arnau G, Lallemand J, Bourgoin M (2003) Fast and reliable strawberry cultivar identification using inter simple sequence repeat (ISSR) amplification. Euphytica 129:69–79CrossRefGoogle Scholar
  2. Bassil N, Gunn M, Folta K, Lewers K (2006) Microsatellite markers for Fragaria from ‘Strawberry Festival’ expressed sequence tags. Mol Ecol Resour 6:473–476CrossRefGoogle Scholar
  3. Bassil N, Davis TM, Zhang H, Ficklin S, Mittmann M, Webster T, Mahoney L, Wood D, Alperin ES, Rosyara UR (2015) Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo- octoploid cultivated strawberry Fragaria × ananassa. BMC Genom 16:155–185CrossRefGoogle Scholar
  4. Bombarely A, Merchante C, Csukasi F, Cruz-Rus E, Caballero JL, Medina-Escobar N, Blanco-Portales R, Botella MA, Muñoz-Blanco J, Sánchez-Sevilla JF (2010) Generation and analysis of ESTs from strawberry (Fragaria × ananassa) fruit and evaluation of their utility in genetic and molecular studies. BMC Genom 11:503–520CrossRefGoogle Scholar
  5. Buti M, Sargent DJ, Mhelembe KG, Delfino P, Tobutt KR, Velasco R (2016) Genotyping-by-sequencing in an orphan plant species Physocarpus opulifolius helps identify the evolutionary origins of the genus Prunus. BMC Res Notes 9:268–276CrossRefGoogle Scholar
  6. Chandler CK, Folta K, Dale A, Whitaker VM, Herrington M (2012) Strawberry. In: Badenes ML, Byrne DH (eds) Fruit breeding. Springer, New York, pp 305–325CrossRefGoogle Scholar
  7. Congiu L, Chicca M, Cella R, Rossi R, Bernacchia G (2000) The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application. Mol Ecol 9:229–232CrossRefGoogle Scholar
  8. Darrow GM (1966) The strawberry: history, breeding, and physiology. Holt, Rinehart and Winston, New York.
  9. Davik J, Sargent DJ, Brurberg MB, Lien S, Kent M, Alsheikh M (2015) A ddRAD based linkage map of the cultivated strawberry, Fragaria × ananassa. PLoS ONE 10:1–10CrossRefGoogle Scholar
  10. Davis TM, DiMeglio LM, Yang R, Styan SM, Lewers KS (2006) Assessment of SSR marker transfer from the cultivated strawberry to diploid strawberry species: functionality, linkage group assignment, and use in diversity analysis. J Am Soc Hortic Sci 131:506–512Google Scholar
  11. Debnath S, Khanizadeh S, Jamieson A, Kempler C (2008) Inter simple sequence repeat (ISSR) markers to assess genetic diversity and relatedness within strawberry genotypes. Can J Plant Sci 88:313–322CrossRefGoogle Scholar
  12. Degani C, Rowland LJ, Saunders JA, Hokanson SC, Ogden EL, Golan-Goldhirsh A, Galletta GJ (2001) A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data. Euphytica 117:1–12CrossRefGoogle Scholar
  13. Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 4:359–361CrossRefGoogle Scholar
  14. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:2611–2620CrossRefGoogle Scholar
  15. Ganal MW, Altmann T, Röder MS (2009) SNP identification in crop plants. Curr Opin Plant Biol 12:211–217CrossRefGoogle Scholar
  16. Garcia M, Ontivero M, Diaz Ricci J, Castagnaro A (2002) Morphological traits and high resolution RAPD markers for the identification of the main strawberry varieties cultivated in Argentina. Plant Breed 121:76–80CrossRefGoogle Scholar
  17. Ge A, Shangguan L, Zhang X, Dong Q, Han J, Liu H, Wang X, Fang J (2013) Deep sequencing discovery of novel and conserved microRNAs in strawberry (Fragaria × ananassa). Physiol Plant 148:387–396CrossRefGoogle Scholar
  18. Gidoni D, Rom M, Kunik T, Zur M, Izsak E, Izhar S, Firon N (1994) Strawberry-cultivar Identification using randomly amplified polymorphic DNA (RAPD) markers. Plant Breed 113:339–342CrossRefGoogle Scholar
  19. Gil-Ariza DJ, Amaya I, Botella MA, Munoz Blanco J, Caballero JL, López-Aranda JM, Valpuesta V, Sánchez-Sevilla JF (2006) EST-derived polymorphic microsatellites from cultivated strawberry (Fragaria × ananassa) are useful for diversity studies and varietal identification among Fragaria species. Mol Ecol Resour 6:1195–1197CrossRefGoogle Scholar
