Molecular Breeding

, 39:5 | Cite as

High-throughput genotyping in onion reveals structure of genetic diversity and informative SNPs useful for molecular breeding

  • Clizia Villano
  • Salvatore Esposito
  • Francesca Carucci
  • Massimo Iorizzo
  • Luigi Frusciante
  • Domenico Carputo
  • Riccardo AversanoEmail author


Onion is an economically important crop cultivated worldwide since ancient times. Over the centuries, domestication and outbreeding have had a significant influence on its genetic pool, leading to a high degree of biodiversity. In this study, using kompetitive allele-specific PCR (KASP) genotyping technology, we explored the genetic variation of 73 onion accessions (including wild species, commercial, and local varieties) from different areas of the world. The SNP dataset inspection returned 375 polymorphic loci with a very low percentage of non-calling sites (0.03%). Eight-nine percent of the onions amplified all polymorphic loci and were considered for a population structure analysis. The ΔK method suggested four populations and enabled the identification of genepools, reflecting the geographical origin of the samples. Through statistical studies, our SNP set has proven to be successful, revealing population-specific alleles and potential candidates for use in future breeding programs. Notably, 74 loci were associated with phenotypic traits (bulbing photoperiod, bulb shape, or bulb color), and 3 loci were identified as putative targets of selection associated with onion improvement. Fifty-three pairs of SNPs were co-inherited, and among them, 17 were both trait-associated and in linkage disequilibrium. In conclusion, the data generated in this study allowed the survey of genetic variability in a heterogeneous and scantily examined germplasm, with repercussions on its exploitation in breeding programs.


Allium sp. KASP SNPs Population structure 



We are grateful to Mr. Raffaele Garramone for his technical assistance and to Marti OhMok Pottorff for manuscript editing. This research was carried out in the frame of the project “Definizione di firme geochimiche e molecolari per la tracciabilità e l’autenticazione di produzioni agrarie di pregio (FIRMA).”

Funding information

This study was funded by Italian Ministry of Agriculture.

Supplementary material

11032_2018_912_MOESM1_ESM.docx (30 kb)
Supplementary Figure 1 The ΔK method result. (DOCX 30 kb)
11032_2018_912_Fig4_ESM.png (105 kb)
Supplementary Figure 2

Genetic variation among populations of onion in the expected and observed groups. For each population, the expected heterozygosity (HE), observed heterozygosity (HO), and fixation index (F) are reported. (PNG 104 kb)

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High Resolution Image (TIF 496 kb)
11032_2018_912_MOESM3_ESM.docx (113 kb)
Supplementary Figure 3 Genetic variation among onion populations of two groups (expected and observed) calculated using a filtered set of markers. For each population, the expected heterozygosity (HE), observed heterozygosity (HO), and fixation index (F) are reported. (DOCX 112 kb)
11032_2018_912_MOESM4_ESM.xlsx (14 kb)
Supplementary Table 1 List of the onion genotypes used in the present study. For each genotype, common name, species, production area, bulbing photoperiod (SD = short day; ID = intermediate day; LD = long day), bulb shape and bulb color are listed. (XLSX 14 kb)
11032_2018_912_MOESM5_ESM.xlsx (142 kb)
Supplementary Table 2 List of SNP markers (Duangjit et al. 2013) used for the present study. For each marker, identification number, assigned function, sequence, position, alleles (X and Y), and linkage group (LG) are reported. (XLSX 141 kb)
11032_2018_912_MOESM6_ESM.xlsx (211 kb)
Supplementary Table 3 SNP results obtained in 73 genotypes of onion species. (XLSX 210 kb)
11032_2018_912_MOESM7_ESM.xlsx (25 kb)
Supplementary Table 4 Statistical analysis of 389 SNPs of 73 different genotypes of Allium spp. For each locus, the major allele frequency (MAF) and polymorphic information content (PIC) values are reported. (XLSX 25 kb)
11032_2018_912_MOESM8_ESM.doc (78 kb)
Supplementary Table 5 List of SNP loci associated with bulbing photoperiod, bulb shape and bulb color. LG = linkage group. (DOC 77 kb)
11032_2018_912_MOESM9_ESM.docx (19 kb)
Supplementary Table 6 List of SNP pairs in linkage disequilibrium and related coefficients. For each pair, the ID of the two co-inherited SNPs (named SNP_A and SNP_B), the linkage group location (LG), the measure of LD (r2), and the coefficient of LD (D′) are reported. (DOCX 19 kb)
11032_2018_912_MOESM10_ESM.docx (14 kb)
Supplementary Table 7 List of SNP pairs in linkage disequilibrium and associated with a phenotypic traits (S = bulb shape; C = bulb color; P = bulbing photoperiod). (DOCX 13 kb)


