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Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression

Abstract

Key message

R1-nj anthocyanin marker inhibition is highly frequent in tropical maize germplasm considerably affecting efficiency of haploid identification. Molecular markers reliably differentiating germplasm with anthocyanin color inhibitor have been identified in this study.

Abstract

The R1-Navajo (R1-nj) color marker facilitates easy and quick identification of haploid kernels at the seed stage during in vivo haploid induction process in maize. However, the Navajo phenotype can be completely suppressed or poorly expressed in some germplasm, making it impossible or inefficient to identify haploids at the seed stage. In this study, we characterized the expression of R1-nj marker in a large array of tropical/subtropical inbred lines, breeding populations and landraces by crossing with the R1-nj-based tropicalized haploid inducer. There was a high frequency of inhibition of the Navajo phenotype in the maize inbred lines, which are used in tropical breeding programs. Genome-wide association mapping showed that the C1 anthocyanin regulatory locus is the most significant genetic factor influencing inhibition of the Navajo phenotype. Molecular marker assays were designed based on polymorphism in the C1 vs C1-I alleles. Analysis of a set of 714 inbred lines demonstrated that a combination of two gene-specific markers—8 bp C1-I InDel and C1-I SNP—could predict with high accuracy the presence of anthocyanin color inhibition in the germplasm analyzed. Information generated in this study aids in making informed decisions on the constitution of source populations for doubled haploid (DH) line development in tropical germplasm, particularly those derived from elite maize lines from CIMMYT. The C1-I gene-specific molecular markers identified and validated will facilitate high-throughput and cost-effective evaluation of a large pool of germplasm for the presence of the dominant color inhibitor in maize germplasm.

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References

  • Altman DG, Bland JM (1994a) Diagnostic tests 2: predictive values. Br Med J 309:102

    CAS  Article  Google Scholar 

  • Altman DG, Bland JM (1994b) Diagnostic tests 3: receiver operating characteristic plots. Br Med J 309:188

    CAS  Article  Google Scholar 

  • Battistelli GM, Von Pinho RG, Justus A et al (2013) Production and identification of doubled haploids in tropical maize. Genet Mol Res 12:4230–4242

    CAS  PubMed  Article  Google Scholar 

  • Brink RA, Greenblatt IM (1954) Diffuse, a pattern gene in Zea Mays epistatic to red pericarp. J Hered 45:47–50

    Google Scholar 

  • Burr FA, Burr B, Scheffler BE et al (1996) The maize repressor-like gene intensifier1 shares homology with the r1/b1 multigene family of transcription factors and exhibits missplicing. Plant Cell 8:1249–1259

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Chaikam V, Prasanna BM (2012) Maternal haploid detection using anthocyanin markers. In: Prasanna BM, Chaikam V, Mahuku G (eds) Doubled haploid technoogy in maize breeding: theory and practice. CIMMYT, Mexico, pp 20–23

    Google Scholar 

  • Chaikam V, Mahuku G, Prasanna BM (2012) Design and implementation of maternal haploid induction. In: Prasanna BM, Chaikam V, Mahuku G (eds) Doubled haploid technoogy in maize breeding: theory and practice. CIMMYT, Mexico, pp 14–19

    Google Scholar 

  • Chase SS (1952) Monoploids in maize. Iowa State College Press, Ames, pp 389–399

    Google Scholar 

  • Chase SS (1969) Monoploids and monoploid-derivatives of maize (Zea mays L.). Bot Rev 35:117–168

    Article  Google Scholar 

  • Chase SS, Nanda DK (1965) Screening for monoploids of maize by use of a purple embryo marker. Maize Gen Coop Newslett 39:59–60

    Google Scholar 

  • Chen S, Li LH (2009) Maize doubled haploid breeding (in Chinese). China Agricultural University Press, Beijing

    Google Scholar 

  • CIMMYT (2005) Laboratory protocols: CIMMYT applied molecular genetics laboratory, 3rd edn. CIMMYT, Mexico

    Google Scholar 

  • Coe EH Jr (1962) Spontaneous mutation of the aleurone color inhibitor in maize. Genetics 47:779

    CAS  PubMed Central  PubMed  Google Scholar 

  • Coe EH, McCormick SM, Modena SA (1981) White pollen in maize. J Hered 72:318–320

