Genome-wide analysis of the R2R3-MYB gene family and identification of candidate genes that regulate isoflavone biosynthesis in red clover (Trifolium pratense)

Red clover (Trifolium pratense) is a perennial legume with high feeding and medicinal value attributed to its abundant isoflavone content. Previous studies reported that R2R3-MYB transcription factors are involved in the biosynthesis of isoflavones; however, their specific role in red clover remains poorly understood. Through comprehensive genome-wide and transcriptome analyses, a total of 138 TpR2R3-MYB genes were identified and classified into 30 distinct subgroups within a phylogenetic tree. Importantly, six of these subgroups showed associations with isoflavone biosynthesis in red clover. The majority of segmental duplication events (Ka/Ks < 1) indicated that the TpR2R3-MYB gene underwent strong purifying selection during evolution. The qRT-PCR analysis demonstrated high expression levels of TpMYB79 and TpMYB53 in Minshan red clover at full flowering stage, consistent with the trend for isoflavone content determination, suggesting that TpMYB79 and TpMYB53 might be important regulators of isoflavone biosynthesis in red clover. Additionally, we observed nucleus and vacuole membrane localization of TpMYB53 and TpMYB79, with TpMYB53 primarily exerting transcriptional activation through its C-terminal activation motifs while TpMYB79 exhibited no transcriptional activity. These findings provided a foundation for the study of the biological function of R2R3-MYB transcription factors in red clover.