nrDNA-ITS 염기서열 분석 및 CAPS marker를 이용한 Zoysia 속 들잔디와 갯잔디의 분자생물학적 동정

Abstract

Zoysia plants are important turf grasses used for school playgrounds, parks, roadsides, golf courses, and athletic fields. Especially, Zoysia japonica and Zoysia sinica species growing near the beaches are not readily identifiable due to their similar morphology. It is thus desirable to have both morphological and molecular identifications. DNA barcoding using specific gene or non-coding DNA as barcode is one of the molecular identification methods for cryptic species. ITS (Internal Transcribed Spacer) is a non-coding DNA involved in nuclear ribosomal DNA, and is a DNA barcode popularly used because of more frequent evolutionary changes at the DNA level than other DNA barcode regions in plastid. In this study, we collected 97 Zoysia turf grasses and classified into 66 Zoysia japonica and 31 Zoysia sinica species according to morphological standards for identification. To classify them based on the molecular method, the nrDNA-ITS regions including ITS1, 5.8S nrDNA, and ITS2 from four different grass species (Zoysia japonica, Zoysia sinica, Agrostis stolonifera, and Poa pratensis) were used for DNA barcoding. The substitutions and InDel (Insert/Deletion) sites in the nrDNA-ITS sequence of Z. japonica and Z. sinica were converted into the restriction endonuclease sites (BglI, StyI, and FauI) for CAPS (Cleaved Amplified Polymorphic Sequence) markers, and applied to collected the Zoysia grasses to verify whether the markers are presenting both seed and plant samples. We successfully identified 43 Zoysia japonica, 25 Zoysia sinica, and 29 hybrids between Zoysia japonica and Zoysia sinica among the 97 collected grass samples. These results suggest that Zoysia japonica and Zoysia sinica can be identified at the species level using nrDNA-ITS barcode region and CAPS markers.