Two mutations in the same MYC-bHLH transcription factor cause segregation of purple coloration of stolons and seed heads in <i>Zoysia japonica</i> x <i>Zoysia matrella</i> F2 and F1 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 <i>Zoysia japonica</i> acc. Meyer × <i>Zoysia matrella</i> acc. PI 231146 F₂ 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 (<i>PP</i> locus). Variant analysis of a candidate gene for <i>PP</i>, <i>Zjn_sc00004.1.g07010.1.sm.mk</i>, which encodes an 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 F₁ population derived from the same parents segregated for seed head color but uniformly displayed purple stolons. Seed head color in the F₁ population comapped with the <i>PP</i> 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 an MYB-dependent manner. The identified mutations can be exploited to develop cultivars with green seed heads and stolons. The high-marker-density interspecific <i>Z. japonica</i> × <i>Z. matrella</i> F₂ genetic map also provides a robust tool for identifying genomic regions and genes of agronomic interest that differentiate the two species.