Skip to main content

Advertisement

SpringerLink
Log in

A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

As more and more re-sequencing genome data in crops were released, SNPs (single nucleotide polymorphisms) are easily achieved for genotyping and show the highest abundance among all kinds of molecular markers. However, high-throughput SNP genotyping methods, such as KASP (Kompetitive Allele Specific PCR), TaqMan, or ARMS (amplification refractory mutation system), are always labor on DNA extraction and depending on expensive equipment. Therefore, most breeders are undergoing the bottleneck of lacking an easy, cost-saving, and stable genotyping system. Here, we report a direct PCR–based medium-throughput SNP marker–assisted selection (D-MAS) system suits available major crops including rice, wheat, maize, and rapeseed. The D-MAS system, which reduces the time of manual operation and result analysis dramatically, contains (1) seedling breeding in greenhouse; (2) high-throughput DNA extraction by alkaline lysis; and (3) gel-free SNP marker detection with 384-well by PARMS (penta-primer amplification refractory mutation) or KASP genotyping system. The stability of alkaline lysis DNA was validated by flexible dilution fold and long storing time under low temperatures. The alkaline lysis DNA from four F2 populations of rice, wheat, maize, and rapeseed showed equal efficiency in SNP calling relative to the ones from cetyl trimethylammonium bromide (CTAB) method. With the alkaline lysis DNA, PAMRS showed denser genotype cluster than KASP. Furthermore, the D-MAS system was adaptive with rice old leaves. The throughput and efficiency of the D-MAS system were validated in the hybrid rice seed purity test with a Xian-Geng-specific SNP marker which is also recommended to select ipa1-2d gene in the rice molecular breeding. In conclusion, we proposed a direct PCR–based SNP calling pipeline, which could be a simple, cheap, and robust standard operation procedure (SOP) of molecular breeding for different crops and get extensive use in most laboratory.

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

  • Batley J (2015) Plant genotyping: methods and protocols. In: Methods in Molecular Biology, vol 1245. Springer, New York

    Google Scholar 

  • Bennetzen JL, Schmutz J, Wang H, Percifield R, Hawkins J, Pontaroli AC, Estep M, Feng L, Vaughn JN, Grimwood J, Jenkins J, Barry K, Lindquist E, Hellsten U, Deshpande S, Wang X, Wu X, Mitros T, Triplett J, Yang X, Ye CY, Mauro-Herrera M, Wang L, Li P, Sharma M, Sharma R, Ronald PC, Panaud O, Kellogg EA, Brutnell TP, Doust AN, Tuskan GA, Rokhsar D, Devos KM (2012) Reference genome sequence of the model plant Setaria. Nat Biotechnol 30(6):555–561. https://doi.org/10.1038/nbt.2196

    Article  CAS  PubMed  Google Scholar 

  • Bukowski R, Guo X, Lu Y, Zou C, He B, Rong Z, Wang B, Xu D, Yang B, Xie C, Fan L, Gao S, Xu X, Zhang G, Li Y, Jiao Y, Doebley JF, Ross-Ibarra J, Lorant A, Buffalo V, Romay MC, Buckler ES, Ware D, Lai J, Sun Q, Xu Y (2018) Construction of the third-generation Zea mays haplotype map. GigaScience 7(4):1–12. https://doi.org/10.1093/gigascience/gix134

    Article  PubMed  Google Scholar 

  • Dayteg C, Tuvesson S, Merker A, Jahoor A, Brantestam KA (2007) Automation of DNA marker analysis for molecular breeding in crops: practical experience of a plant breeding company. Plant Breed 126(4):410–415. https://doi.org/10.1111/j.1439-0523.2007.01306.x

    Article  Google Scholar 

  • Eathington SR, Crosbie TM, Edwards MD, Reiter RS, Bull JK (2007) Molecular markers in a commercial breeding program. Crop Sci 47(Supplement_3):S-154

    Article  Google Scholar 

  • Heim M, Meyer UA (1990) Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 336(8714):529–532. https://doi.org/10.1016/0140-6736(90)92086-W

