Construction of a new improvement system using DNA molecular technique in the forest tree species

为了利用DNA分子技术构建有效的林木品种改良体系，我们开发了一种新的DNA分子标记，并尝试应用于实际育种。(1)一种新的用于品种/无性系鉴定的DNA分子标记：以马占相思和柳杉为材料，利用随机扩增多态性DNA（RAPD）片段设计了18个以上的SCAR（sequence characterized amplified regions）标记。利用Multiplex-PCR技术，将这些SCAR标记组合起来，构建了一个具有特异性的多标记复合PCR体系MuPS。26万个基因型。(2)对黑松种内叶绿体DNA的多态性进行了研究：通过对黑松叶绿体DNA的BESS（碱基切除序列扫描）、CFLP（切割酶片段长度多态性）和SSR（简单重复序列）分析，发现了8个基因间隔区的突变。这些叶绿体DNA标记可用于后代花粉亲本的鉴定。(3)MuPS系统在杉品种和优树无性系分类中的应用：确定了九州杉77个品种和589个优树无性系的MuPS类型，并建立了它们的MuPS类型数据库。MuPS分析结果表明，有相当一部分优树无性系来源于品种或未知的扦插无性系。这些重复无性系应从九州地区的优树种群中剔除。

For constructing an effective improvement system of forest tree species using DNA molecular technique, we developed a new DNA molecular marker and tried to apply the actual breeding.(1) A new DNA molecular marker for identification of cultivars/clones : In Acacia mangium and Cryptomeria japonica (Sugi), more than 18 SCAR (sequence characterized amplified regions) markers were designed from RAPD (random amplified polymorphic DNA) fragments in each species. By combining these SCAR markers, a MuPS (multiplex-PCR of SCAR markers) system was constructed using Multiplex-PCR.This system possesses the discrimination power to identify ca. 260,000 genotypes.(2) Survey of the intra-specific chloroplast DNA polymorphism in Pinus thunbergii : BESS (base excision sequence scanning), CFLP (cleavase fragment length polymorphism), and SSR (simple sequence repeats) analysis of the chloroplast DNA revealed the eight mutations in the intergenic spacer regions. These chloroplast DNA markers were useful for ascent the pollen parent of progenies.(3) Application MuPS system into the classification of Sugi cultivars and plus tree clones : We determined the MuPS types of 77 cultivars and 589 plus tree clone of Kyushu and constructed the data base of their MuPS types. As the result of MuPS analysis, quite a few plus tree clones originated from some cultivars or some unknown cutting clones. These duplicated clones should be eliminated from the plus trees population of Kyushu region.