Two mutations in the same MYC-bHLH transcription factor cause segregation of purple coloration of stolons and seed heads in Zoysia japonica ×  Zoysia matrella F₂ and F₁ populations

Abstract

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 ×  Zoysia matrella 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 (PP locus). Variant analysis of a candidate gene for PP, Zjn_sc00004.1.g07010.1.sm.mk, 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 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 an 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 ×  Z. matrella F₂ genetic map also provides a robust tool for identifying genomic regions and genes of agronomic interest that differentiate the two species.

Acknowledgements

This research was supported by award #1915919 from the National Science Foundation Plant Genome Research Program to K.M.D., and award 2019-51181-30472 from the National Institute of Food and Agriculture (NIFA)—Speciality Crop Research Initiative (SCRI) to K.M.D., S.M-.L., and B.M.S. We thank J. Ajello, C. Greene, G. Sidhu, and G. Pillai for assistance with tissue collection and phenotyping.

Author contributions

S.P., J.J.S., and K.M.D. designed the experiments. S.P. conducted the GBS analyses, generated the genetic maps, conducted most of the phenotyping, carried out the QTL analyses and sequenced the parents. J.Z. conducted the Y2H experiments. E.M.B. conducted the PCR analyses demonstrating the presence of two independent mutations in MYC-bHLH. B.M.S. generated the Meyer × PI 231146 F₁ and F₂ populations. J.C. validated the F₁ progeny. X.Y. and S.M-.L. generated the GBS data for the F₁ population. S.K. scored the F₁ and F₂ populations in the Tifton nursery for seed head color. K.M.D. assisted with data interpretation. S.P. and K.M.D. co-wrote the manuscript. All authors edited and approved the manuscript.

Data availability

Data supporting the findings of this work are presented within the main text, and as Supplementary Tables, Figures, and Files. The whole-genome shotgun Illumina reads for Z. japonica acc. Meyer, Z. matrella acc. PI 231146 and F₁-19-TZ-14321, and the GBS Illumina reads for Meyer, PI 231146 and the 530 F₂ progeny used in the generation of the genetic map have been deposited to NCBI-SRA (Project PRJNA1235172).

Conflict of interest statement

The authors have no conflict of interest to declare.

Supplementary data

Supplementary data is available at Horticulture Research online.

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