    Google Scholar 

  • Coe EH Jr, Nueffer MG, Hoisington DA (1988) The genetics of corn. In: Sprague GF, Dudley JW (eds) Corn andcorn improvement, 3rd edn. Madison, Wisconsin, pp 81–258

    Google Scholar 

  • Cone KC, Burr FA, Burr B (1986) Molecular analysis of the maize anthocyanin regulatory locus C1. Proc Natl Acad Sci 83:9631–9635

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Della Vedova CB, Lorbiecke R, Kirsch H et al (2005) The dominant inhibitory chalcone synthase allele C2-Idf (Inhibitor diffuse) from Zea mays L. acts via an endogenous RNA silencing mechanism. Genetics 170:1989–2002

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Dooner HK, Robbins TP, Jorgensen RA (1991) Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet 25:173–199

    CAS  PubMed  Article  Google Scholar 

  • Elshire RJ, Glaubitz JC, Sun Q et al (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 6:e19379

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Fisher RA (1922) On the interpretation of χ 2 from contingency tables, and the calculation of P. J R Stat Soc 85:87–94

    Article  Google Scholar 

  • Flint-Garcia SA, Thuillet A-C, Yu J et al (2005) Maize association population: a high-resolution platform for quantitative trait locus dissection. Plant J 44:1054–1064

    CAS  PubMed  Article  Google Scholar 

  • Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375

    CAS  PubMed  Article  Google Scholar 

  • Franken P, Niesbach-Klösgen U, Weydemann U et al (1991) The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene in. EMBO J 10:2605

    CAS  PubMed Central  PubMed  Google Scholar 

  • Geiger HH (2009) Doubled haploids. Handbook of maize. Springer, New York, pp 641–657

    Book  Google Scholar 

  • Geiger HH, Gordillo GA (2009) Doubled haploids in hybrid maize breeding. Maydica 54:485–489

    Google Scholar 

  • Goff SA, Cone KC, Fromm ME (1991) Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins. Genes Dev 5:298–309

    CAS  PubMed  Article  Google Scholar 

  • Greenblatt IM, Bock M (1967) A commercially desirable procedure for detection of monoploids in maize. J Hered 58:9–13

    Google Scholar 

  • Kang HM, Sul JH, Service SK et al (2010) Variance component model to account for sample structure in genome-wide association studies. Nat Genet 42:348–354

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Kebede AZ, Dhillon BS, Schipprack W et al (2011) Effect of source germplasm and season on the in vivo haploid induction rate in tropical maize. Euphytica 180:219–226

    Article  Google Scholar 

  • Li L, Xu X, Jin W, Chen S (2009) Morphological and molecular evidences for DNA introgression in haploid induction via a high oil inducer CAUHOI in maize. Planta 230:367–376

    CAS  PubMed  Article  Google Scholar 

  • Lübberstedt T, Frei UK (2012) Application of doubled haploids for target gene fixation in backcross programmes of maize. Plant Breed 131:449–452

    Article  Google Scholar 

  • Melchinger AE, Schipprack W, Würschum T et al (2013) Rapid and accurate identification of in vivo-induced haploid seeds based on oil content in maize. Sci Rep 3:2129

    PubMed Central  PubMed  Article  Google Scholar 

  • Melchinger AE, Schipprack W, Friedrich Utz H, Mirdita V (2014) In vivo haploid induction in maize: identification of haploid seeds by their oil content. Crop Sci 54:1497–1504

    Article  Google Scholar 

  • Nanda DK, Chase SS (1966) An embryo marker for detecting monoploids of maize (Zea mays L.). Crop Sci 6:213–215

    Article  Google Scholar 

  • Paz-Ares J, Ghosal D, Saedler H (1990) Molecular analysis of the C1-I allele from Zea mays: a dominant mutant of the regulatory C1 locus. EMBO J 9:315

    CAS  PubMed Central  PubMed  Google Scholar 

  • Prasanna BM (2012) Doubled haploid technology in maize breeding:an overview. In: Prasanna BM, Chaikam V, Mahuku G (eds) Doubled haploid technoogy in maize breeding: theory and practice. CIMMYT, Mexico, pp 1–8

