WADA, M.; OTSUKA, R.; NISHIUMI, I.; Tokyo Med. Dent. Univ., Col. Lib. Arts Sci. ; Tokyo Med. Dent. Univ., Col. Lib. Arts Sci. ; National Sci. Museum, Dept. Zool.: Establishment of microsatellite probes and paternity analysis in polygynous Japanese bush warblers

The Japanese bush warblers, Cettia diphone, are common species in Japan and have polygynous breeding system. In the breeding ground some males have their own territories with more than one female and male floaters are also not uncommon. To understand breeding strategy, exact parentage should be elucidated. In order to obtain a tool for this purpose, we constructed a genomic library and develop primers for the species. DNA for library construction was obtained from blood samples of birds captured in Chichibu which was digested. Fragments ranging from 400 to 800 bp were ligated into pUC118BamHI/BAP and transformed into Escherichia coli JM109 Electro-Cells by electroporation. Out of approximately 10,000 colonies screened, we found 34 positive colonies, 12 clones contained a microsatellite sequence, and primers were designed for each locus. These primers were tested in Japanese bush warblers and successfully amplified. In analyses of 49 unrelated individuals, allelic diversity ranged from 2 to 22, and observed heterozygosity ranged from 0.27 to 0.854 except for two loci. These polymorphic loci were tested in other 4 related species on the same conditions. In Short-tailed bush warbler Cettia squameiceps, Taczanowski�s grasshopper warbler Locustella pleskei and Ijima�s willow warbler Phylloscopus ijimae, no loci were amplified. In the Great reed warbler Acrocephalus arundinaceus, one locus was amplified and polymorphic, but the other 11 microsatellite loci failed to be amplified. Paternity analysis of juveniles captured in Chichibu was made using these microsatellite loci and we found that both owners and floaters have their offspring.