Development of a New Cultivar-Discrimination Method Based on DNA Polymorphism in a Vegetatively Propagated Crop

Abstract

Restriction landmark genome scanning (RLGS) is a high-speed genome analysis method to detect DNA polymorphism, and is suitable to develop useful DNA markers for cultivar-discrimination of agricultural plants. The mat-rush, Juncus effusus is a vegetatively propagated crop that has been cultivated for over 500 years in Japan as the material of "tatami-omote," a surface of Japanese conventional mats. In recent years, Japanese mat-rush cultivars are grown in foreign countries and reimported to Japan in violation of the Seeds and Seedlings Law, economically damaging domestic production. The largest problem of illegal mat-rush reimportation has been with cv. Hinomidori. We developed a cultivar-discrimination DNA marker of mat-rush using RLGS, and detected the RLGS spot markers that didn't exist specifically in Hinomidori. The spot marker can be used with the RLGS method to identify Hinomidori accurately among cultivars.

A Sequence-tagged Site Marker for Identifying the Japanese Mat Rush (<i>Juncus effusus</i>) Cultivar ‘Hinomidori’

“…Eighteen Japanese mat rush (J. effusus) cultivars including 'Hinomidori' were analyzed in the present study as well as in our previous study (Okamoto et al 2006). The relationships between the cultivars ('Shimomasuda,' 'Hinomidori,' 'Senchou,' 'Asanagi,' 'Setonami,' 'Fukunami,' 'Bunsei,' 'Isonami,' 'Kiyonami,' 'Chikugomidori,' 'Ohara-4,' 'Okayama-3,' 'Kumagawa,' 'Takasu,' 'Sazanami,' 'Shiranui,' 'Okayama F,' and 'Okayamamidori') are described in Okamoto et al (2006).…”

“…The RLGS method is understood to be effective for the detection of polymorphism in vegetatively propagated plants that have either not been subjected to advanced genome analysis or have very low genetic diversity (Jacob 1996). We applied the RLGS method to mat rush (Okamoto et al 2006) and detected polymorphic spots among 18 cultivars, which are well known as genetic resources in Japan.…”

“…In the present study, we aimed to develop a cultivar identification STS marker specific to 'Hinomidori' using the polymorphic spot "A2" detected by the RLGS method (hereafter, "spot A2") to distinguish it among other mat rush cultivars (Okamoto et al 2006). …”

Application of RLGS Method for Detection of Alteration in Tissue Cultured Plants

“…Briefly, the genomic DNA (0.1 µg) was digested by NotI and HpaII at 37ºC for 1 h. Then, the sample was mixed with DNA fragments labeled at the NotI sites and digested with HpaII. Thereafter, 2-D electrophoresis was carried out as described by Okamoto et al 11 and Takamiya et al 12 . The target RLGS spots were punched out from the dried gel and soaked in Tris-EDTA (TE) buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).…”

“…We carried out RLGS with restriction enzyme combination, NotI-HpaII-BamHI (NEB, Beverly, MA, USA), described in Okamoto et al 11 and Takamiya et al 12 .…”