Development and validation of multiplex-PCR assay to simultaneously detect favourable alleles of shrunken2, opaque2, crtRB1 and lcyE genes in marker-assisted selection for maize biofortification

Abstract

Sweet corn has emerged as a popular vegetable worldwide. Commercial shrunken2 (sh2)-based sweet corn lacks lysine, tryptophan and provitamin-A, deficiency of which cause various health problems. To overcome these problems, marker-assisted stacking of sh2, opaque2 (o2), β-carotene hydroxylase (crtRB1) and lycopene epsilon cyclase (lcyE) was attempted to combine nutritional quality traits in maize endosperm. Gene-based simple sequence marker (SSR) specific to o2, InDel-based marker for crtRB1 and lcyE, and SSR linked to sh2 were used in marker-assisted selection. Four backcross populations segregating for the alleles of crtRB1, lcyE, o2 and sh2 were developed. The favourable alleles of sh2 and o2 produced 103 and 150 bp amplicons, while the unfavourable alleles were 96 and 160 bp, respectively. CrtRB1 produced 543 bp favourable and 296 bp unfavourable allele, while the same for lcyE was 650 and 300 bp, respectively. To detect desirable alleles of the target genes simultaneously, multiplex-PCR (Mx-PCR) assay was developed and validated in BC2F2 populations. Mx-PCR assay successfully identified the favourable alleles of four genes simultaneously, and validated the results of individual uniplex-PCR assays. Mx-PCR assay saved 68% of cost and approximately 63% of time. This is the first report of multiplexing sh2, o2, crtRB1 and lcyE genes in maize. The information generated here would be immensely useful in molecular breeding programme.