Identification of a candidate gene responsible for the G locus in onion
A gene encoding a laccase enzyme was proposed as a strong candidate gene for the G locus controlling the chartreuse bulb color in this study. Since Agrobacterium-mediated genetic transformation is hardly applicable in onion, a complementation test using genetic transformation could not be performed to prove that AcLAC12 was responsible for the chartreuse bulb color. However, several evidences presented in this study supported a role of AcLAC12 in the appearance of the chartreuse bulb color. First, a perfect linkage between LAC12 markers and G locus in segregating populations and diverse accessions was demonstrated in this study. Perfect linkage disequilibrium among diverse breeding lines implies that no recombinant has been produced during breeding periods of such accessions and that both AcLAC12 and G loci might be identical to each other.
Second, chartreuse alleles of AcLAC12 harbored critical mutations leading to complete inactivation of gene products. The 4-bp deletion in exon5 was a frame-shift mutation that created a premature stop codon before two highly conserved copper-binding domains, L3 and L4 (Fig. 3A). Significantly reduced transcripts of AcLAC12 in chartreuse onions were probably caused by a mechanism of nonsense-mediated RNA decay (NMD). Since 2.5 kb putative promoter regions showed no significant polymorphic sequence between yellow and chartreuse alleles, reduced transcripts might be resulted from degradation of mRNA by the NMD mechanism. NMD is conserved among eukaryotes. It plays an essential role in maintaining the quality of transcriptome by eliminating mutant transcripts containing premature stop codons (Nickless et al. 2017).
Another novel mutant AcLAC12 allele harboring an LTR-retrotransposon was identified from nine chartreuse breeding lines. Since the 6,449-bp LTR-retrotransposon was transposed at the upstream sequence of a 4-bp deletion in exon 5 (Fig. 3A), this mutant allele must be inactive as well. Nucleotide sequences of 954-bp LTRs were identical to each other. A 3,891-bp intact ORF encoded a polyprotein containing three major domains (GAG, INT, and RT) of LTR-retrotransposons. These features imply that this LTR-retrotransposon might be active and recently transposed into the AcLAC12 gene. Organization of integrase (INT) and reverse transcriptase (RT) domains indicated that this element belonged to Copia superfamily (Wicker et al. 2007). Interestingly, an unusually long 9-bp target site duplication (TSD) was identified in this LTR-retrotransposon. In general, the length of TSD ranges from four to six base pairs (Wicker et al. 2007).
Underlying relationship between inactivation of AcLAC12 and appearance of chartreuse bulb color in onion
Laccase, a member of the multicopper oxidase superfamily, is widely distributed in all kingdoms of life ranging from bacteria to mammals. Although laccase was first discovered from Japanese lacquer tree almost 138 years ago, its precise biochemical and physiological roles in plants remain mostly unresolved. Their wide taxonomic distribution and broad substrate specificity might be obstacles to resolve precise roles of laccases (Turlapati et al. 2011; Janusz et al. 2020). Plant laccases are known to be involved in diverse processes such as lignification, defense responses, wound healing, iron metabolism, and polymerization of phenolic compounds (Janusz et al. 2020).
Seventeen genes coding for laccases were found in Arabidopsis genome. However, physiological functions of only two genes (AtLAC3 and AtLAC15) have been elucidated (Pourcel et al. 2005; Zhuang et al. 2020). Regarding mutants of AtLAC12 showing the highest homology with AcLAC12, no apparent mutant phenotype was observed (Cai et al. 2006). In the case of AtLAC3, this gene is known to be involved in directing Casparian strip formation in Arabidopsis (Zhuang et al. 2020). Casparian strip is a ring-like water-impermeable lignin polymer positioned in the endodermis of root (Barbosa et al. 2019). Meanwhile, Arabidopsis AtLAC15 gene was revealed to be the causal gene for transparent testa10 (tt10) mutant showing delayed browning of seed coats. The AtLAC15 gene has been shown to be involved in oxidative polymerization of flavonoids (Pourcel et al. 2005). Likewise, AcLAC12 might be involved in the modification of flavonoid derivatives in onions since laccases have a broad spectrum of substrate specificity (Fig. 1).
The G locus determining chartreuse bulb color in onion was first proposed by El-Shafie and Davis (1967). They hypothesized that the G locus might be involved in sequential pigment synthesis pathway. It acted after the C locus and before the R (DFR-A) locus based on the fact that chartreuse color was dominant over recessive white and recessive to yellow. However, since quercetin derivatives were detected in chartreuse onions (Fig. 7), genes coding for enzymes from chalcone synthase (CHS) to flavonol synthase (FLS) should be active in chartreuse onions. Therefore, the G locus is likely to be involved in later steps than FLS in the flavonoid biosynthesis pathway (Fig. 1).
If we assume that chartreuse color would appear only when both R and G loci are homozygous recessive, inheritance patterns of chartreuse color could be clearly explained (Fig. 1). Indeed, all chartreuse breeding lines analyzed in this study contained four different kinds of inactive DFR-A alleles (Supplementary Table 1). The AcLAC12 gene might be involved in the modification of quercetin derivatives. Since Arabidopsis AtLAC15 catalyzes oxidative polymerization of flavonoids, AcLAC12 might also catalyze polymerization of quercetin derivatives (Fig. 1), although polymers of flavonoid compounds in onions have not been reported yet. Involvement of a laccase enzyme in the production of onion flavonoids will provide an important clue to resolve precise compounds responsible for chartreuse and yellow bulb colors. Furthermore, chartreuse onion will be a precious material to elucidate the role of laccase enzyme in plant pigmentation.
Application of molecular markers linked to the G locus in onion breeding programs
Compared with white, yellow, and red bulb colors, chartreuse bulb color has been rarely used to produce commercial cultivars. The complex inheritance of the G locus might have partly contributed to the rare usage of chartreuse bulb color. Linkage between the G and R loci might also complicate inheritance. We showed that the R (DFR-A) locus was linked to the G locus in the same chromosome (Fig. 4). Information about the chromosomal location of the G locus and molecular markers linked to the G locus will be useful for developing commercial chartreuse cultivars in onion breeding programs. In particular, if AcLAC12 is the genuine causal gene for the G locus, LAC12 markers would be functional markers developed based on critical mutations (Andersen and Lübberstedt 2003). Therefore, prediction of AcLAC12 genotypes could be accurate without any errors.