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Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of crtRB1 and lcyE governing higher accumulation of provitamin A in maize

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Abstract

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC1F1, BC2F1 and BC2F2 revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.

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Abbreviations

MAS:

Marker-assisted selection

PCR:

Polymerase chain reaction

TE:

Transposable element

UTR:

Untranslated region

VAD:

Vitamin A deficiency

References

  • Babu R, Rojas NP, Gao S, Yan J, Pixley K (2013) Validation of the effects of molecular marker polymorphisms in lcyE and crtRB1 on provitamin A concentrations for 26 tropical maize populations. Theor Appl Genet 26:389–399

    Article  Google Scholar 

  • Choudhary M, Hossain F, Muthusamy V, Thirunavukkarasu N, Saha S, Pandey N, Jha SK, Gupta HS (2015) Microsatellite marker-based genetic diversity analyses of novel maize inbreds possessing rare allele of β-carotene hydroxylase (crtRB1)for their utilization in β-carotene enrichment. J Plant Biochem Biotechnol. https://doi.org/10.1007/s13562-015-0300-3

    Google Scholar 

  • Frey JE, Frey B, Sauer C, Kellerhals M (2004) Efficient low-cost DNA extraction and multiplex fluorescent PCR method for marker-assisted selection in breeding. Plant Breed 123:554–557

    Article  CAS  Google Scholar 

  • Fu ZY, Chai YC, Zhou Y, Yang XH, Warburton ML, Xu S, Cai Y, Zhang D, Li J, Yan J (2013) Natural variation in the sequence of PSY1 and frequency of favorable polymorphisms among tropical and temperate maize germplasm. Theor Appl Genet 126:923–935

    Article  CAS  PubMed  Google Scholar 

  • Giuliano G (2014) Provitamin A biofortification of crop plants: a gold rush with many miners. Curr Opin Biotechnol 44:169–180

    Article  Google Scholar 

  • Gupta HS, Babu R, Agrawal PK, Mahajan V, Hossain F, Nepolean T (2013) Accelerated development of quality protein maize hybrid through marker-assisted introgression of opaque-2 allele. Plant Breed 132:77–82

    Article  CAS  Google Scholar 

  • Gupta HS, Hossain F, Muthusamy V (2015) Biofortification of maize: an Indian perspective. Indian J Genet 75:1–22

    Google Scholar 

  • Harjes CE, Rocheford TR, Bai L, Brutnell TP, Kandianis CB, Sowinski SG, Stapleton AE, Vallabhaneni R, Williams M, Wurtzel ET, Yan J, Buckler ES (2008) Natural genetic variation in Lycopene Epsilon Cyclase tapped for maize biofortification. Science 319:330–333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • International Food Policy Research Institute (2016) Global Hunger Index. http://www.ifpri.org/publication/2016-global-hunger-index-getting-zero-hunger

  • Liu L, Jeffers D, Zhang Y, Ding M, Chen W, Kang MS, Fan X (2015) Introgression of the crtRB1 gene into quality protein maize inbred lines using molecular markers. Mol Breed 35:154

    Article  PubMed  PubMed Central  Google Scholar 

  • Markoulatos P, Siafakas N, Moncany M (2002) Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 16:47–51

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muthusamy V, Hossain F, Nepolean T, Choudhary M, Saha S, Bhat JS, Prasanna BM, Gupta HS (2014) Development of β-carotene rich maize hybrids through marker-assisted introgression of β-carotene hydroxylase allele. PLoS ONE 9(12):e113583

    Article  PubMed  PubMed Central  Google Scholar 

  • Muthusamy V, Hossain F, Nepolean T, Saha S, Agrawal PK, Guleria SK, Gupta HS (2015a) Genetic variability and inter-relationship of kernel carotenoids among indigenous and exotic maize (Zea mays L.) inbreds. Cereal Res Commun. https://doi.org/10.1556/0806.43.2015.012

    Google Scholar 

  • Muthusamy V, Hossain F, Thirunavukkarasu N, Pandey N, Vishwakarma AK, Saha S, Gupta HS (2015b) Molecular characterization of exotic and indigenous maize inbreds for biofortification with kernel carotenoids. Food Biotechnol 29:276–295

    Article  CAS  Google Scholar 

  • Muthusamy V, Hossain F, Thirunavukkarasu N, Saha S, Gupta HS (2015c) Allelic variations for lycopene-ε-cyclase and β-carotene hydroxylase genes in maize inbreds and their utilization in β-carotene enrichment programme. Cogent Food Agric 1:1033141

