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森林树种改良体系，我们开发了一种新的DNA分子标记，并尝试在实际育种中应用。(1)建立了一种新的品种/无性系鉴定DNA分子标记：在相思（Acacia mangium）和柳杉（Cryptomeria japonica, Sugi）中，利用RAPD（随机扩增多态性DNA）片段设计了18个以上的SCAR （sequence characteristic amplification regions）标记。将这些SCAR标记组合在一起，利用多重pcr技术构建了SCAR标记多重pcr （multiplex-PCR）体系。该系统具有识别约26万个基因型的能力。(2)松种内叶绿体DNA多态性调查：BESS（碱基切除序列扫描）、CFLP（裂解酶片段长度多态性）和SSR（简单序列重复）分析揭示了8个基因间隔区突变。这些叶绿体DNA标记有助于后代花粉亲本的提升。(3)将MuPS系统应用于杉木品种和正树无性系的分类：确定了九州地区77个杉木品种和589个正树无性系的MuPS类型，并构建了其MuPS类型数据库。MuPS分析结果表明，相当多的正树无性系来源于某些品种或一些未知的扦插无性系。这些重复无性系应从九州地区正树种群中消除。

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.