Use of different seed tissues for separate biparentage identification of dispersed seeds in conifers: Confirmations and practices for gene flow in Pinus densiflora

To investigate how accurately biparentage assignments for coniferous seeds can be improved by using different kinds of seed tissues, we assigned biparentage to dispersed seeds in a natural stand of Pinus densiflora Siebold & Zucc. (288 mature trees) using two procedures: with or without megagametophyte haplotype data ("MH" and "ordinary" procedures, respectively). Using archived clones, we confirmed the conifer-specific modes of inheritance of three kinds of seed tissues from certain maternal trees using six microsatellite markers. In the natural stand, under the MH procedure, 39.2% of male parents and 77.0% of female parents for a total of 204 seeds analyzed were assigned to at least one mature tree within the study area. At that time, the proportion of exactly matching seeds out of seeds with one matching parent under the MH procedure was significantly larger than that of the seeds under the ordinary procedure. The biparentage assignments under the ordinary procedure corresponded to only 53.7% of the accurate separate assignments under the MH procedure. It is suggested that analyzing different seed tissues is effective for exact and accurate biparentage assignments in investigations of biparentally mediated gene flow in coniferous populations, particularly at the seed-dispersal stage.