Development of novel gene-based markers for waxy1 gene and their validation for exploitation in molecular breeding for enhancement of amylopectin in maize

Waxy corn possessing high amylopectin is widely employed as an industrial product. Traditional corn contains ~ 70–75% amylopectin, whereas waxy corn with the mutant waxy1 (wx1) gene possesses ~ 95–100% amylopectin. Marker-assisted breeding can greatly hasten the transfer of the wx1 allele into normal corn. However, the available gene-based marker(s) for wx1 are not always polymorphic between recipient and donor parents, thereby causing a considerable delay in the molecular breeding program. Here, a 4800 bp sequence of the wx1 gene was analyzed among seven wild-type and seven mutant inbreds employing 16 overlapping primers. Three polymorphisms viz., 4 bp InDel (at position 2406 bp) in intron-7 and two SNPs (C to A at position 3325 bp in exon-10 and G to T at position 4310 bp in exon-13) differentiated the dominant (Wx1) and recessive (wx1) allele. Three breeder-friendly PCR markers (WxDel4, SNP3325_CT1, and SNP4310_GT2) specific to InDel and SNPs were developed. WxDel4 amplified 94 bp among mutant-type inbreds, while 90 bp was amplified among wild-type inbreds. SNP3325_CT1 and SNP4310_GT2 revealed the presence-absence polymorphisms with an amplification of 185 bp and 189 bp of amplicon, respectively. These newly developed markers showed 1:1 segregation in both BC1F1 and BC2F1 generations, while 1:2:1 segregation was observed in BC2F2. The recessive homozygotes (wx1wx1) of BC2F2 identified by the markers possessed significantly higher amylopectin (97.7%) compared to the original inbreds (Wx1Wx1: 72.7% amylopectin). This is the first report of novel wx1 gene-based markers. The information generated here would help in accelerating the development of waxy maize hybrids.