Skip to main content

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes

Functional & Integrative Genomics volume 14, pages 643–655 (2014)Cite this article

Abstract

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium™ array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3

References

  • Ahmad R, Parfitt DE, Fass J et al (2011) Whole genome sequencing of peach (Prunus persica L.) for SNP identification and selection. BMC Genomics 12:569

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Akhunov E, Nicolet C, Dvorak J (2009) Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina Golden Gate assay. Theor Appl Genet 119:507–517

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    CAS  PubMed  Article  Google Scholar 

  • Anithakumari AM, Tang JF, Van Eck HJ et al (2010) A pipeline for high throughput detection and mapping of SNPs from EST databases. Mol Breed 26:65–75

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Azam S, Thakur V, Ruperao P et al (2012) Coverage-based consensus calling (CBCC) of short sequence reads and comparison of CBCC results to identify SNPs in chickpea (Cicer arietinum; Fabaceae), a crop species without a reference genome. Am J Bot 99:186–192

    CAS  PubMed  Article  Google Scholar 

  • Bachlava E, Taylor CA, Tang S et al (2012) SNP discovery and development of a high-density genotyping array for sunflower. PLoS One 7:e29814

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Barbazuk WB, Emrich SJ, Chen HD, Li L, Schnable PS (2007) SNP discovery via 454 transcriptome sequencing. Plant J 51:910–918

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Batley J, Edwards D (2007) SNP applications in plants. In: Oraguzie NC, Rikkerink EHA, Gardiner SE, De Silva HN (eds) Association mapping in plants. Springer, New York, pp 95–102

    Chapter  Google Scholar 

  • Bekele WA, Wieckhorst S, Friedt W, Snowdon RJ (2013) High-throughput genomics in sorghum: from whole-genome resequencing to a SNP screening array. Plant Biotechnol J 11(9):1112–1125

    CAS  PubMed  Article  Google Scholar 

  • Bus A, Hecht J, Huettel B, Reinhardt R, Stich B (2012) High-throughput polymorphism detection and genotyping in Brassica napus using next-generation RAD sequencing. BMC Genomics 13:281

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Cavanagh CR, Chao S, Wang S et al (2013) Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars. Proc Natl Acad Sci U S A 110:8057–8062

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Ccc (2010) Canola Council of Canada - Official definition of canola. http://www.canolacouncil.org/ind_definition.aspx On-line [URL] Year/Electronic Resource Number

  • Chagné D, Crowhurst RN, Troggio M et al (2012) Genome-wide SNP detection, validation, and development of an 8 K array for apple. PLoS One 7:e31745

    PubMed Central  PubMed  Article  Google Scholar 

  • Clarke WE, Parkin IA, Gajardo HA et al (2013) Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP) in Brassica napus L. PLoS One 8

  • Cowling WA (2007) Genetic diversity in Australian canola and implications for crop breeding for changing future environments. Field Crop Res 104:103–111

    Article  Google Scholar 

  • Davey JW, Cezard T, Fuentes-Utrilla P, Eland C, Gharbi K, Blaxter ML (2013) Special features of RAD sequencing data: implications for genotyping. Mol Ecol 22:3151–3164

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Delourme R, Falentin C, Fomeju BF et al (2013) High-density SNP-based genetic map development and linkage disequilibrium assessment in Brassica napus L. BMC Genomics 14:120

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Duran C, Appleby N, Vardy M, Imelfort M, Edwards D, Batley J (2009) Single nucleotide polymorphism discovery in barley using autoSNPdb. Plant Biotechnol J 7:326–333

    CAS  PubMed  Article  Google Scholar 

  • Durstewitz G, Polley A, Plieske J et al (2010) SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napus. Genome 53:948–956

    CAS  PubMed  Article  Google Scholar 

  • Eckert AJ, Van Heerwaarden J, Wegrzyn JL et al (2010) Patterns of population structure and environmental associations to aridity across the range of loblolly pine (Pinus taeda L., Pinaceae). Genetics 185:969–982

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Edwards D, Forster J, Chagné D, Batley J (2007) What are SNPs? In: Oraguzie N, Rikkerink E, Gardiner S, Silva H (eds) Association mapping in plants. Springer, New York, pp 41–52

    Chapter  Google Scholar 

  • Felcher KJ, Coombs JJ, Massa AN et al (2012) Integration of two diploid potato linkage maps with the potato genome sequence. PLoS One 7:e36347

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Ferguson ME, Hearne SJ, Close TJ et al (2012) Identification, validation and high-throughput genotyping of transcribed gene SNPs in cassava. Theor Appl Genet 124:685–695

    CAS  PubMed  Article  Google Scholar 

  • Friedt W, Snowdon R (2009) Oilseed rape. In: Vollmann J, Rajcan I (eds) Oil crops. Springer, New York, pp 91–126