  1. Baldwin S, Pither-Joyce M, Wright K, Chen L, McCallum J (2012) Development of robust genomic simple sequence repeat markers for estimation of genetic diversity within and among bulb onion (Allium cepa L.) populations. Mol Breed 30(3):1401–1411CrossRefGoogle Scholar
  2. Baldwin S, Revanna R, Pither-Joyce M, Shaw M, Wright K, Thomson S, Moya L, Lee R, Macknight R, McCallum J (2014) Genetic analyses of bolting in bulb onion (Allium cepa L.). Theor Appl Genet 127(3):535–547CrossRefGoogle Scholar
  3. Bölter B, Nada A, Fulgosi H, Soll J (2006) A chloroplastic inner envelope membrane protease is essential for plant development. FEBS Letters 580(3):789-794Google Scholar
  4. Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am J Hum Genet 32:314–331PubMedPubMedCentralGoogle Scholar
  5. Boutet G, Carvalho SA, Falque M, Peterlongo P, Lhuillier E, Bouchez O, Baranger A (2016) SNP discovery and genetic mapping using genotyping by sequencing of whole genome genomic DNA from a pea RIL population. BMC Genomics 17(1):121–134CrossRefGoogle Scholar
  6. Brewster JL (2008) Onions and other vegetable alliums (no 15). CABI, OxfordshireCrossRefGoogle Scholar
  7. Caruso G, Conti S, Villari G, Borrelli C, Melchionna G, Minutolo M, Amalfitano C (2014) Effects of transplanting time and plant density on yield, quality and antioxidant content of onion (Allium cepa L) in southern Italy. Sci Hortic 166:111–120CrossRefGoogle Scholar
  8. Casanova JE (2007) Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors. Traffic 8(11):1476–1485CrossRefGoogle Scholar
  9. Cavanagh CR, Chao S, Wang S, Huan BE, Stephen S, Kiani S, See D (2013) Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars. Proc Natl Acad Sci 110(20):8057–8062CrossRefGoogle Scholar
  10. Chandra S, Singh D, Pathak J, Kumari S, Kumar M, Poddar R, Mukhopadhyay K (2017) SNP discovery from next-generation transcriptome sequencing data and their validation using KASP assay in wheat (Triticum aestivum L). Mol Breed 37(7):92–107CrossRefGoogle Scholar
  11. Corrado G, Caramante M, Piffanelli P, Rao R (2014) Genetic diversity in Italian tomato landraces: implications for the development of a core collection. Sci Hortic 168:138–144CrossRefGoogle Scholar
  12. Cui C, Mei H, Liu Y, Zhang H, Zheng Y (2017) Genetic diversity, population structure, and linkage disequilibrium of an association-mapping panel revealed by genome-wide SNP markers in sesame. Front Plant Sci 8:1189CrossRefGoogle Scholar
  13. de Lima Castro SA, Gonçalves-Vidigal MC, Gilio TAS, Lacanallo GF, Valentini G, Martins VDSR, Pastor-Corrales MA (2017) Genetics and mapping of a new anthracnose resistance locus in Andean common bean Paloma. BMC Genomics 18(1):306CrossRefGoogle Scholar
  14. Del Carpio DP, Basnet RK, De Vos RC, Maliepaard C, Visser R, Bonnema G (2011) The patterns of population differentiation in a Brassica rapa core collection. Theor Appl Genet 122(6):1105–1118CrossRefGoogle Scholar
  15. Duangjit J, Bohanec B, Chan AP, Town CD, Havey MJ (2013) Transcriptome sequencing to produce SNP-based genetic maps of onion. Theor Appl Genet 126(8):2093–2101CrossRefGoogle Scholar
  16. 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):359–361CrossRefGoogle Scholar
  17. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14(8):2611–2620CrossRefGoogle Scholar
  18. Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164(4):1567–1587PubMedPubMedCentralGoogle Scholar
  19. Fletcher JC (2001) The ULTRAPETALA gene controls shoot and floral meristem size in Arabidopsis. Development 128(8):1323–1333PubMedGoogle Scholar
  20. Foll M, Gaggiotti O (2008) A genome-scan method to identify selected loci appropriate for both dominant and codominant markers: a Bayesian perspective. Genetics 180:977–993CrossRefGoogle Scholar
