[논문]Development of Gene Based STS Markers in Wheat

Lee, Sang-Kyu; Heo, Hwa-Young; Kwon, Young-Up; Lee, Byung-Moo

doi:10.7740/kjcs.2012.57.1.071

Development of Gene Based STS Markers in Wheat 원문보기

Korean journal of crop science = 韓國作物學會誌, v.57 no.1, 2012년, pp.71 - 77

Lee, Sang-Kyu (Department of Plant Biotechnology, Dongguk University-Seoul) , Heo, Hwa-Young (Department of Plant Sciences and Plant Pathology, Montana State University-Bozeman) , Kwon, Young-Up (National Institute of Crop Science, RDA) , Lee, Byung-Moo (Department of Plant Biotechnology, Dongguk University-Seoul)

Abstract ▼ AI-Helper

The objective of this study is to develop the gene based sequence tagged site (STS) markers in wheat. The euchromatin enriched genomic library was constructed and the STS primer sets were designed using gene based DNA sequence. The euchromatin enriched genomic (EEG) DNA library in wheat was constructed using the $Mcr$A and $Mcr$BC system in $DH5{\alpha}$ cell. The 2,166 EEG colonies have been constructed by methylated DNA exclusion. Among the colonies, 606 colonies with the size between 400 and 1200 bp of PCR products were selected for sequencing. In order to develop the gene based STS primers, blast analysis comparing between wheat genetic information and rice genome sequence was employed. The 227 STS primers mainly matched on $Triticum$ $aestivum$ (hexaploid), $Triticum$ $turgidum$ (tetraploid), $Aegilops$ (diploid), and other plants. The polymorphisms were detected in PCR products after digestion with restriction enzymes. The eight STS markers that showed 32 polymorphisms in twelve wheat genotypes were developed using 227 STS primers. The STS primers analysis will be useful for generation of informative molecular markers in wheat. Development of gene based STS marker is to identify the genetic function through cloning of target gene and find the new allele of target trait.

주제어

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문제 정의

The objective of this study was to develop gene tagging marker by using euchromatin enriched genomic DNA library. In order to develop the gene based STS marker, the effective genome specific primers were designed from genetic information of genomic DNA library.

제안 방법

, 1984). After STS primers were designed, PCR was undertaken with twelve wheat genotypes. PCR amplification was done in volumes of 50 ㎕, each containing 0.
With the sequencing data of individual colonies, specific primers were designed using the Primer 3 program. The primers were designed with melting temperatures between 55 and 61℃, GC contents between 40 and 60, and lengths between 18 and 24 bases.

이론/모형

Template DNA used for PCR analysis was extracted from fresh leaf tissues by the CTAB method (Saghai-Maroof et al., 1984). After STS primers were designed, PCR was undertaken with twelve wheat genotypes.
The reference DNA of wheat in this study was ‘Keumkang’, a Korean variety. The DNA was extracted by the CTAB method (Saghai-Maroof et al., 1984). The DNA was digested with restriction enzyme, Spe I and was ligated with pGEM7zf(+) vector treated with Xba I.

성능/효과

The STS primers, KG 7, KG 25, KG 30, KG 75 and KG 171 showed polymorphism by one restriction enzyme; KG 54 primer showed polymorphism by two restriction enzymes; KG 92 and KG 203 primers showed polymorphism by three restriction enzymes. In conclusion, the eight STS markers that showed 32 polymorphisms in twelve wheat genotypes were developed using 227 STS primers (Table 5).

참고문헌 (18)

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