    Chapter  Google Scholar 

  • Fulton TM, Chunwongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Report 13:207–209

    CAS  Article  Google Scholar 

  • Ganal MW, Durstewitz G, Polley A et al (2011) A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome. PLOS One 6

  • Geraldes A, Difazio SP, Slavov GT et al (2013) A 34 K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species. Mol Ecol Resour 13:306–326

    CAS  PubMed  Article  Google Scholar 

  • Hackett CA, Mclean K, Bryan GJ (2013) Linkage analysis and QTL mapping using SNP dosage data in a tetraploid potato mapping population. PLoS One 8:e63939

    PubMed Central  PubMed  Article  Google Scholar 

  • Hamilton JP, Hansey CN, Whitty BR et al (2011) Single nucleotide polymorphism discovery in elite North American potato germplasm. BMC Genomics 12

  • Haseneyer G, Schmutzer T, Seidel M et al (2011) From RNA-seq to large-scale genotyping—genomics resources for rye (Secale cereale L.). BMC Plant Biol 11:131

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Hayward A, Mason A, Dalton-Morgan J, Zander M, Edwards D, Batley J (2012) SNP discovery and applications in Brassica napus. J Plant Biotechnol 39:49–61

    Article  Google Scholar 

  • Hu Z, Hua W, Huang S et al (2012) Discovery of pod shatter-resistant associated SNPs by deep sequencing of a representative library followed by bulk segregant analysis in rapeseed. PLoS One 7:e34253

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Huang X, Wei X, Sang T et al (2010) Genome-wide association studies of 14 agronomic traits in rice landraces. Nat Genet 42:961–967

    CAS  PubMed  Article  Google Scholar 

  • Hyten DL, Song Q, Choi IY et al (2008) High-throughput genotyping with the GoldenGate assay in the complex genome of soybean. Theor Appl Genet 116:945–952

    CAS  PubMed  Article  Google Scholar 

  • Hyten DL, Song QJ, Fickus EW et al (2010) High-throughput SNP discovery and assay development in common bean. BMC Genomics 11

  • Imelfort M, Duran C, Batley J, Edwards D (2009) Discovering genetic polymorphisms in next-generation sequencing data. Plant Biotechnol J 7:312–317

    CAS  PubMed  Article  Google Scholar 

  • Iorizzo M, Senalik DA, Grzebelus D et al (2011) De novo assembly and characterization of the carrot transcriptome reveals novel genes, new markers, and genetic diversity. BMC Genomics 12

  • Kumar S, You FM, Cloutier S (2012) Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries. BMC Genomics 13:684

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Liu S, Liu Y, Yang X et al (2014) The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat Commun 5:3930

    PubMed  Google Scholar 

  • Lorenc MT, Hayashi S, Stiller J et al (2012) Discovery of single nucleotide polymorphisms in complex genomes using SGSautoSNP. Biology 1:370–382

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Myles S, Chia JM, Hurwitz B et al (2010) Rapid genomic characterization of the genus vitis. PLoS One 5(1):e8219

    PubMed Central  PubMed  Article  Google Scholar 

  • Novaes E, Drost DR, Farmerie WG et al (2008) High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome. BMC Genomics 9:312

    PubMed Central  PubMed  Article  Google Scholar 

  • Raman H, Dalton-Morgan J, Diffey S et al (2014) SNP markers-based map construction and genome-wide linkage analysis in Brassica napus. Plant Biotechnol J. doi:10.1111/pbi.12186

  • Rostoks N, Mudie S, Cardle L et al (2005) Genome-wide SNP discovery and linkage analysis in barley based on genes responsive to abiotic stress. Mol Gen Genomics 274:515–527

    CAS  Article  Google Scholar 

  • Shin JH, Blay S, Mcneney B, Graham J (2006) LDheatmap: an R function for graphical display of pairwise linkage disequilibria between single nucleotide polymorphisms. J Stat Soft 16 Code Snippet 3

  • Sim SC, Durstewitz G, Plieske J et al (2012a) Development of a large SNP genotyping array and generation of high-density genetic maps in tomato. PLoS One 7(7):e40563

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Sim SC, Van Deynze A, Stoffel K et al (2012b) High-density SNP genotyping of tomato (Solanum lycopersicum L.) reveals patterns of genetic variation due to breeding. PLoS One 7:e45520

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Song Q, Hyten DL, Jia G et al (2013) Development and evaluation of SoySNP50K, a high-density genotyping array for soybean. PLoS One 8:e54985

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Syvänen A-C (2001) Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet 2:930–942

    PubMed  Article  Google Scholar 

  • Trick M, Long Y, Meng JL, Bancroft I (2009) Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencing. Plant Biotechnol J 7:334–346

    CAS  PubMed  Article  Google Scholar 

  • Verde I, Bassil N, Scalabrin S et al (2012) Development and evaluation of a 9 K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm. PLoS One 7

