Isolation and characterization of microsatellite loci in a Phylloscopus warbler.

have been successfully used to identify differences between closely related species (Taylor et al. 1994), subspecies (Paetkau & Strobeck 1994) and populations of the same species (Bowcock et al. 1994). Roy et al. (1994) presented a thorough analysis at all three levels for wolves and their close relatives. Gotelli et al. (1994) were able to reveal hybridization between the Ethiopian wolf and domestic dogs, and others have used microsatellite variation to suggest recent population bottlenecks (Paetkau & Strobeck 1994). As more accurate models to analyse population structure data of microsatellite loci become available (Slatkin 1995) it appears likely that the study of microsatellite variation will contribute significantly to the fields of population genetics and conservation. In this paper we present information on the isolation and characterization of microsatellite loci in the warbler Phylloscopus occipitalis. Our research aims at investigating genetic structuring along an altitudinal gradient in its Himalayan breeding quarters and how this is related to morphological differentiation. Genomic DNA isolated from blood of an adult female large crowned leaf warbler Phylloscopus occipitalis was digested with MboI and electrophoresed in a 1.5% agarose gel. DNA fragments in the size range of 300–600 bp were excised from the gel and isolated by electroelution. A size selected library was then constructed by ligating the DNA into the vector M13mp18 (opened with BamHI), followed by transfection into E. coli DH5αF' by electroporation (E. coli Pulser, Bio-Rad). According to the ratio of blue and white colonies 80% of the vectors contained inserted DNA. A total of ï¿¿ 40 000 colonies were screened using the oligonucleotides CA 15 and GA 15 end-labelled with γ 32 P-ATP. Nine positive clones were detected and sequenced (Table 1). The sequence of POCC3 happened to be identical to clone POCC2 although they were found on different plates. Primers were designed to have a Tm of 60 ï¿¿ C. The primers were used on putatively unrelated individuals of 16 Phylloscopus occipitalis sampled at Manali, Himachal Pradesh, India. In addition, we checked a family (male, female and four chicks) to look for inheritance patterns. Initial PCR amplifications were performed under the following conditions: 30 s at 94 ï¿¿ C, 30 s at 55 ï¿¿ C, 30 s at 72 ï¿¿ C (28 cycles). Before the cyclic reactions the samples were incubated at 94 ï¿¿ C for 3 min, and after completion at 72 ï¿¿ C for 5 min. Reactions of 10 …