Fine Mapping and Candidate Gene Analysis of a Major Locus Controlling Black Seed Coat Color in Mung Bean (Vigna radiata L.)

Abstract

Seed coat color is an important quality trait in mung bean (Vigna radiata) and is closely associated with seed appearance, commercial value, and phytochemical composition. To investigate the genetic basis of black seed coat formation, six F2 populations were derived from reciprocal crosses between the black-seeded accession LZL115 and the green-seeded accession LZL156. Among 755 F2 plants, 559 produced black-coated seeds and 196 produced green-coated seeds, conforming to a 3:1 segregation ratio (χ2 = 0.37, p = 0.54). These results indicated that black seed coat color was dominant and consistent with the control by a single dominant locus, designated VrScL115, in the LZL115 × LZL156 genetic background. Bulked segregant analysis sequencing (BSA-seq) initially mapped VrScL115 to an approximately 2.90 Mb region on chromosome 4. Using newly developed KASP markers and recombinant screening in expanded F2 populations, the locus was further delimited to a 121.79 kb interval between markers LS_K3333 and LS_K3379. Of the 11 annotated genes within this interval, LOC106758748 was the only gene containing high-confidence coding-sequence variants between the parents. This gene encodes a putative R2R3-MYB transcription factor homologous to MYB90. Comparative sequence analysis identified several allelic variants potentially associated with black seed coat color, and protein structure prediction indicated local structural differences between the parental proteins. LOC106758748 showed consistently higher expression in the developing seed coats of LZL115 than in those of LZL156 at 10, 15, and 20 days after pollination, with expression peaking at 15 days. Haplotype analysis of 246 mung bean accessions showed that the LZL115-associated allele combination at LS_K3352, LS_K3365, LS_K3367, and LS_K3370 was present in 21 of 27 black-seeded accessions (77.8%) and absent from all 219 non-black accessions, corresponding to a specificity of 100% and a false-negative rate of 22.2%. These findings support LOC106758748 as the leading candidate gene for VrScL115; however, direct in vivo functional validation is still required to confirm its causal role in black seed coat formation. The four-marker combination may be useful for identifying germplasm carrying the LZL115-associated allele, although further validation in independent germplasm populations is required.