Bulked sequence analysis identifies two loci controlling anthocyanin accumulation in a red-purple cultivar of Japanese leaf vegetable komatsuna (Brassica rapa L.)

We investigated leaf color and anthocyanin content in cross-progeny of two Japanese Brassica rapa crops, a red-purple cultivar of komatsuna (Japanese mustard spinach) and a green cultivar of nabana (young flowering stems of Brassica eaten as a vegetable). The phenotype of all F1 progeny was intermediate between the parents. High-performance liquid chromatography analysis revealed the similarity of the anthocyanin compositions between the parent komatsuna and the F1 plants, indicating that the shade of purple depends on the total amount of anthocyanins. One hundred and one F2 plants segregated into green and purple-tinted phenotypes at a ratio of 43:58, suggesting the presence of at least two loci controlling anthocyanin accumulation in the komatsuna parent. Bulked sequence analysis of genomic DNA bulked by F2 color identified two loci controlling anthocyanin accumulation around 23 Mb on chromosome A07 (a07-23) and 45 Mb on A09 (a09-45) within the B. rapa reference genome v. 3.0. Genotyping by DNA markers flanking the loci indicated that a07-23 from the red-purple komatsuna is likely identical to a known locus harboring several MYB genes involved in the anthocyanin biosynthesis pathway in pigmented B. rapa vegetables, and that a09-45 is a minor locus complementarily working under certain genetic backgrounds. Comparative RNA-seq analysis between red-purple komatsuna and green nabana cultivars confirmed that many anthocyanin genes, including two MYB genes within the a07-23 region, were upregulated in the red-purple cultivar, suggesting that these are responsible for constitutive anthocyanin accumulation in the pigmented B. rapa vegetable.