Identification of two novel inactive DFR-A alleles responsible for failure to produce anthocyanin and development of a simple PCR-based molecular marker for bulb color selection in onion (Allium cepa L.)

Abstract

Two novel inactive alleles of Dihydroflavonol 4-reductase-A (DFR-A) were identified in yellow onion (Allium cepa L.) cultivars and breeding lines from Korea and Japan. Unlike the previously reported inactive yellow DFR-A allele, designated as DFR-A ( TRN ) , in which the 3' portion of the coding sequences was deleted, an allele containing a premature stop codon, DFR-A ( PS ) , was isolated from the majority of cultivars. Co-segregation of DFR-A ( PS ) and color phenotypes in the F(2) population from a cross between yellow and red parents showed that inactivation of DFR-A was responsible for lack of anthocyanin in these yellow onions. In addition, RT-PCR analysis of F(2) population showed that the transcription level of the DFR-A ( PS ) allele was significantly reduced owing to non-sense-mediated mRNA decay. A 20-bp deletion of a simple sequence repeat in the promoter region of the DFR-A ( PS ) allele was used to develop a simple PCR-based molecular marker for selection of the DFR-A ( PS ) allele. All genotypes of 138 F(2) individuals were clearly distinguished by this molecular marker. In addition to the DFR-A ( PS ) allele, another DFR-A allele, DFR-A ( DEL ) , was identified in some cultivars. In case of the DFR-A ( DEL ) allele, no PCR products were amplified throughout DFR-A sequences including promoter regions, suggesting deletion of the entire DFR-A gene. Co-segregation of the absence of DFR-A and color phenotypes was confirmed in another F(2) population. Furthermore, RT-PCR results showed that no DFR-A transcript was detected in any yellow F(2) individuals.

Identification of a Novel Mutant <i>pAMT</i> Allele Responsible for Low-pungency and Capsinoid Production in Chili Pepper: Accession ‘No. 4034’ (<i>Capsicum chinense</i>)

“…The mechanism exists in all eukaryotes, and the involvement of NMD in the reduction of gene expression in plants also has also reported in inactive alleles of anthocyanin biosynthetic genes. The expression level of inactive anthocyanin biosynthetic gene alleles containing premature stop codons was significantly reduced in the morning glory and onion (Hoshino et al, 2003;Kim et al, 2009).…”

Development of Functional Markers for Detection of Inactive DFR-A Alleles Responsible for Failure of Anthocyanin Production in Onions (Allium cepa L.)

“…Two F 2 populations originating from the crosses between red and yellow breeding lines were selected from 11 F 2 populations produced in a previous study (Kim et al, 2009 …”

“…, harboring a premature stop codon was identified from yellow onion cultivars bred in Korea and Japan in a previous study (Kim et al, 2009). A 20-bp deletion in the 5' non-coding region was initially used for detection of the DFR-A PS allele, but this 20-bp deletion in the DFR-A PS allele was also found in another active DFR-A allele (GenBank accession: AY221250) identified from several red onion breeding lines or cultivars (data not shown).…”

“…The third inactive DFR-A allele in which the entire DFR-A coding region might be deleted was previously reported (Kim et al, 2009). To obtain more extended flanking sequences of the DFR-A coding regions, multiple cycles of genome walking were carried out, and an 839-bp additional 5' end sequence of the previously reported DFR-A sequence (AY221250) and 2,642-bp 3' end sequences were newly obtained.…”

“…Because of complete linkage between polymorphic sequence motifs and phenotypic variations, a functional marker is the most ideal marker to be utilized in marker-assisted selection. On the contrary, the previously developed molecular marker for detection of the DFR-A PS allele developed on the basis of the 20-bp indel located at the 5' nod-coding sequence (Kim et al, 2009) is considered to be a gene-targeted marker according to the definition by Andersen and Lübberstedt (2003). Despite the tight linkage between markers and mutations affecting phenotypic variation, a gene-targeted marker has limitations compared with a functional marker.…”