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

为了利用DNA分子技术构建林木树种的有效改良体系，我们开发了一种新的DNA分子标记，并尝试将其应用于实际育种。(1)一种新的用于品种/无性系鉴定的DNA分子标记：在马占相思和柳杉中，从每个物种的随机扩增多态DNA片段中设计了18个以上的序列特征扩增区(SCAR)标记。将这些SCAR标记组合在一起，构建了多重-PCRSCAR标记的MUPS系统，该系统具有识别约26万个基因型的能力。(2)黑松种内叶绿体DNA多态性调查：碱基切除序列扫描、CFLP(裂解酶片段长度多态性)和SSR(简单序列重复序列)分析揭示了基因间隔区的8个突变。应用MUPS系统对九州77个品种和589个优树无性系的MUPS类型进行了测定，并建立了它们的MUPS类型数据库。MUPS分析的结果表明，相当多的优树无性系来源于一些品种或一些未知的插穗无性系。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.