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

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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.