Skip to main content
SpringerLink
Log in

Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in Brassica rapa

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Allen AM, Barker GLA, Berry ST (2011) Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.) Plant Biotechnol J 9:1086–1099

    Article  CAS  PubMed  Google Scholar 

  • Chen YP, Cao JS, Miao Y, Ye WZ (2000) Analysis of genetic polymorphism in vegetable crops of Brassica campestris by RAPD markers. J Zhejiang Univ (Agric Life Sci) 26:131–136

    CAS  Google Scholar 

  • Cortés AJ, Chavarro MC, Blair MW (2011) SNP marker diversity in common bean (Phaseolus vulgaris L.) Theor Appl Genet 123:827–845

    Article  PubMed  Google Scholar 

  • Earl DA, Vonholdt BM (2011) Structure harvester: a website and program for visualizing structure output and implementing the evanno method. Conserv Genet Resour 4(2):359–361

    Article  Google Scholar 

  • Edgington ES (1995) Randomization tests. CRC Press, Boca Raton, FL

    Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479–491

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fan JB, Oliphant A, Shen R, Kermani BG, Garcia F, Gunderson KL, Hansen M, Steemers F, Butler SL, Deloukas P, Galver L, Hunt S, McBride C, Bibikova M, Rubano T, Chen J, Wickham E, Doucet D, Chang W, Campbell D, Zhang B, Kruglyak S, Bentley D, Haas J, Rigault P, Zhou L, Stuelpnagel J, Chee MS (2003) Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol 68:69–78

    Article  CAS  PubMed  Google Scholar 

  • Federico L, Amy V, Thomas C (2008) Development of a set of public SSR markers derived from genomic sequence of a rapid cycling Brassica oleracea L. genotype. Theor Appl Genet 117:977–985

    Article  Google Scholar 

  • Grünwald NJ, Hoheisel G-A (2006) Hierarchical analysis of diversity, selfing, and genetic differentiation in populations of the oomycete aphanomyces euteiches. Phytopathology 96(10):1134–1141

    Article  PubMed  Google Scholar 

  • Guo JX, Zhou NY, Ma RC, Cao MQ (2002) Genetic diversity in Brassica rapa revealed by AFLP molecular markers. J Agric Biotechnol 10:138–143

    Google Scholar 

  • He YT, Chen BY, Fu TD, Li DR, Tu JX (2003) Origins and evolution of Brassica campestris L. in China. Acta Genet Sin 30:1003–1012

    CAS  Google Scholar 

  • Hirai M, Matsumoto S (2007) Brassica rapa. In: Vegetables. Springer, Berlin, pp 185–190

  • Hiremath PJ, Kumar A, Penmetsa RV (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 10:716–732

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang XH, Kurata N, Wei XH, Wang ZX, Wang A, Zhao Q, Zhao Y, Liu KY, Lu HY, Li WJ, Han B (2012) A map of rice genome variation reveals the origin of cultivated rice. Nature 490:497–501

    Article  CAS  PubMed  Google Scholar 

  • Hufford MB, Xu X, van Heerwaarden J, Pyhäjärvi T, Chia J-M, Cartwright RA, Elshire RJ, Glaubitz JC, Guill KE, Kaeppler SM, Lai J, Morrell PL, Shannon LM, Song C, Springer NM, Swanson-Wagner RA, Tiffin P, Wang J, Zhang G, Doebley J, McMullen MD, Ware D, Buckler ES, Yang S, Ross-Ibarra J (2012) Comparative population genomics of maize domestication and improvement. Nat Genet 44(7):808–811

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ke GL (2010) Chinese cabbage breeding. China Agricluture Press, Beijing, pp 18–24

    Google Scholar 

  • Labana KS, Gupta ML (1993) Importance and origin in breeding oilseed Brassicas Springer-Verlag. Berlin, Germany, pp 1–20

    Google Scholar 

  • Livak KJ, Flood SJA, Marmaro J, Giusti W, Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. Genome Res 4:357–362

    Article  CAS  Google Scholar 

  • McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nagaharu U (1935) Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jap J Bot 7:389–452

    Google Scholar 

  • Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in excel population genetic software for teaching and research. Mol Ecol Notes 6:288–295

    Article  Google Scholar 

  • Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalaski A (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2(3):225–238

    Article  CAS  Google Scholar 

  • Pritchard JK, Stephens P, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959

    CAS  PubMed  PubMed Central  Google Scholar 

  • Qi JJ, Liu X, Shen D, Miao H, Xie BY, Li XX, Zeng P, Wang SH, Shang Y, Gu XF, Du YC, Huang SW (2013) A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nature Genet 45:1510–1515

    Article  CAS  PubMed  Google Scholar 

  • Roorkiwal M, Sawargaonkar SL, Chitikineni A, Thudi M, Saxena RK, Upadhyaya HD, Vales MI, Riera-Lizarazu O, Varshney RK (2013) Single nucleotide polymorphism genotyping for breeding and genetics applications in chickpea and pigeonpea using the beadxpress platform. The Plant Genome 6(2):494–494