  21. Goddard KA, Hopkins PJ, Hall JM, Witte JS (2000) Linkage disequilibrium and allele-frequency distributions for 114 single-nucleotide polymorphisms in five populations. Am J Hum Genet 66(1):216–234CrossRefGoogle Scholar
  22. Gurushidze M, Mashayekhi S, Blattner FR, Friesen N, Fritsch RM (2007) Phylogenetic relationships of wild and cultivated species of Allium section Cepa inferred by nuclear rDNA ITS sequence analysis. Plant Syst Evol 269(3):259–269CrossRefGoogle Scholar
  23. Haudry A, Cenci A, Ravel C, Bataillon T, Brunel D, Poncet C, David J (2007) Grinding up wheat: a massive loss of nucleotide diversity since domestication. Mol Biol Evol 24(7):1506–1517CrossRefGoogle Scholar
  24. Havey MJ, Ghavami F (2018) Informativeness of single nucleotide polymorphisms and relationships among onion populations from important world production regions. J Am Soc Hortic Sci 143(1):34–44CrossRefGoogle Scholar
  25. Hiremath PJ, Kumar A, Penmetsa RV, Farmer A, Schlueter JA, Chamarthi SK, Kishor K (2012) Large-scale development of cost-effective SNP marker assays for diversity assessment and genetic mapping in chickpea and comparative mapping in legumes. Plant Biotechnol J 10(6):716–732CrossRefGoogle Scholar
  26. Huang K, Whitlock R, Press MC, Scholes JD (2012) Variation for host range within and among populations of the parasitic plant Striga hermonthica. Heredity 108(2):96–104CrossRefGoogle Scholar
  27. Hufford MB, Xu X, Van Heerwaarden J, Pyhäjärvi T, Chia JM, Cartwright RA, Lai J (2012) Comparative population genomics of maize domestication and improvement. Nat Genet 44(7):808–811CrossRefGoogle Scholar
  28. Iorizzo M, Senalik DA, Ellison SL, Grzebelus D, Cavagnaro PF, Allender C, Simon PW (2013) Genetic structure and domestication of carrot (Daucus carota subsp sativus) (Apiaceae). Am J Bot 100(5):930–938CrossRefGoogle Scholar
  29. Jakse J, Martin W, McCallum J, Havey MJ (2005) Single nucleotide polymorphisms, indels, and simple sequence repeats for onion cultivar identification. J Am Soc Hortic Sci 130(6):912–917Google Scholar
  30. Khar A, Jakse J, Havey MJ (2008) Segregations for onion bulb colors reveal that red is controlled by at least three loci. J Am Soc Hortic Sci 133(1):42–47Google Scholar
  31. Khosa JS, Dhatt AS (2015) Genetic diversity for morphological and biochemical traits in bulb onion. Indian J Hort 72(1):143–146CrossRefGoogle Scholar
  32. Khosa JS, McCallum J, Dhatt AS, Macknight RC (2016) Enhancing onion breeding using molecular tools. Plant Breed 135(1):9–20CrossRefGoogle Scholar
  33. Kisha TJ, Cramer CS (2011) Determining redundancy of short-day onion accessions in a germplasm collection using microsatellite and targeted region amplified polymorphic markers. J Am Soc Hortic Sci 136(2):129–134Google Scholar
  34. Kitakura S, Adamowski M, Matsuura Y, Santuari L, Kouno H, Arima K, Tanaka H (2017) BEN3/BIG2 ARF GEF is involved in brefeldin A-sensitive trafficking at the trans-Golgi network/early endosome in Arabidopsis thaliana. Plant Cell Physiol 58(10):1801–1811CrossRefGoogle Scholar
  35. Lischer HEL, Excoffier L (2012) PGDSpider: an automated data conversion tool for connecting population genetics and genomics programs. Bioinformatics 28:298–299CrossRefGoogle Scholar
  36. Liu K, Muse SV (2005) PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21(9):2128–2129CrossRefGoogle Scholar
  37. Lohse M, Nagel A, Herter T, May P, Schroda M, Zrenner R, Usadel B (2014) Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ 37(5):1250–1258CrossRefGoogle Scholar
  38. Martin WJ, McCallum J, Shigyo M, Jakse J, Kuhl JC, Yamane N, Havey MJ (2005) Genetic mapping of expressed sequences in onion and in silico comparisons with rice show scant collinearity. Mol Gen Genomics 274(3):197–204CrossRefGoogle Scholar
  39. McCallum J, Pither-Joyc M, Shaw M, Kenel F, Davis S, Butler R, Havey MJ (2007) Genetic mapping of sulfur assimilation genes reveals a QTL for onion bulb pungency. Theor Appl Genet 114(5):815–822CrossRefGoogle Scholar
