Identification of a candidate gene responsible for the G locus determining chartreuse bulb color in onion (Allium cepa L.) using bulked segregant RNA-Seq.

KEY MESSAGE: A gene encoding a laccase responsible for chartreuse onion bulb color was identified. Markers tagging this gene showed perfect linkage with bulb colors among diverse germplasm. To identify a casual gene for the G locus determining chartreuse bulb color in onion (Allium cepa L.), bulked segregant RNA-Seq (BSR-Seq) was performed using yellow and chartreuse individuals of a segregating population. Through single nucleotide polymorphism (SNP) and differentially expressed gene (DEG) screening processes, 163 and 143 transcripts were selected, respectively. One transcript encoding a laccase-like protein was commonly identified from SNP and DEG screening. This transcript contained four highly conserved copper-binding domains known to be signature sequences of laccases. This gene was designated AcLAC12 since it showed high homology with Arabidopsis AtLAC12. A 4-bp deletion creating a premature stop codon was identified in exon 5 of the chartreuse allele. Another mutant allele in which an intact LTR-retrotransposon was transposed in exon 5 was identified from other chartreuse breeding lines. Genotypes of molecular markers tagging AcLAC12 were perfectly matched with bulb color phenotypes in segregating populations and diverse breeding lines. All chartreuse breeding lines contained inactive alleles of DFR-A gene determining red bulb color, indicating that chartreuse color appeared when both DFR-A and AcLAC12 genes were inactivated. Linkage maps showed that AcLAC12 was positioned at the end of chromosome 7. Transcription levels of structural genes encoding enzymes in anthocyanin biosynthesis pathway were generally reduced in chartreuse bulk compared with yellow bulk. Concentrations of total quercetins were also reduced in chartreuse onion. However, significant amounts of quercetins were detected in chartreuse onion, implying that AcLAC12 might be involved in modification of quercetin derivatives in onion.