    Article  CAS  PubMed  Google Scholar 

  • Huang X, Wei X, Sang T, Zhao Q, Feng Q, Zhao Y, Li C, Zhu C, Lu T, Zhang Z, Li M, Fan D, Guo Y, Wang A, Wang L, Deng L, Li W, Lu Y, Weng Q, Liu K, Huang T, Zhou T, Jing Y, Li W, Lin Z, Buckler ES, Qian Q, Zhang QF, Li J, Han B (2010) Genome-wide association studies of 14 agronomic traits in rice landraces. Nat Genet 42(11):961–967. https://doi.org/10.1038/ng.695

    Article  CAS  PubMed  Google Scholar 

  • Huang X, Yang S, Gong J, Zhao Y, Feng Q, Gong H, Li W, Zhan Q, Cheng B, Xia J, Chen N, Hao Z, Liu K, Zhu C, Huang T, Zhao Q, Zhang L, Fan D, Zhou C, Lu Y, Weng Q, Wang ZX, Li J, Han B (2015) Genomic analysis of hybrid rice varieties reveals numerous superior alleles that contribute to heterosis. Nat Commun 6:6258. https://doi.org/10.1038/ncomms7258

    Article  CAS  PubMed  Google Scholar 

  • Huang X, Yang S, J-Y G, Zhao Q, Feng Q, Zhan Q, Zhao Y, Li W, Cheng B, Xia J, Chen N, Huang T, Zhang L, Fan D, Chen J, Zhou C, Lu Y, Weng Q, Han B (2016) Genomic architecture of heterosis for yield traits in rice. Nature 537. https://doi.org/10.1038/nature19760

    Article  CAS  PubMed  Google Scholar 

  • International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436(7052):793–800. https://doi.org/10.1038/nature03895

    Article  CAS  Google Scholar 

  • International Wheat Genome Seqencing Consortium(IWGSC) (2014) A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 345(6194):1251788. https://doi.org/10.1126/science.1251788

    Article  CAS  Google Scholar 

  • Ma C, Ma X, Yao L, Liu Y, Du F, Yang X, Xu M (2017) qRfg3, a novel quantitative resistance locus against Gibberella stalk rot in maize. Theor Appl Genet 130(8):1723–1734. https://doi.org/10.1007/s00122-017-2921-5

    Article  CAS  PubMed  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Myakishev MV, Khripin Y, Hu S, Hamer DH (2001) High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genome Res 11(1):163–169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17(7):2503–2516. https://doi.org/10.1093/nar/17.7.2503

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nuovo GJ, Hohman RJ, Nardone GA, Nazarenko IA (1999) In situ amplification using universal energy transfer-labeled primers. J Histochem Cytochem 47(3):273–279. https://doi.org/10.1177/002215549904700301

    Article  CAS  PubMed  Google Scholar 

  • Pan Q, Xu Y, Li K, Peng Y, Zhan W, Li W, Li LAND, Yan J (2017) The genetic basis of plant architecture in 10 maize recombinant inbred line populations. Plant Physiol 175:858–873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H, Wang X, Wicker T, Bharti AK, Chapman J, Feltus FA, Gowik U, Grigoriev IV, Lyons E, Maher CA, Martis M, Narechania A, Otillar RP, Penning BW, Salamov AA, Wang Y, Zhang L, Carpita NC, Freeling M, Gingle AR, Hash CT, Keller B, Klein P, Kresovich S, McCann M, Ming R, Peterson DG, Mehboob-ur-Rahman, Ware D, Westhoff P, Mayer KF, Messing J, Rokhsar DS (2009) The Sorghum bicolor genome and the diversification of grasses. Nature 457(7229):551–556. https://doi.org/10.1038/nature07723

    Article  CAS  PubMed  Google Scholar 

  • Rasheed A, Wen W, Gao F, Zhai S, Jin H, Liu J, Guo Q, Zhang Y, Dreisigacker S, Xia X, He Z (2016) Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theor Appl Genet 129(10):1843–1860. https://doi.org/10.1007/s00122-016-2743-x

    Article  CAS  PubMed  Google Scholar 

  • Rosas JE, Bonnecarrère V, Pérez de Vida F (2014) One-step, codominant detection of imidazolinone resistance mutations in weedy rice (Oryza sativa L.). Electron J Biotechnol 17:95–101

