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A new identification system of sugi (Cryptomeria japonica) cultivars based on DNA typing

基于DNA分型的杉树品种新鉴定系统

基本信息

批准号：
06556024
负责人：
SHIRAISHI Susumu
金额：
$ 6.14万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

This research subject was carried out for three years. The traditional local cutting cultivars and the plus-tree clones in sugi (Cryptomeria japonica) were reclassified based on DNA type by RAPD (random amplified polymorphic DNA) analysis. Furthermore, the new DNA markers called microsatellite DNA that possessed high variability and repeatability were developed.1.Reclassification of cutting cultivars : RAPD was used to discriminate the cutting cultivars in Kyushu region. About 550 individuals from 84 cultivars were analyzed. It was clarified that 53 cultivars were monoclonal and remaining 31 cultivar were clone-complex from the DNA type of RAPD.About 620 plus-tree clones in Kyushu region were also investigated to reveal the relation to the traditional cutting cultivars.2.Development of the new identification system : The microsatellite DNA regions, (GT) n, (CT) n, (TAT) n, and (AAT) n, were screened from genomic DNA using RAMP (random amplified microsatellite polymorphism) method. Thirty-one reliable markers were selected from the first screening pool. High heterozygosity (>0.5) was observed in twelve markers, and it was confirmed that microsatellite DNAs were very variable. The DNA sequences of 8 highly variable markers were determined, and the primers were designed for PCR (polymerase chain reaction). These markers are available to a new identification system using microsatellite DNA.

这项研究课题进行了三年。通过RAPD（随机扩增多态性DNA）分析，根据DNA类型对杉（Cryptomeria japonica）传统的地方扦插品种和加树无性系进行了重新分类。此外，还开发了具有高变异性和重复性的新的DNA标记，即微卫星DNA。1.插条品种的重新分类：采用RAPD技术对九州地区的插条品种进行判别。对来自 84 个品种的约 550 个个体进行了分析。从RAPD的DNA类型中明确了53个品种是单克隆的，其余31个品种是克隆复合体。还对九州地区的约620个加树克隆进行了研究，以揭示与传统切割品种的关系。2.开发新的识别系统：微卫星DNA区域，（GT）n，（CT）n，（TAT）n和（AAT） n，是使用RAMP（随机扩增微卫星多态性）方法从基因组DNA中筛选。从第一批筛选池中选出了 31 个可靠的标记。在 12 个标记中观察到高杂合性 (>0.5)，并证实微卫星 DNA 变化很大。确定了8个高度可变标记的DNA序列，并设计了PCR（聚合酶链式反应）引物。这些标记可用于使用微卫星 DNA 的新识别系统。