Zoysia japonica, Z. matrella and Z. pacifica are native turfgrass species in Japan and are the most economically important species in the Zoysia genus. To characterize the genetic systems of Zoysia and to analyze their structural and functional differences in individual species and accessions, we characterized morphological traits and determined genetic variation and population structure among our collected 248 Zoysia accessions using microsatellite markers. Based on the resulted genotypes, we detected high genetic diversity among the accessions. Moreover, both PCoA and Bayesian-based Structure analysis revealed two distinct populations represented by Z. japonica and Z. pacifica, while both ancestries were found in Z. matrella. On the other hand, we sequenced the genomes of each representative Zoysia species using NGS platforms. We generated a high-quality draft sequence of the genome of Z. japonica ‘Nagirizaki’ (334 Mb) as a reference sequence for Zoysia species. In parallel, draft genome sequences of Z. matrella ‘Wakaba’ and Z. pacifica ‘Zanpa’ were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions were accumulated, and aligned against the reference genome of ‘Nagirizaki’. As a result, we detected single-nucleotide polymorphisms and short indels among these species. Most of the SNPs and polymorphic indels among the three Zoysia species are between Z. japonica and Z. pacifica, and these sites are often heterozygous in Z. matrella. Based on these microsatellite and genome-wide studies, we suggest that Z. matrella might represent an interspecific hybrid between Z. japonica and Z. pacifica.