    Google Scholar 

  • Muthusamy V, Hossain F, Thirunavukkarasu N, Saha S, Agrawal PK, Gupta HS (2016) Genetic analyses of kernel carotenoids in novel maize genotypes possessing rare allele of β-carotene hydroxylase gene. Cereal Res Commun 44:669–680

    Article  CAS  Google Scholar 

  • Pixley K, Palacios-Rojas N, Babu R, Mutale R, Surles R, Simpungwe E (2013) Carotenoids and human health. In: Tanumihardjo S (ed) Biofortification of maize with Provitamin A carotenoids. Springer, New York Publishers, pp 271–292

    Google Scholar 

  • Shiferaw B, Prasanna BM, Hellin J, Banziger M (2011) Crops that feed the world 6. Past successes and future challenges to the role played by maize in global food security. Food Secur 3:307–327

    Article  Google Scholar 

  • Tanumihardjo SA, Anderson C, Kaufer-Horwitz M, Bode L, Emenaker NJ, Haqq AM, Satia JA, Silver H, Stadler DD (2007) Poverty, obesity and malnutrition: an international perspective recognizing the paradox. J Am Diet Assoc 107:1966–1972

    Article  PubMed  Google Scholar 

  • Vallabhaneni R, Gallagher CE, Licciardello N, Cuttriss AJ, Quinlan RF, Wurtzel ET (2009) Metabolite sorting of a germplasm collection reveals the hydroxylase3 locus as a new target for maize provitamin A biofortification. Plant Physiol 151:1635–1645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vignesh M, Hossain F, Nepolean T, Saha S, Agrawal PK, Guleria SK, Prasanna BM, Gupta HS (2012) Genetic variability for kernel β-carotene and utilization of crtRB1 3’TE gene for biofortification in maize (Zea mays L.). Indian J Genet 72:189–194

    CAS  Google Scholar 

  • Vignesh M, Nepolean T, Hossain F, Singh AK, Gupta HS (2013) Sequence variation in 3′UTR region of crtRB1 gene and its effect on β-carotene accumulation in maize kernel. J Plant Biochem Biotechnol 22:401–408

    Article  CAS  Google Scholar 

  • Yan J, Kandianis BC, Harjes EC, Bai L, Kim HE, Yang X, Skinner DJ, Fu Z, Mitchell S, Li Q, Fernandez GSM, Zaharoeva M, Babu R, Fu Y, Palacios N, Li J, DellaPenna D, Brutnell T, Buckler SE, Warburton LM, Rocheford T (2010) Rare genetic variation at Zea mays crtRB1 increases beta carotene in maize grain. Nat Genet 42:322–327

    Article  CAS  PubMed  Google Scholar 

  • Zunjare RU, Hossain F, Muthusamy V, Baveja A, Chauhan HS, Thirunavukkarasu N, Saha S, Gupta HS (2017) Influence of rare alleles of β-carotene hydroxylase and lycopene epsilon cyclase genes on accumulation of provitamin A carotenoids in maize kernels. Plant Breed. https://doi.org/10.1111/pbr.12548

    Google Scholar 

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Acknowledgements

RUZ is grateful to University Grants Commission (UGC) for awarding Rajiv Gandhi National Fellowship for his PhD programme. The funding from ICAR-IARI, New Delhi, and Department of Biotechnology (DBT) through the project entitled “Enrichment of nutritional quality in maize through molecular breeding” (SAN No. BT/PR10922/AGII/106/944/2014) is duly acknowledged. We thank Dr. Natalia Palacios-Rojas, Dr. Raman Babu, and Dr. Bodupalli M. Prasanna from CIMMYT, Mexico for providing proA rich inbreds. We also thank the breeders of CCS-HAU, Uchani Centre for sharing their inbred lines.

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  1. ICAR-Indian Agricultural Research Institute, New Delhi, India

    Rajkumar Uttamrao Zunjare, Rashmi Chhabra, Firoz Hossain, Vignesh Muthusamy, Aanchal Baveja & Hari Shanker Gupta

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  1. Rajkumar Uttamrao Zunjare
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  2. Rashmi Chhabra
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  3. Firoz Hossain
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  4. Vignesh Muthusamy
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  6. Hari Shanker Gupta
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Correspondence to Firoz Hossain.

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Zunjare, R.U., Chhabra, R., Hossain, F. et al. Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of crtRB1 and lcyE governing higher accumulation of provitamin A in maize. J. Plant Biochem. Biotechnol. 27, 208–214 (2018). https://doi.org/10.1007/s13562-017-0432-8

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  • DOI: https://doi.org/10.1007/s13562-017-0432-8

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