Multivariate colorimetric phenotyping reveals genetic loci associated with soybean seed coat pigmentation and epicatechin accumulation

Seed coat pigmentation in soybean is controlled by complex genetic mechanisms involving structural and regulatory genes in the flavonoid biosynthetic pathway. Although brown seed coats are often associated with epicatechin (EC) accumulation, visual classification alone cannot reliably predict EC content. To quantitatively characterize seed coat coloration and its relationship with EC accumulation, we evaluated multivariate colorimetric traits (L*, a*, and b* values in the CIELAB color space) in 235 recombinant inbred lines (RILs) derived from Jinpung (yellow seed coat) and IT109098 (greenish-brown seed coat). Principal component analysis (PCA) of L*, a*, and b* values revealed that genotypes with detectable EC were confined to specific regions of the multivariate color space, indicating that EC accumulation is associated with coordinated color balance rather than overall pigmentation intensity. RILs with high EC content showed significantly lower L* (32.76 ± 2.49) and b* (13.18 ± 2.66) values and higher a* values (5.47 ± 1.31) than those with low EC content. Quantitative trait loci (QTL) mapping identified thirteen loci associated with L*, a*, b*, and principal component scores across chromosomes 01, 05, 06, 08, and 19 A major locus on chromosome 08 near the classical I locus explained a large proportion of phenotypic variance in pigmentation traits. In addition, loci on chromosomes 06 and 19 were associated with integrated color components, suggesting quantitative modulation of EC accumulation. Candidate genes within these regions included flavonoid 3′-hydroxylase and transcription factors such as MYB117, MYB60, and TCP5, supported by sequence variation and differential expression analyses. These findings demonstrate that multivariate colorimetric traits provide a useful phenotyping framework for dissecting seed coat pigmentation and EC accumulation and for pre-selecting high-EC soybean lines.