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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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.
KeywordsProvitamin A Multiplex-PCR Uniplex-PCR Marker-assisted selection
Polymerase chain reaction
Vitamin A deficiency
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.
Compliance with ethical standards
Conflict of interest
Authors declare that they have no conflict of interest.
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