Identification of the Male Parents of Half-sib Progeny from Japanese Black Pine (Pinus thunbergii Parl.) Clonal Seed Orchard using RAPD Markers

Abstract

Random amplified polymorphic DNA (RAPD) markers were used to identify the male parents of the half-sib progeny from a single clone within a Japanese black pine (Pinus thunbergii Parl.) clonal seed orchard consisting of 16 pinewood-nematode [Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle] resistant clones. Twenty-one primers, with 28 polymorphic and reproducible fragments, were selected from 320 primers examined. The genotypes of orchard clones at each fragment were fingerprinted by RAPD analyses of both diploid needles and haploid tissues of megagametophytes from each clone. We used all 28 RAPD markers to identify the male parents of the half-sib progeny of a single orchard clone, Tanabe-(t)54. By comparing the genotype of orchard clones with the phenotype in the seedling at each RAPD locus, we were able to identify the male parents of 82 out of 85 seedlings. Pollen contamination and natural selfing occurred at rates of only 2.4% and 1.2%, respectively. Mating with the clone deviated significantly from panmixia. More than half of the progeny of the clone were produced through fertilization by only two clones. Seven clones did not contribute at all to fertilization as the male parent. Deviations from panmixia have been caused by a difference in the distance between clones, variation in the amounts of pollen, and phenological synchrony between clones. The possibility and limitations of RAPD markers for paternity analysis in conifer seed orchards are discussed.