  20. Gil-Ariza DJ, Amaya I, López-Aranda JM, Sánchez-Sevilla JF, Botella MÁ, Valpuesta V (2009) Impact of plant breeding on the genetic diversity of cultivated strawberry as revealed by expressed sequence tag-derived simple sequence repeat markers. J Am Soc Hortic Sci 134:337–347Google Scholar
  21. Govan C, Simpson D, Johnson A, Tobutt K, Sargent D (2008) A reliable multiplexed microsatellite set for genotyping Fragaria and its use in a survey of 60 F. × ananassa cultivars. Mol Breed 22:649–661CrossRefGoogle Scholar
  22. Graham J, McNicol R, McNicol J (1996) A comparison of methods for the estimation of genetic diversity in strawberry cultivars. Theor Appl Genet 93:402–406CrossRefGoogle Scholar
  23. Hadonou A, Sargent D, Wilson F, James C, Simpson D (2004) Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping. Genome 47:429–438CrossRefGoogle Scholar
  24. Hancock JF, Lavín A, Retamales J (1999) Our southern strawberry heritage: Fragaria chiloensis of Chile. HortScience 34:814–816Google Scholar
  25. Hirakawa H, Shirasawa K, Kosugi S, Tashiro K, Nakayama S, Yamada M, Kohara M, Watanabe A, Kishida Y, Fujishiro T (2014) Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species. DNA Res 21:169–181CrossRefGoogle Scholar
  26. Hokanson K, Smith M, Connor A, Luby J, Hancock JF (2006) Relationships among subspecies of New World octoploid strawberry species, Fragaria virginiana and Fragaria chiloensis, based on simple sequence repeat marker analysis. Botany 84:1829–1841Google Scholar
  27. Hummer KE, Nathewet P, Yanagi T (2009) Decaploidy in Fragaria iturupensis (Rosaceae). Am J Bot 96:713–716CrossRefGoogle Scholar
  28. Hussein TS, Tawfik AA, Khalifa MA (2008) Molecular identification and genetic relationships of six strawberry varieties using ISSR markers. Int J Agric Biol 10:677–680Google Scholar
  29. Hwang J, Li J, Liu WY, An SJ, Cho H, Her NH, Yeam I, Kim D, Kang BC (2009) Double mutations in eIF4E and eIFiso4E confer recessive resistance to Chilli veinal mottle virus in pepper. Mol Cells 27:329–336CrossRefGoogle Scholar
  30. Jung HJ, Veerappan K, Natarajan S, Jeong N, Hwang I, Nagano S, Shirasawa K, Isobe S, Nou IS (2017) A system for distinguishing octoploid strawberry cultivars using high-throughput SNP genotyping. Trop Plant Biol 10:68–76CrossRefGoogle Scholar
  31. Kaur S, Francki MG, Forster JW (2012) Identification, characterization and interpretation of single-nucleotide sequence variation in allopolyploid crop species. Plant Biotechnol J 10:125–138CrossRefGoogle Scholar
  32. Keniry A, Hopkins CJ, Jewell E, Morrison B, Spangenberg GC, Edwards D, Batley J (2006) Identification and characterization of simple sequence repeat (SSR) markers from Fragaria × ananassa expressed sequences. Mol Ecol Resour 6:319–322CrossRefGoogle Scholar
  33. Lerceteau-Köhler E, Guerin G, Laigret F, Denoyes-Rothan B (2003) Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (Fragaria × ananassa) using AFLP mapping. Theor Appl Genet 107:619–628CrossRefGoogle Scholar
  34. Mahoney LL, Sargent DJ, Abebe-Akele F, Wood DJ, Ward JA, Bassil NV, Hancock JF, Folta KM, Davis TM (2016) A high-density linkage map of the ancestral diploid strawberry, constructed with single nucleotide polymorphism markers from the IStraw90 Array and genotyping by sequencing. Plant Genome 9:1–14CrossRefGoogle Scholar
  35. Milella L, Saluzzi D, Lapelosa M, Bertino G, Spada P, Greco I, Martelli G (2006) Relationships between an Italian strawberry ecotype and its ancestor using RAPD markers. Genet Resour Crop Evol 53:1715–1720CrossRefGoogle Scholar
  36. Nagano S, Shirasawa K, Hirakawa H, Maeda F, Ishikawa M, Isobe SN (2017) Discrimination of candidate subgenome-specific loci by linkage map construction with an S1 population of octoploid strawberry (Fragaria × ananassa). BMC Genom 18:374–384CrossRefGoogle Scholar
