Sequence characterization of T, Bip, and Phbw demonstrates the role of MYB-bHLH-WD40 complexes and temperature in common bean seed color pattern formation

Seed colors and color patterns are critical for the survival of wild plants and the consumer appeal of crops. In common bean, a major global staple, these patterns are also critical for determining market classes, yet the genetic and environmental control of many pigmentation patterns remains unresolved. In this study, we genetically mapped variation for three important seed pattern loci, T , Bip , and phbw , which co-segregated completely with PvTTG1 , PvMYC1 , and PvTT8 , respectively. Proteins encoded by these genes are predicted to work together in MYB-bHLH-WD40 (MBW) complexes, propagating flavonoid biosynthesis across the seed coat. Whole-genome sequencing of 37 diverse accessions identified putative mutations in each gene, including seven unique parallel mutations in T ( PvTTG1 ) and a non-synonymous SNP in a conserved residue in bipana ( PvMYC1 ). A 612 bp intron deletion in phbw (PvTT8) eliminated motifs conserved since the origins of the Papilionoidea and corresponded to a 20-fold reduction in transcript abundance. Mutations in MBW candidate genes for Z ( PvTT2 ) and Sellatus (WDR) were also identified. In multi-location field trials with seven varieties with partial seed coat patterning, pigmented seed coat area was highly unstable and correlated with temperature, with up to 11-fold differences in pigmented area between the warmest and the coolest environments. In controlled growth chamber conditions, an increase of 4 °C was sufficient to cause pigmentation on an additional 21% of the seed coat area. Our results shed light on the fundamental activation of flavonoid biosynthesis in common bean and will be instrumental for maximizing consumer appeal in this nutritious staple crop. Summary - Seed colors and patterns are critical for the survival of wild plants, and are important in differentiating crop market classes, but the genetic control of these in the staple crop common bean ( Phaseolus vulgaris ) is largely unknown. - The genetic, transcriptional, and environmental basis of common bean seed color patterning was explored through QTL mapping, whole-genome sequencing, RT-qPCR, and automated pigmentation quantification of seed grown in multi-location field trials and growth chamber environments. - MYB-bHLH-WD40 complex-forming genes PvTTG1 , PvMYC1 , and PvTT8 co-segregated completely with the color patterning genes T , Bip , and phbw . Mutations were identified in each gene, including seven unique parallel mutations in T ( PvTTG1 ), a non-synonymous SNP in a conserved residue in bipana ( PvMYC1 ), and an intron deletion in phbw (PvTT8) eliminating highly conserved motifs and corresponding to 20-fold lower PvTT8 transcript abundance. Mutations in MBW candidate genes Z ( PvTT2 ) and Sellatus (WDR) were also identified. In multi-location field trials, pigmented seed coat area was highly unstable and corresponded to temperature. In growth chamber conditions, an increase of 4 °C caused pigmentation on an additional 21% of the seed coat area. - Our results highlight the critical interaction between MYB-bHLH-WD40 complex components and temperature in establishing seed pattern diversity. ### Competing Interest Statement The authors have declared no competing interest.