    Google Scholar 

  • Prasanna BM, Chaikam V, Mahuku G (2012) Doubled haploid technoogy in maize breeding: theory and practice. CIMMYT, Mexico

    Google Scholar 

  • Prigge V, Sánchez C, Dhillon BS et al (2011) Doubled haploids in tropical maize: I. Effects of inducers and source germplasm on in vivo haploid induction rates. Crop Sci 51:1498–1506

    Article  Google Scholar 

  • Prigge V, Schipprack W, Mahuku G et al (2012) Development of in vivo haploid inducers for tropical maize breeding programs. Euphytica 185:481–490

    Article  Google Scholar 

  • Rober FK, Gordillo GA, Geiger HH (2005) In vivo haploid induction in maize-performance of new inducers and significance of doubled haploid lines in hybrid breeding. Maydica 50:275

    Google Scholar 

  • Rotarenco VA, Kirtoca IH, Jacota AG (2007) The possibility of identifying kernels with haploid embryos using oil content. Maize Gen Coop NewsLett 81:11

    Google Scholar 

  • Rotarenco VA, Dicu G, State D, Fuia S (2010) New inducers of maternal haploids in maize. Maize Genet Coop Newslett 84:15

    Google Scholar 

  • Schmidt W (2004) Hybrid maize breeding at KWS Saat AG. Vereinigung der Pflanzenzüchter und Saatgutkaufleute Österreichs, St. Pölten, pp 1–6

    Google Scholar 

  • Seitz G (2005) The use of doubled haploids in corn breeding. Proceedings 41st Annual Illinois Corn Breeders’ School 2005. Urbana–Champaign, Illinois, pp 1–7

  • Stinard PS, Sachs MM (2002) The identification and characterization of two dominant r1 haplotype-specific inhibitors of aleurone color in Zea mays. J Hered 93:421–428

    CAS  PubMed  Article  Google Scholar 

  • Strigens A, Schipprack W, Reif JC, Melchinger AE (2013) Unlocking the genetic diversity of maize landraces with doubled haploids opens new avenues for breeding. PLoS One 8:e57234

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Weber DF (2014) Today’s use of haploids in corn plant breeding. In: Sparks DL (ed) Advances in agronomy. Academic press, Dublin, pp 123–144

    Google Scholar 

  • Wilde K, Burger H, Prigge V et al (2010) Testcross performance of doubled-haploid lines developed from European flint maize landraces. Plant Breed 129:181–185

    CAS  Article  Google Scholar 

  • Yu J, Buckler ES (2006) Genetic association mapping and genome organization of maize. Curr Opin Biotechnol 17:155–160

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the financial support received from the CGIAR Research Program (CRP) MAIZE, Limagrain, and the MASAGRO project funded by the Secretariat of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA). The R1-nj-based tropical haploid inducers used in this study were the products of collaborative research efforts between CIMMYT and the University of Hohenheim, Germany. The assistance provided by Juan Burgueno for data analysis, and Miguel Mellado for the preparation of figures, is gratefully acknowledged.

Conflict of interest

The authors declare that no competing interests exist.

Ethical standards

The authors declare that the experiments comply with the laws of Mexico.

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Correspondence to Prasanna M. Boddupalli.

Additional information

Communicated by Xianchun Xia.

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Supplementary Table 1 Data on R1-nj marker expression in endosperm and embryo in the progenies of tropical inbreds crossed with a R1-nj-based inducer

Supplementary Table 2 List of inbred lines with complete anthocyanin color inhibition and the corresponding C1-I marker genotypes

Supplementary Fig. 1 Genome-wide association study (GWAS) based on ~410 K GBS-SNPs in 897 lines for (a) endosperm color intensity; (b) endosperm area marked; (c) embryo inhibition; and (d) embryo color intensity established C1 as the major gene controlling color expression. The most significant SNP (S9_9741377) on chromosome 9 was localized within the C1 locus.

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Chaikam, V., Nair, S.K., Babu, R. et al. Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression. Theor Appl Genet 128, 159–171 (2015). https://doi.org/10.1007/s00122-014-2419-3

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  • DOI: https://doi.org/10.1007/s00122-014-2419-3

Keywords

  • Inbred Line
  • Doubled Haploid
  • Color Expression
  • True Negative Rate
  • Haploid Inducer