RLGS Spot Cloning

Watanabe, Sachihiko; Hayashizaki, Yoshihide

doi:10.1007/978-4-431-67953-0_4

Cited by 2 publications

(1 citation statement)

References 8 publications

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“…RLGS spot cloning was conducted according to Takamiya et al (2008) and Watanabe & Hayashizaki (1997). Briefly, a target RLGS spot was punched out of the dried gel of the mat rush cultivar 'Shimomasuda,' which is the mater-nal parent of 'Hinomidori,' and soaked with TE (pH 8.0).…”

Section: Rlgs Spot Cloningmentioning

confidence: 99%

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

Noguchi

Hosobuchi

Takamiya

et al. 2017

JARQ

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We have developed a genetic marker that can identify a registered variety of mat rush in Japan. A vegetatively propagated plant, mat rush is cultivated and used as the material for the surface layer of tatami mats in Japan. Because it has been difficult to detect DNA polymorphism among mat rush cultivars, we applied restriction landmark genome scanning (RLGS) to discriminate mat rush cultivars. RLGS is a genome analysis technique that can detect many DNA polymorphisms as spots separated by two-dimensional electrophoresis. By cloning the DNA of spots specific to the superior mat rush cultivar 'Hinomidori' detected by RLGS, we developed a sequence-tagged site (STS) marker for polymerase chain reaction (PCR) analyses. This STS marker makes it possible to distinguish 'Hinomidori' specifically from other mat rush cultivars. The strategy of developing the STS marker in this study is applicable to other vegetatively propagated plants that are characterized by difficult DNA polymorphism detection.

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Section: Rlgs Spot Cloningmentioning

confidence: 99%

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

Noguchi

Hosobuchi

Takamiya

et al. 2017

JARQ

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Restriction landmark genome scanning method using isoschizomers (MspI/HpaII) for DNA methylation analysis

et al. 2006

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Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.

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RLGS Spot Cloning

Cited by 2 publications

References 8 publications

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

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

Restriction landmark genome scanning method using isoschizomers (MspI/HpaII) for DNA methylation analysis

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