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

本研究课题进行了三年。利用RAPD技术对日本柳杉传统扦插品种和优树无性系进行了DNA类型的分类。1.扦插品种的再分类：利用RAPD技术对九州地区的扦插品种进行了分类。共分析了84个品种的550个单株。通过RAPD分析，确定了53个品种为单克隆品种，31个品种为无性系复合体品种，并对九州地区近620个无性系进行了调查，以揭示其与传统扦插品种的亲缘关系。采用随机扩增微卫星多态性（RAMP）方法从基因组DNA中筛选出（GT）n、（CT）n、（达特）n和（AAT）n微卫星DNA区域。从第一次筛选池中选择了31个可靠的标记。在12个标记中观察到了高杂合性（>0.5），证实了微卫星DNA的变异性很大。测定了8个高度变异标记的DNA序列，并设计引物进行PCR（聚合酶链反应）。这些标记可用于使用微卫星DNA的新鉴定系统。

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.