Identification of two novel mutant ANS alleles responsible for inactivation of anthocyanidin synthase and failure of anthocyanin production in onion (Allium cepa L.)

Abstract

Two genes (DFR-A and ANS) encoding dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) enzymes in the anthocyanin biosynthesis pathway, respectively, are complementarily involved in anthocyanin production in onion (Allium cepa L.). Eleven inactive DFR-A alleles have been reported, with only a single inactive ANS allele previously identified. Two additional inactive ANS alleles are reported in this study. A mutant ANS allele containing a 4-bp insertion at the end of exon1 was identified from yellow bulbs of the F2 population in which the DFR-A genotype was homozygous for an active allele. The 4-bp insertion caused a frame-shift mutation and resulted in creation of a premature stop codon at the start of exon2. This mutant ANS allele was designated ANS PS allele. RT-PCR results showed that transcripts of the ANS PS allele were almost undetectable in yellow F2 bulbs, implying the involvement of nonsense-mediated mRNA decay. A cleaved amplified polymorphic sequence marker was developed for detection of the ANS PS allele. Another inactive ANS allele was identified from the light-red F1 populations showing complementation between DFR-A and ANS genes. A critical amino acid change of the strictly conserved serine residue into leucine was found in this mutant allele designated ANS S188L . In addition, seven variants of active ANS alleles were identified from diverse onion germplasm. A stepwise process consisting of PCR amplification and sequencing of PCR products was devised to identify three inactive (ANS PS , ANS S188L , and ANS G229R ), one leaky (ANS P ), and two active ANS alleles (ANS L and ANS h1 ).