An evaluation system of resistance ability of Japanese black pine clones against pine wood nematode based on parentage diagnoses using DNA molecular markers

为建立商品林黑松无性系抗松材线虫病能力的评价体系，以家系诊断为基础技术，对黑松无性系抗松材线虫病能力进行了评价。由于叶绿体DNA（cpDNA）是父系遗传的针叶树种，有用的cpDNA标记被开发用于确定父系的后代。结果如下：1.为了筛选cpDNA的序列多态性，在16个克隆中测定了由52个非编码区（13，360 bp）和编码区（5，209 bp）构建的18，569 bp序列。其长度约为整个cpDNA的七分之一。结果，在5个非编码区中识别出7个变异。其中2个突变为单核苷酸多态性（SNP），3个为简单重复序列多态性（SSR），1个为插入，1个为插入/缺失（indel）.为了评价cpDNA SSR的长度多态性，对12个SSR区域进行了研究。在五个区域中发现了11种变异。遗传自父本的叶绿体DNA标记已实际应用于种子园生产种子的亲子鉴定。分别对从种子中分离的胚和胚乳的DNA进行了分析，并从其cpDNA类型中检测出母本和父本。从亲子诊断数据估计每个克隆的贡献。结果表明，少数无性系在种子园中产生下一代种子。这是种子果园管理中必须克服的一个严重问题.将这些cpDNA标记与核SSR标记相结合，建立了16个无性系的抗病性评价体系。

To establish the evaluation system in commercial forests of the strongness of resistance Japanese black pine (Pinus thunbergii) clones against pine wood nematode, a parentage diagnosis was developed as a basic technique for the system. Since chloroplast DNA (cpDNA) is paternally inherited in coniferous species, useful cpDNA markers were developed for the determination of the paternal parent of the progenies. The results were as follows ;1. For screening sequence polymorphisms of cpDNA, 18,569 bp-sequence constructed with 52 non-coding regions (13,360 bp) and coding regions (5,209 bp) were sequenced in 16 clones. The length is one seventh of the whole cpDNA. As the results, seven variations in five non-coding regions were recognized. Two mutations of them were single nucleotide polymorphism (SNP), three were simple sequence repeat polymorphism (SSR), one was insertion, and one was insertion / deletion (indel).2. To evaluate the length polymorphism in cpDNA SSR, twelve SSR regions were investigated. Eleven variations were recognized in the five regions.3. Chloroplast DNA markers inherited from its paternal parent were actually applied into the parentage diagnosis of the seeds produced in seed orchards. The DNA types of the embryo and endosperm isolated from a seed were analyzed separately and the maternal parent and paternal parent were detected from their cpDNA types. The contribution of each clone was estimated from the parentage diagnosis data. The result indicated that a few clones product the next generation (seeds) in seed orchards. This suggests a serious problem that must be overcome in seed orchard management.4. An evaluation system of resistance ability of 16 clones is available by using these cpDNA markers and nuclear SSRs together.