Bulb color in onions (Allium cepa) is an important trait and has been used as a major criterion for classifying cultivars, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study indicated that five major loci are involved in the determination of qualitative color difference in onion bulb, and that these loci are closely related to a pigment biosynthesis pathway, where the red color in onion bulb has been attributed to anthocyanin derivatives. In a previous study by these authors they showed that the lack of dihydroflavonol 4-reductase (DFR) transcription (a enzyme in the anthocyanin biosynthesis pathway) in yellow onion is responsible for the color difference between yellow and red onions. However, they were unable to develop reliable molecular markers for the selection of yellow and red DFR alleles, possibly due to the huge genome size of the onion which is 107-fold larger then that of Arabidopsis, and the existence of multiple genes with a very high degree of sequence homology.
The objectives of the present study were the identification of the critical mutation in the DFR gene (DFR-A) and the development of a PCR-based marker for allelic selection.
The researches examined three homologous onion DFR genes and the promoter region from two of these genes in an attempt to find a unique sequence that could be used to detect the DFR gene of interest. Based on the unique sequence of the promoter regions, they were able to develop a reliable co-dominant PCR-based molecular marker and subsequently used this marker to show perfect co-segregation of marker and color phenotypes in the F2 population originating from the cross between yellow and red onions.
Onion genomic DNA was used as a template for PCR amplification of the DFR gene. The result showed three different isoforms of this gene. The normally transcribed onion DFR gene labeled DFR-A, and another two homolog DFR-B and DFR-C. Both genes shared more then 95% nucleotide sequence identity with the DFR-A gene. The most conspicuous difference between the DFR-A and DFR-B gene was the length of poly-A stretch in the 5'UTR. Between the DFR-A and DFR-C gene it was a deletion of 499-bp. The researches believed the DFR-B and DFR-C genes to be a pseudo-gene since no transcripts were detected in the red onion cDNA pool.
It was possible to specifically amplify only DFR-A gene using primers designed to anneal to unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800-bp deletion within the 3' region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a deletion mutation in the yellow DFR-A gene results in the lack of anthocyanin production in yellow onions.
The PCR-based marker developed in this study is a direct marker for gene causing the different phenotypes. Molecular markers like this one, for important traits are especially useful in biennial crops such as onions in order to reduce the breeding period. In edition for efficient selection of desirable colors, the combined use of molecular markers for the major genes is required. The PCR-based marker for allelic selection of the DFR-A gene developed in this study would be the first marker for the complete system and a valuable tool in onion breeding programs.