Inactivation of DFR (Dihydroflavonol 4-reductase) gene transcription results in blockage of anthocyanin production in yellow onions (Allium cepa)

Abstract

Anthocyanin, one of the flavonoids, is a primary determinant of red color in onions. Inheritance studies indicate that a single gene determines the color difference between yellow and red onions. In order to establish which gene might be responsible for this color difference, full-length cDNAs of five structural genes: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS) were cloned using degenerate PCR and RACE (Rapid Amplification of cDNA Ends). RT-PCR was carried out for these five genes to examine differential expression between yellow and red colored bulbs. Accumulation of the DFR gene transcript only occurred in red onions. In F3 populations which originated from the cross between yellow and red parents, DFR transcript was detected only in red F3 lines. To design molecular markers for selection of yellow and red DFR alleles, the DFR gene was sequenced from genomic DNA isolated from both types of onions. The genomic DNA sequence revealed the DFR gene consists of six exons and five introns. A PCR-RFLP marker was designed based on 2% polymorphic nucleotide sequence of the DFR gene between yellow and red onions. The co-segregation of markers and red color were observed in F2 segregating populations, supporting the conclusion that color difference in red and yellow onions is likely to be due to the lack of an active DFR gene.