Comparative Analysis between the ITS TaqMan SNP Genotyping Assay and CAPS for the Rapid Molecular Identification of Zoysia japonica and Zoysia sinica, Related Hybrid Lines, and the Habitat Distribution of Each Species

Abstract

In this study, zoysiagrasses found in a major province of the Korean peninsula were collected and identified by molecular techniques. The zoysiagrasses used in this study were collected from mountainous national parks; ‘oreum’ areas; and from inland, coastal and island areas, including marine and coastal national parks on the west, east, and south coasts. Molecular identification was done by means of a cleaved amplified polymorphic sequence (CAPS) marker analysis based on the nrDNA-internal transcribed spacer (ITS) method established by our research team. Additionally, in this study the ITS-based TaqMan single-nucleotide polymorphism (SNP) genotyping assay was developed and used to perform molecular identification more rapidly and accurately. In total, 450 collected zoysiagrass plants were classified into three lines (234 Zoysia japonica plants, 62 Zoysia sinica plants, and 154 Z. japonica and Z. sinica hybrid plants) by molecular identification based on the ITS-based CAPS marker analysis and ITS-based TaqMan SNP genotyping assays. The results of the ITS-based TaqMan SNP genotyping assay were consistent with those of the ITS-based CAPS marker analysis. Both the ITS-based CAPS marker analysis and the ITS-based TaqMan SNP genotyping assay were found to be efficient molecular methods to identify the three species of zoysiagrasses. Specifically, the ITS-based TaqMan SNP genotyping assay method developed in this study produced results more rapidly compared to the ITS-based CAPS marker analysis method. The habitat distributions of each of the molecularly identified zoysiagrass plants investigated in this study were also assessed. The Z. japonica plants were mainly distributed in mountainous national parks and oreum areas as well as inland and coastal areas. The Z. sinica plants were distributed along the coast and on rocks and stones frequently submerged in seawater. The Z. japonica and Z. sinica hybrid plants were distributed more in the inland, coastal, and island areas than in the mountainous national parks and oreum areas. Findings related to the leaf blade width as a main external morphological classification characteristic of the molecularly identified 450 zoysiagrass plants are described below. The leaf blade width ranged from 4 to 5 mm for the Z. japonica plants, from 2 to 4 mm for the Z. sinica plants, and from 3 to 4 mm for the Z. japonica and Z. sinica hybrid plants. Additionally, some Z. japonica and Z. sinica plants and Z. japonica and Z. sinica hybrid plants with leaf blade widths of 2 to 3, 4 to 5, and from 2 to 3 and 4 to 5 mm, respectively, were also present. Because the ranges of the leaf blade width of each species overlapped, it was difficult to identify the hybrid lines using this metric. The feasibility of the developed molecular identification method for these three species of zoysiagrasses was confirmed in this study.