  • Wang XW, Wang HZ, Wang J et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–U157

    CAS  PubMed  Article  Google Scholar 

  • Wang W, Huang S, Liu Y et al (2012) Construction and analysis of a high-density genetic linkage map in cabbage (Brassica oleracea L. var. capitata). BMC Genomics 13:523

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Warnes G, Gorjanc G, Leisch F, Man M (2012) Genetics: population genetics V. R package version 1.3.7

  • Westermeier P, Wenzel G, Mohler V (2009) Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (Brassica napus L.). Theor Appl Genet 119:1301–1311

    CAS  PubMed  Article  Google Scholar 

  • Wu XL, Ren CW, Joshi T, Vuong T, Xu D, Nguyen HT (2010) SNP discovery by high-throughput sequencing in soybean. BMC Genomics 11

  • Würschum T, Langer SM, Longin CF et al (2013) Population structure, genetic diversity and linkage disequilibrium in elite winter wheat assessed with SNP and SSR markers. Theor Appl Genet 126:1477–1486

    PubMed  Article  Google Scholar 

  • Yamamoto T, Nagasaki H, Yonemaru J et al (2010) Fine definition of the pedigree haplotypes of closely related rice cultivars by means of genome-wide discovery of single-nucleotide polymorphisms. BMC Genomics 11

  • You FM, Huo N, Deal KR et al (2011) Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence. BMC Genomics 12:59

    CAS  PubMed Central  PubMed  Article  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge funding support from the Australian Research Council (Projects LP0882095, LP0883462, LP0989200, LP110100200 and DP0985953). Support from the Australian Genome Research Facility (AGRF), the Queensland Cyber Infrastructure Foundation (QCIF) and the Australian Partnership for Advanced Computing (APAC) is gratefully acknowledged. Rowan Bunch is gratefully acknowledged for HiScanSQ 6 K SNP chip scanning.

Author information

Affiliations

  1. Centre for Integrative Legume Research and School of Agriculture and Food Sciences, University of Queensland, Brisbane, Australia

    Jessica Dalton-Morgan, Alice Hayward, Salman Alamery, Reece Tollenaere, Annaliese S. Mason, Emma Campbell, Dhwani Patel, Michał T. Lorenc, David Edwards & Jacqueline Batley

  2. Australian Centre for Plant Functional Genomics, School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, 4072, Australia

    Michał T. Lorenc & David Edwards

  3. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Bin Yi, Yan Long & Jinling Meng

  4. NSW Agricultural Genomics Centre, NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, PMB, Wagga Wagga, NSW, 2650, Australia

    Rosy Raman & Harsh Raman

  5. Illumina Inc., Hayward, CA, USA

    Cindy Lawley

  6. School of Plant Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia

    Jacqueline Batley

Authors
  1. Jessica Dalton-Morgan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alice Hayward
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Salman Alamery
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Reece Tollenaere
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Annaliese S. Mason
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Emma Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dhwani Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michał T. Lorenc
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bin Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yan Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jinling Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rosy Raman
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Harsh Raman
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Cindy Lawley
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. David Edwards
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Jacqueline Batley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jacqueline Batley.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Suppl. Figure 1
figure 4

Representative samples from the observed cluster types with Brassica napus SNPs based on the GenomeStudio software. Genotype calls are represented by different coloured regions: AA is red, BB is blue and AB is purple. (a) Locus produces well defined clusters which segregates on the A genome and fails on the C genome. (b) Locus produces well defined clusters which segregates on the C genome and fails on the A genome. (c) Locus is monomorphic within all samples run on 6 K chip. (d) Locus is monomorphic on C genome and has failed on the A genome. Individuals from diploid species are identified by the colour scheme: B. rapa: green; B. oleracea:cyan; B. nigra: pink. (GIF 334 kb)

Full size image
Suppl. Figure 2
figure 5

Principle component analysis on the 71 informative B. napus genotypes plus individuals from the progenitor species (46 B. rapa, four B. oleracea). (GIF 17 kb)

Full size image
Suppl. Figure 3
figure 6

Principle component analysis on the 71 informative B. napus genotypes. (GIF 16 kb)

Full size image

High resolution image (TIFF 761 kb)

High resolution image (TIFF 3439 kb)

High resolution image (TIFF 3439 kb)

Suppl. Figure 4

Heatmap displaying pairwise Linkage disequilibrium between polymorphic SNPs (PNG 1420 kb)

Table S1

(XLSX 26.6 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dalton-Morgan, J., Hayward, A., Alamery, S. et al. A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes. Funct Integr Genomics 14, 643–655 (2014). https://doi.org/10.1007/s10142-014-0391-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10142-014-0391-2

Keywords

  • Single-nucleotide polymorphism (SNP)
  • Illumina
  • Infinium
  • Genotyping
  • Brassica