    Article  Google Scholar 

  • Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang XC, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463(7278):178–183. https://doi.org/10.1038/nature08670

    Article  CAS  PubMed  Google Scholar 

  • Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, Chen W, Yan L, Higginbotham J, Cardenas M, Waligorski J, Applebaum E, Phelps L, Falcone J, Kanchi K, Thane T, Scimone A, Thane N, Henke J, Wang T, Ruppert J, Shah N, Rotter K, Hodges J, Ingenthron E, Cordes M, Kohlberg S, Sgro J, Delgado B, Mead K, Chinwalla A, Leonard S, Crouse K, Collura K, Kudrna D, Currie J, He R, Angelova A, Rajasekar S, Mueller T, Lomeli R, Scara G, Ko A, Delaney K, Wissotski M, Lopez G, Campos D, Braidotti M, Ashley E, Golser W, Kim H, Lee S, Lin J, Dujmic Z, Kim W, Talag J, Zuccolo A, Fan C, Sebastian A, Kramer M, Spiegel L, Nascimento L, Zutavern T, Miller B, Ambroise C, Muller S, Spooner W, Narechania A, Ren L, Wei S, Kumari S, Faga B, Levy MJ, McMahan L, van Buren P, Vaughn MW, Ying K, Yeh CT, Emrich SJ, Jia Y, Kalyanaraman A, Hsia AP, Barbazuk WB, Baucom RS, Brutnell TP, Carpita NC, Chaparro C, Chia JM, Deragon JM, Estill JC, Fu Y, Jeddeloh JA, Han Y, Lee H, Li P, Lisch DR, Liu S, Liu Z, Nagel DH, McCann M, SanMiguel P, Myers AM, Nettleton D, Nguyen J, Penning BW, Ponnala L, Schneider KL, Schwartz DC, Sharma A, Soderlund C, Springer NM, Sun Q, Wang H, Waterman M, Westerman R, Wolfgruber TK, Yang L, Yu Y, Zhang L, Zhou S, Zhu Q, Bennetzen JL, Dawe RK, Jiang J, Jiang N, Presting GG, Wessler SR, Aluru S, Martienssen RA, Clifton SW, McCombie W, Wing RA, Wilson RK (2009) The B73 maize genome: complexity, diversity, and dynamics. Science 326(5956):1112–1115. https://doi.org/10.1126/science.1178534

    Article  CAS  PubMed  Google Scholar 

  • Thomson JM (2014) High-throughput SNP genotyping to accelerate crop improvement. Plant Breed Biotechnol 2(3):195–212

    Article  Google Scholar 

  • Wang K, Wang Z, Li F, Ye W, Wang J, Song G, Yue Z, Cong L, Shang H, Zhu S, Zou C, Li Q, Yuan Y, Lu C, Wei H, Gou C, Zheng Z, Yin Y, Zhang X, Liu K, Wang B, Song C, Shi N, Kohel RJ, Percy RG, Yu JZ, Zhu YX, Yu S (2012) The draft genome of a diploid cotton Gossypium raimondii. Nat Genet 44(10):1098–1103. https://doi.org/10.1038/ng.2371

    Article  CAS  PubMed  Google Scholar 

  • Wang W, Mauleon R, Hu Z, Chebotarov D, Tai S, Wu Z, Li M, Zheng T, Fuentes RR, Zhang F, Mansueto L, Copetti D, Sanciangco M, Palis KC, Xu J, Sun C, Fu B, Zhang H, Gao Y, Zhao X, Shen F, Cui X, Yu H, Li Z, Chen M, Detras J, Zhou Y, Zhang X, Zhao Y, Kudrna D, Wang C, Li R, Jia B, Lu J, He X, Dong Z, Xu J, Li Y, Wang M, Shi J, Li J, Zhang D, Lee S, Hu W, Poliakov A, Dubchak I, Ulat VJ, Borja FN, Mendoza JR, Ali J, Li J, Gao Q, Niu Y, Yue Z, Naredo MEB, Talag J, Wang X, Li J, Fang X, Yin Y, Glaszmann JC, Zhang J, Li J, Hamilton RS, Wing RA, Ruan J, Zhang G, Wei C, Alexandrov N, McNally K, Li Z, Leung H (2018) Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature 557(7703):43–49. https://doi.org/10.1038/s41586-018-0063-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wing RA, Purugganan MD, Zhang Q (2018) The rice genome revolution: from an ancient grain to Green Super Rice. Nat Rev Genet 19(8):505–517. https://doi.org/10.1038/s41576-018-0024-z

