Diversity in flower colorations of Ranunculus asiaticus L. revealed by anthocyanin biosynthesis pathway in view of gene composition, gene expression patterns, and color phenotype.

Anthocyanin biosynthesis is one of the best studied secondary metabolisms. However, related pathways were generally concluded based on anthocyanin components; most studies focused on the backbone forming of anthocyanidins (cyanidin, delphinidin, and pelargonidin) of model or commercial plants, while anthocyanin modification was less discussed, and non-model plants with abundant colorations were less researched either. Ranunculus asiaticus L. has great diversity in flower colorations, not only indicating its value in researching anthocyanin biosynthesis but also implying it is unique in this regard. Based on transcriptome sequencing and gene annotation of three varieties (10 samples) of Ranunculus asiaticus L., 176 unigenes from 151,136 unigenes were identified as involved in anthocyanin biosynthesis, among which, 74 unigenes were related to anthocyanin modification; 61 unigenes were responsible for glycosylation at C3 and C5 with 3-monosaccharides of glucose, 3-biosides of rutinose, sophorose, or sambubiose to form 3Gly-, 3Gly5Gly-, 3Gly3'Gly-, 3Gly2''Gly-, 3Gly2''Xly-, 3Gly2''Rly-glycosylated anthocyanins, etc.; 2 unigenes transferred -CH; 11 unigenes of BAHD family catalyzd the aromatic or malonyl acylation at 6'' / 6''''position of 3/5-O-glucoside. Based on gene composition, a putative pathway was established. The pathway was validated by flower colorations, and gene expression patterns where F3H, F3'H, 3GT, 5GT, and FMT2 were highly expressed in varieties colored as lateritious and carmine, while variety with purple flowers had high expression of F3'5'H and 3MAT. In view of anthocyanin biosynthesis pathway of Ranunculus asiaticus L., great diversity in its flower colorations was illustrated via the complete branches (F3H, F3'H and F3'5'H) as well as complete modifications (glycosylation, methylation, and acylation), and besides, via the higher percentage of C3 glycosylation than C5 glycosylation.