Polymorphic microsatellites in the blue tit Parus caeruleus and their cross‐species utility in 20 songbird families

© 2000 Blackwell Science Ltd, Molecular Ecology, 9, 1919–1952 Using the Primer 3 computer program (Rozen & Skaletsky 1997), primers could be designed to microsatellite-flanking regions of 37 clones. PCR was performed in 25 μL volumes containing 1.6 mm MgCl2, 0.2 mm dCTP, dGTP and dTTP, 0.02 mm dATP, 0.06 μL [α32P]dATP, 75 mm Tris–HCl (pH 9), 20 mm (NH4)2SO4, 0.01% Tween-20, 5 pmol of each primer, 0.5 units Taq DNA polymerase (Eurogentec) and 10 ng of template DNA. After initial denaturation (95 °C, 20 s), PCR was run for 35 cycles (94 °C for 20 s, 53 °C for 25 s, 67 °C for 23 s) in a Perkin-Elmer 2400 thermocycler. Products were separated on 6% sequencing gels and autoradiographed (Sambrook et al. 1989). Primer functionality was initially tested on three A. rabiei isolates, and one isolate of A. fabae. Using an annealing temperature of 53 °C, single bands of the expected size were obtained for 26 loci. Twenty-four primer pairs also amplified one or more products from A. fabae. Whether these fragments contain microsatellites and/or are polymorphic within A. fabae remains to be examined. Twenty marker loci were selected to genotype 22 A. rabiei isolates from the USA, Pakistan, Syria, Turkey and Tunisia. Each of these constituted a unique clonal lineage as defined by its fingerprint haplotype (Geistlinger et al. 1997). The Popgene computer package (Yeh & Boyle 1997) was used to calculate allele frequencies and to determine Nei’s expected genetic diversity valves for haploid organisms (Nei 1987; Tenzer et al. 1999; Table 1). All loci were polymorphic, with 2–14 alleles and genetic diversity values of 0.17–0.90. With up to 14 alleles among 22 isolates, these microsatellites are more variable than other types of molecular markers applied to A. rabiei. Locus-specific primers not only ensure a high reproducibility of results, but also allow the analysis of mixed samples. Groppe & Boller (1997) have specifically amplified a microsatellite locus from the fungal endophyte Epichloe in the presence of contaminating host DNA. Following a similar strategy, microsatellite markers could be used to type A. rabiei isolates directly from individual lesions. The distribution of A. rabiei pathotypes in the field could then be monitored more efficiently, avoiding the lengthy procedures of single-sporing and culturing.