    Article  CAS  PubMed  Google Scholar 

  • Yang G, Chen S, Chen L, Sun K, Huang C, Zhou D, Huang Y, Wang J, Liu Y, Wang H (2019) Development of a core SNP arrays based on the KASP method for molecular breeding of rice. Rice 12:21. https://doi.org/10.1186/s12284-019-0272-3

    Article  PubMed  PubMed Central  Google Scholar 

  • Zeng D, Tian Z, Rao Y, Dong G, Yang Y, Huang L, Leng Y, Xu J, Sun C, Zhang G, Hu J, Zhu L, Gao Z, Hu X, Guo L, Xiong G, Wang Y, Li J, Qian Q (2017) Rational design of high-yield and superior-quality rice. Nat Plants 3:17031. https://doi.org/10.1038/nplants.2017.31

    Article  PubMed  Google Scholar 

  • Zhang J, Chen L-L, Xing F, Kudrna DA, Yao W, Copetti D, Mu T, Li W, Song J-M, Xie W, Lee S, Talag J, Shao L, An Y, Zhang C-L, Ouyang Y, Sun S, Jiao W-B, Lv F, Du B, Luo M, Maldonado CE, Goicoechea JL, Xiong L, Wu C, Xing Y, Zhou D-X, Yu S, Zhao Y, Wang G, Yu Y, Luo Y, Zhou Z-W, Hurtado BEP, Danowitz A, Wing RA, Zhang Q (2016) Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63. Proc Natl Acad Sci U S A 113(35):E5163–E5171. https://doi.org/10.1073/pnas.1611012113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang L, Yu H, Ma B, Liu G, Wang J, Gao R, Li J, Liu J, Xu J, Zhang Y, Li Q, Huang X, Qian Q, Han B, He Z (2017) A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice. Nat Commun 8:14789. https://doi.org/10.1038/ncomms14789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Q, Li J, Xue Y, Han B, Deng XW (2008) Rice 2020: a call for an international coordinated effort in rice functional genomics. Mol Plant 1(5):715–719. https://doi.org/10.1093/mp/ssn043

    Article  CAS  PubMed  Google Scholar 

  • Zhao J, Wang Z, Liu H, Li T, Hou J, Zhang X, Hao C (2019) Global status of 47 major wheat loci controlling yield, quality, adaptation and stress resistance selected over the last century. BMC Plant Biol 19(1):5. https://doi.org/10.1186/s12870-018-1612-y

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Dr. Haitao Li, Dr. YiKe Liu, and Mr. Duo Wang for their kindly providing rapeseed, wheat, and maize F2 population. We gratefully acknowledge Dr. Huihui Yu for the advice and revision of this manuscript.

Funding information

This research was supported by the Fundamental Research Funds for the Central Universities of China (Grant No. 2662017QD033), Hubei Provincial Natural Science Foundation of China (Grant No. 2019CFA061), Special Major Projects for Technological Innovation in Hubei Province (2019ABA104), National Natural Science Foundation of China (Grant No.31600983), and the Earmarked Fund for the China Agriculture Research System of China (Grant No. CARS-01-05).

Author information

Authors and Affiliations

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

    Jun Lu, Yidan Ouyang, Chao Mao, Meng Han, Lei Wang, Jinghua Xiao & Xu Li

  2. Wuhan Gentides Biotech Co., Ltd., Wuhan, 430074, China

    Jing Hou, Hui Luo, Jianhua Zhao & Yanyu Yang

Authors
  1. Jun Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yidan Ouyang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hui Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jianhua Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chao Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Meng Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jinghua Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yanyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yanyu Yang or Xu Li.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM1

(DOCX 19 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lu, J., Hou, J., Ouyang, Y. et al. A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops. Mol Breeding 40, 9 (2020). https://doi.org/10.1007/s11032-019-1091-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11032-019-1091-3

Keywords

Search

Navigation