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

基于DNA分子标记亲子鉴定的黑松无性系抗松材线虫能力评价体系

基本信息

批准号：
12556024
负责人：
SHIRAISHI Susumu
金额：
$ 7.1万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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.

为了建立商品林日本黑松（Pinus thunbergii）无性系对松材线虫的抗性评价体系，采用亲本诊断作为评价体系的基础技术。由于叶绿体DNA （cpDNA）在针叶树属植物中具有父系遗传特性，因此开发了有用的cpDNA标记来确定后代的父系亲本。实验结果如下：1.实验结果表明：为筛选cpDNA序列多态性，共对16个克隆的18569 bp序列进行测序，该序列由52个非编码区（13360 bp）和编码区（5209 bp）组成。长度是整个cpDNA的七分之一。结果，识别出5个非编码区域的7个变异。其中2个突变为单核苷酸多态性（SNP）， 3个突变为简单序列重复多态性（SSR）， 1个突变为插入型，1个突变为插入/缺失型（indel）。为了评估cpDNA SSR的长度多态性，对12个SSR区域进行了研究。在5个地区发现了11种变异。从父本遗传的叶绿体DNA标记实际应用于种子园生产的种子亲本鉴定。分别分析了一颗种子的胚和胚乳的DNA类型，并从其cpDNA类型中检测了母本和父本。每个克隆的贡献是根据亲子诊断数据估计的。结果表明，少数无性系在种子园中产生下一代（种子）。这表明在种子园管理中必须克服一个严重的问题。将这些cpDNA标记与核ssr结合使用，建立了16个无性系的抗性评价体系。