The allelic variation of anthocyanidin reductase underlies anthocyanin biosynthesis and purple leaf trait in Brassica napus.

key messageBnaC04.ANR, encoding anthocyanidin reductase, was identified as a novel candidate gene conferring the purple leaf trait in Brassica napus. This mutation reduces the content of epicatechins, disrupts proanthocyanidin polymerization, and leads to significant accumulation of anthocyanins. Purple leaves are rich in health-protecting anthocyanins and provide ornamental value and may enhance stress resistance, but the causal genes and molecular mechanisms underlying purple leaves remain largely unknown in Brassica napus. In the current study, a novel rapeseed mutant CL620 with an extreme deep purple leaf color was identified, displaying a total anthocyanin content 54.7-fold higher than that of the green leaf cultivar ZhongShuang11. Using bulked segregant analysis (BSA)-based gene mapping and kompetitive allele-specific PCR (KASP) markers, a single dominant anthocyanidin reductase gene, BnaC04.ANR, was verified to confer the purple leaf. Gene cloning and sequence comparison detected three exon substitutions, among which only two caused non-synonymous mutations and resulted in two amino acid changes of Q to R (position 112) and K to E (position 332) between purple and green leaf parental lines. These substitutions greatly reduced the content of epicatechins, a downstream product of BnaC04.ANR, and interfered with the polymerization of proanthocyanidins. Transcriptome profiling and co-expression network uncovered the entire anthocyanin synthesis pathway were further enhanced, with 38 upregulating genes in the purple mutant, and two key regulators of BnaC09.TT8 and BnaA05.MYB5 in MYB-bHLH-WD40 complexes were directly connected with BnaC04.ANR. These changes ultimately led to the accumulation of excess anthocyanins in vacuoles, ultimately producing purple leaves. Additionally, a KASP marker based on the SNPs was successfully developed and further validated for the alleles of BnaC04.ANR. Our findings demonstrate for the first time that BnaC04.ANR regulates anthocyanin accumulation in B. napus, and provide new insights into leaf color production and breeding novel dual-purpose rapeseed varieties for oil production and vegetable use.