Two mutations in the same MYC-bHLH transcription factor cause segregation of purple coloration of stolons and seed heads in Zoysia japonica x Zoysia matrella F ₂ and F ₁ populations

Anthocyanins play diverse roles in plants, including attracting pollinators and protecting cells from oxidative damage. In zoysiagrass, a warm season turfgrass, their accumulation in seed heads and stolons can decrease the aesthetic appeal. In this study, a high-density genetic map with ∼8000 single nucleotide polymorphism (SNP) markers organized into 20 linkage groups was generated in a Zoysia japonica acc. Meyer x Zoysia matrella acc. PI 231146 F ₂ population. Using this genetic map, a large-effect quantitative trait locus (QTL) for anthocyanin variation in stolons and seed heads was mapped to chromosome 12 ( PP locus). Variant analysis of a candidate gene for PP , Zjn_sc00004.1.g07010.1.sm.mk , which encodes a MYC-bHLH transcription factor that regulates anthocyanin biosynthesis, revealed a SNP at an exon-intron boundary in Meyer that led to intron retention. Interestingly, an F ₁ population derived from the same parents segregated for seed head color but uniformly displayed purple stolons. Seed head color in the F ₁ population co-mapped with the PP locus which, combined with genotypic and yeast two-hybrid analyses, revealed that a SNP in PI 231146 leading to an Ala163Ser substitution in the MYB-interacting N-terminal domain of the same MYC-bHLH transcription factor was likely causal. The Ala163Ser substitution affected interaction of MYC-bHLH with MYB in a MYB-dependent manner. The identified mutations can be exploited to develop cultivars with green seed heads and stolons. The high-marker-density interspecific Z. japonica x Z. matrella F ₂ genetic map also provides a robust tool for identifying genomic regions and genes of agronomic interest that differentiate the two species.