  40. McCallum J, Thomson S, Pither-Joyce M, Kenel F, Clarke A, Havey MJ (2008) Genetic diversity analysis and single–nucleotide polymorphism marker development in cultivated bulb onion based on expressed sequence tag–simple sequence repeat markers. J Am Soc Hortic Sci 133:810–818Google Scholar
  41. Mengistu DK, Kidane YG, Catellani M, Frascaroli E, Fadda C, Pè ME, Dell’Acqua M (2016) High-density molecular characterization and association mapping in Ethiopian durum wheat landraces reveals high diversity and potential for wheat breeding. Plant Biotechnol J 14(9):1800–1812CrossRefGoogle Scholar
  42. Monfared MM, Carles CC, Rossignol P, Pires HR, Fletcher JC (2013) The ULT1 and ULT2 trxG genes play overlapping roles in Arabidopsis development and gene regulation. Mol Plant 6(5):1564–1579CrossRefGoogle Scholar
  43. Nishimura K, Kato Y, Sakamoto W (2017) Essentials of proteolytic machineries in chloroplasts. Mol Plant 10:4–19CrossRefGoogle Scholar
  44. Peakall PE, Smouse R (2012) GenAlEx 65: genetic analysis in excel population genetic software for teaching and research-an update. Bioinformatics 28:2537–2539CrossRefGoogle Scholar
  45. Pérez-Figueroa A, García-Pereira MJ, Saura M, Rolán-Alvarez E, Caballero A (2010) Comparing three different methods to detect selective loci using dominant markers. J Evol Biol 23:2267–2276CrossRefGoogle Scholar
  46. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2):945–959PubMedPubMedCentralGoogle Scholar
  47. Pu L, Liu MS, Kim SY, Chen LFO, Fletcher JC, Sung ZR (2013) EMBRYONIC FLOWER1 and ULTRAPETALA1 act antagonistically on Arabidopsis development and stress response. Plant Physiol 162(2):812–830CrossRefGoogle Scholar
  48. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Sham PC (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81(3):559–575CrossRefGoogle Scholar
  49. Rashid MHA, Massiah AJ, Thomas B (2016) Genetic regulation of day length adaptation and bulb formation in onion (Allium cepa L). In: VII International Symposium on Edible Alliaceae, vol 1143, pp 7–14Google Scholar
  50. Saxena RK, Varma Penmetsa R, Upadhyaya HD, Kumar A, Carrasquilla-Garcia N, Schlueter JA, Farmer A, Whaley AM, Sarma BK, May GD, Cook DR, Varshney RK (2012) Large-scale development of cost-effective single-nucleotide polymorphism marker assays for genetic mapping in pigeonpea and comparative mapping in legumes. DNA Res 19(6):449–461CrossRefGoogle Scholar
  51. Semagn K, Babu R, Hearne S, Olsen M (2014) Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Molecular Breeding 33(1):1-14.
  52. Steele KA, Quinton-Tulloch MJ, Amgai RB, Dhakal R, Khatiwada SP, Vyas D, Heine M, Witcombe JR (2018) Accelerating public sector rice breeding with high-density KASP markers derived from whole genome sequencing of indica rice. Mol Breed 38(4):38CrossRefGoogle Scholar
  53. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739CrossRefGoogle Scholar
  54. Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P, Selbig J, Müller LA, Rhee SY, Stitt S (2004) mapman: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. The Plant Journal 37(6):914-939.
  55. Villano C, Carputo D, Frusciante L, Santoro X, Aversano R (2014) Use of SSR and retrotransposon-based markers to interpret the population structure of native grapevines from southern Italy. Mol Biotechnol 56(11):1011–1020CrossRefGoogle Scholar
  56. Zhou X, Stephens M (2012) Genome-wide efficient mixed-model analysis for association studies. Nat Genet 44(7):821–824CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Agricultural SciencesUniversity of Naples Federico IIPorticiItaly
  2. 2.Plants for Human Health Institute, Department of Horticultural SciencesNorth Carolina State UniversityKannapolisUSA

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