  37. Njuguna W (2010) Development and use of molecular tools in Fragaria. Oregon State University, OregonGoogle Scholar
  38. Peng M, Zong X, Wang C, Meng F (2015) Genetic diversity of strawberry (Fragaria ananassa Duch.) from the Motuo County of the Tibet Plateau determined by AFLP markers. Biotechnol Biotechnol Equip 29:876–881CrossRefGoogle Scholar
  39. Perrier X, Flori A, Bonnot F (2003) Methods for data analysis. Genetic diversity of cultivated tropical plants. Science Publishers, Inc. and CIRAD, Montpellier, FRA, pp 31-63Google Scholar
  40. Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94–100CrossRefGoogle Scholar
  41. Rho IR, Woo JG, Jeong HJ, Jeon HY, Lee CH (2012) Characteristics of F1 hybrids and inbred lines in octoploid strawberry (Fragaria × ananassa Duchesne). Plant Breed 131:550–554CrossRefGoogle Scholar
  42. Sánchez-Sevilla JF, Horvath A, Botella MA, Gaston A, Folta K, Kilian A, Denoyes B, Amaya I (2015) Diversity Arrays Technology (DArT) marker platforms for diversity analysis and linkage mapping in a complex crop, the octoploid cultivated strawberry (Fragaria × ananassa). PLoS ONE 10:e0144960CrossRefGoogle Scholar
  43. Sargent D, Davis T, Tobutt K, Wilkinson M, Battey N, Simpson D (2004) A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria. Theor Appl Genet 109:1385–1391CrossRefGoogle Scholar
  44. Sargent D, Clarke J, Simpson D, Tobutt K, Arus P, Monfort A, Vilanova S, Denoyes-Rothan B, Rousseau M, Folta K (2006) An enhanced microsatellite map of diploid Fragaria. Theor Appl Genet 112:1349–1359CrossRefGoogle Scholar
  45. Sargent D, Fernandéz-Fernandéz F, Ruiz-Roja J, Sutherland B, Passey A, Whitehouse A, Simpson D (2009) A genetic linkage map of the cultivated strawberry (Fragaria × ananassa) and its comparison to the diploid Fragaria reference map. Mol Breed 24:293–303CrossRefGoogle Scholar
  46. Sargent DJ, Kuchta P, Girona EL, Zhang H, Davis TM, Celton JM, Marchese A, Korbin M, Folta KM, Shulaev V (2011) Simple sequence repeat marker development and mapping targeted to previously unmapped regions of the strawberry genome sequence. Plant Genome 4:165–177CrossRefGoogle Scholar
  47. Sargent D, Yang Y, Šurbanovski N, Bianco L, Buti M, Velasco R, Giongo L, Davis T (2016) HaploSNP affinities and linkage map positions illuminate subgenome composition in the octoploid, cultivated strawberry (Fragaria × ananassa). Plant Sci 242:140–150CrossRefGoogle Scholar
  48. Spigler RB, Lewers KS, Johnson AL, Ashman TL (2010) Comparative mapping reveals autosomal origin of sex chromosome in octoploid Fragaria virginiana. J Hered 101:S107–S117CrossRefGoogle Scholar
  49. Vining KJ, Salinas N, Tennessen JA, Zurn JD, Sargent DJ, Hancock J, Bassil NV (2017) Genotyping-by-sequencing enables linkage mapping in three octoploid cultivated strawberry families. PeerJ Preprints 5:e2975v1Google Scholar
  50. Yoon MY, Moe KT, Kim DY, Rho IR, Kim S, Kim KT, Won MK, Chung JW, Park YJ (2012) Genetic diversity and population structure analysis of strawberry (Fragaria × ananassa Duch.) using SSR markers. Electron J Biotechnol 15:6–6Google Scholar
  51. Zorrilla-Fontanesi Y, Cabeza A, Torres AM, Botella MA, Valpuesta V, Monfort A, Sánchez-Sevilla JF, Amaya I (2011) Development and bin mapping of strawberry genic-SSRs in diploid Fragaria and their transferability across the Rosoideae subfamily. Mol Breed 27:137–156CrossRefGoogle Scholar

Copyright information

© Korean Society for Horticultural Science 2018

Authors and Affiliations

  • Jinhee Kim
    • 1
    Email author
  • Sun Yi Lee
    • 1
  • Dosun Kim
    • 1
  • Eun-Su Lee
    • 1
  • Hye-Eun Lee
    • 2
  • Koen Han
    • 2
  • Byoung-Cheorl Kang
    • 2
  1. 1.Vegetable Research DivisionNational Institute of Horticultural and Herbal Science, RDAWanju-gunKorea
  2. 2.Research Institute of Agriculture and Life ScienceSeoul National UniversitySeoulKorea

Personalised recommendations