    Article  Google Scholar 

  • Saxena RK, Varma Penmetsa R, Upadhyaya HD, Kumar AS, Carrasquilla-Garcia NO, Schlueter JA, Farmer AN, 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):449–461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song M, Osborn TC, Williams PH (1988) Brassica taxonomy based on nuclear restriction fragment length polymorphism (RFLPs). 2. Preliminary analysis of sub-species within B. rapa (syn campestris) and B. oleracea. Theor Appl Genet 76:593–600

    Article  CAS  PubMed  Google Scholar 

  • Steemers FJ, Gunderson KL (2007) Whole genome genotyping technologies on the BeadArray ™ platform. Biotechnol J 2:41–49

    Article  CAS  PubMed  Google Scholar 

  • Su TB, Yu SC, Wang J, Zhang FL, Yu TJ, Zhang DS, Zhao XY, Wang WH (2014) Loss of function of the carotenoid isomerase gene BrCRTISO confers Orange color to the inner leaves of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant Mol Biol Rep 33:648–659. https://doi.org/10.1007/s11105-014-0779-0

    Article  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tian H-L, Wang F-G, Zhao J-R, Yi H-M, Wang L, Wang R, Yang Y, Song W (2015) Development of maizeSNP3072, a high-throughput compatible SNP array, for DNA fingerprinting identification of Chinese maize varieties. Mol Breed 35(6):136

    Article  PubMed  PubMed Central  Google Scholar 

  • Tobias W, Simon M, Longin C, Korzun V, Akhunov E, Ebmeyer E, Schachschneider R, Schacht J, Kazman E, Jochen CR (2013) Population structure, genetic diversity and linkage disequilibrium in elite winter wheat assessed with SNP and SSR markers. Theor Appl Genet 126(6):1477–1486

    Article  Google Scholar 

  • Wang X, Wang H, Wang J et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039

    Article  CAS  PubMed  Google Scholar 

  • Wang WH, Zhang DE, Yu SC, Liu J, Wang D, Zhang FL, Yu YJ, Zhao XY, Lu GX, Su TB (2014) Mapping the BrPur gene for purple leaf color on linkage group A03 of Brassica rapa. Euphytica 199:293–302

    Article  CAS  Google Scholar 

  • Wright S (1978) Evolution and genetics of populations, variability within and among natural populations, vol 4. The University of Chicago Press, Chicago

    Google Scholar 

  • Yu SC, Zhang FL, Zhao XY, Yu YJ, Zhang DS (2011) Sequence-characterized amplified region and simple sequence repeat markers for identifying the major quantitative trait locus responsible for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis). Plant Breed 130:580–583

    Article  CAS  Google Scholar 

  • Zhang SJ, Yu SC, Zhang FL, Zhao XY, Yu YJ, Zhang DS (2012) Inheritance of downy mildew resistance at different developmental stages in Chinese cabbage via the leaf disk test. Hort Environ Biotechnol 3(5):397–403

    Article  Google Scholar 

Download references

Funding

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101701), the National Plan for Science and Technology Support (2014BAD01B08), the Technology Innovation Program, BAAFS (KJCX20161503), the earmarked fund for China Agrculture Research System (CARS-23-A-05), and the National Natural Science Foundation of China (Nos. 31772297 and 31772307).

Author information

Author notes

  1. Tongbing Su and Peirong Li have contributed equally to this work.

Authors and Affiliations

  1. Beijing Vegetable Research Center (BVRC), Beijing Academy of Agricultural and Forestry Sciences (BAAFS), Beijing, 100097, China

    Tongbing Su, Peirong Li, Jingjing Yang, Guanglei Sui, Yangjun Yu, Deshuang Zhang, Xiuyun Zhao, Weihong Wang, Changlong Wen, Shuancang Yu & Fenglan Zhang

  2. Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Beijing, 100097, China

    Tongbing Su, Peirong Li, Jingjing Yang, Guanglei Sui, Yangjun Yu, Deshuang Zhang, Xiuyun Zhao, Weihong Wang, Changlong Wen, Shuancang Yu & Fenglan Zhang

  3. Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing, 100097, China

    Tongbing Su, Peirong Li, Jingjing Yang, Guanglei Sui, Yangjun Yu, Deshuang Zhang, Xiuyun Zhao, Weihong Wang, Changlong Wen, Shuancang Yu & Fenglan Zhang

Authors
  1. Tongbing Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peirong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingjing Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guanglei Sui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yangjun Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Deshuang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiuyun Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Weihong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Changlong Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Shuancang Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Fenglan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Shuancang Yu or Fenglan Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Supplementary Fig. 1

3-D plot of principal coordinate analysis (PCoA) of the 231 accessions. (DOCX 120 kb)

Supplementary Fig. 2

Delta K calculation (DOCX 50 kb)

Supplementary Table 1

(XLSX 25 kb)

Supplementary Table 2

(XLSX 12 kb)

Supplementary Table 3

(XLSX 142 kb)

Supplementary Table 4

(XLSX 52 kb)

Supplementary Table 5

(XLSX 8 kb)

Supplementary Table 6

(XLSX 9 kb)

Supplementary Table 7

(XLSX 695 kb)

Supplementary Table 8

(XLSX 9 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Su, T., Li, P., Yang, J. et al. Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in Brassica rapa. Mol Breeding 38, 42 (2018). https://doi.org/10.1007/s11032-018-0795-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11032-018-0795-0

Keywords

Search

Navigation