Genomic structural variation underlies cell type-specific betacyanin variegation in Chenopodium quinoa

The allotetraploid crop quinoa (Chenopodium quinoa) accumulates red/violet betacyanins, which function as vital stress-mitigating antioxidants. We investigated the genetic basis of red/green variegation observed in the aerial organs of the P0429 accession. We demonstrated that this color mosaic is primarily localized to epidermal bladder cells (EBCs), with red EBCs accumulating betacyanin levels ~ 50-fold higher than colorless EBCs. Cell-type-specific RNA-sequencing of EBCs identified the cytochrome P450 gene Cqu0091301 (CYP76ADα) as the dominant and rate-limiting factor, exhibiting strong upregulation in red EBCs. This high pigmentation requires a specific structural variation in the P0429 accession: a ~ 4-kb genomic insertion that restores the full functionality of Cqu0091301, which is otherwise truncated and non-functional in common reference genomes. Genomic analysis reveals that Cqu0091301 is part of a multicopy CYP76ADα-DODA gene cluster. Notably, expression analysis revealed functional divergence between the quinoa subgenomes, with B-subgenome CYP76ADα genes highly dominant in EBCs, while A-subgenome homologs were preferentially expressed in other tissues. Our results establish a clear link between structural genomic variation and cell-type-specific betalain biosynthesis, providing molecular insight into pigment regulation and subgenome specialization in allotetraploid quinoa.