Primers for genotyping single nucleotide polymorphisms and microsatellites in the pathogenic fungus Coccidioides immitis

The pathogenic fungus Coccidioides immitis is endemic to semi-arid soils of southwestern United States, Central and South America (Pappagianis 1988). Until recently, C. immitis had been considered a single species; however, recent molecular analysis has shown that C. immitis consists of a species pair, now referred to as the Californian and non-California types (Zimmermann et al. 1994; Burt et al. 1997; Koufopanou et al. 1997). These species are estimated to have been reproductively isolated from one another for the past 11 million years (Koufopanou et al. 1997; Koufopanou et al. 1998). A study by Burt et al. (1997) used a panel of single nucleotide polymorphisms (SNPs) to infer the mode of reproduction and the amounts of gene flow between populations of the non-Californian species of C. immitis. However, due to the time of separation between the two species, these SNP markers had all gone to fixation within the California species and were uninformative in population genetic studies. In this study, we describe the characterization and isolation of two classes of polymorphic genetic markers from the California species of C. immitis: (i) SNPs and (ii) microsatellites. Due to their low mutation rates, SNP markers were isolated to test hypotheses about the mode of reproduction in California C. immitis. The multi-allelic nature of microsatellites lends them to clinical diagnostic uses in both species of C. immitis simultaneously, and were isolated to this end. To search for SNPs, genomic DNA was amplified from seven tester strains of Californian C. immitis using pairwise combinations of arbitrary primers and low-stringency conditions as described by Burt et al. (1994). A second amplication was performed with the exception that 0.1 µL of [α35S]-thio-dATP (12.5 mCi/mL, 1 mCi = 37 Mbq) was added and the amplicons electrophoresed on a polyacrylamide gel (AT Biochem, Malvern, PA, USA) to reveal single-strand conformational polymorphisms (SSCPs). Polymorphic amplicons were cycle sequenced using fluorescent dNTPs (Applied Biosystems) to determine the genetic basis of the variation and specific primers designed to amplify the locus (Table 1). We also used this technique to screen C. immitis sequences from GenBank: (i) CTS1; (ii) pyrG (OR); (iii) ITS; (iv) bl and unpublished sequences to HSP 60 (HSP) and BGL2 kindly provided by G. Cole (Medical College of Ohio, Toledo). For each locus, a single restriction endonuclease was chosen to score each polymorphic site in a restriction assay (Table 1). If no suitable restriction site existed, then SSCP was used to score the polymorphism (locus BGL2). Microsatellite loci were isolated as described by Rassmann et al. (1991). Libraries containing 300–500 bp inserts in the vector pGEM 4Z (Promega) were made for both C. immitis species. Colony lifts and replicas were fixed to Hybond-N (Amersham) following the manufacturer's instructions. (AC)15 and (GA)15 oligonucleotides (Operon) were end-labelled using T4 polynucleotide kinase (Promega) and [γ32P]-ATP (5000 Ci/mmol, Amersham), and used independently to screen the libraries. Filters were hybridized in 10% w/v Dextran sulphate (Sigma), 1% w/v sodium dodecyl sulphate (SDS, Sigma), 1 m NaCl at 60 °C, with the probe at a final concentration of 10 ng/ml and washed twice in 2× SSPE (0.36 m NaCl, 20 mm Na2HP04, 2 mm ethylenediaminetetraacetic acid), 0.1% w/v SDS at 50 °C. Putative positive clones were rescreened in the same way and the inserts amplified using M13 forward and reverse primers. These amplified inserts were then cycle sequenced using the same primers. Using this method 12 simple sequence repeat motifs were identified, seven from the Californian C. immitis library and five from the non-Californian library. Primers to eight of these loci were designed (Table 2) and the forward primers labelled with either FAM, ROX or HEX fluorescent dyes. PCR amplifications were performed for 30 cycles of (94 °C, 30 s; annealing temperature (Ta °C), 30 s; 72 °C, 45 s) followed by a final extension for 7 min at 72 °C. PCR products were electrophoresed through a 6% denaturing polyacrylamide gel with a TAMRA-labelled internal size standard using an automated sequencer (Applied Biosystems). All microsatellite-containing loci amplified successfully and were polymorphic within the C. immitis species from which they were developed. Furthermore, all loci could be amplified from their sister taxa showing that the flanking sequences of these loci are sufficiently conserved to allow amplification from both the known taxa of C. immitis. Only a single allele was found within each C. immitis strain, confirming that the fungus is haploid. The numbers of alleles at each polymorphic locus and their range in allele size are shown in Table 2. Here, we have described the isolation of two sets of polymorphic genetic markers, one based on SNPs and the other on microsatellites, from the pathogenic fungus C. immitis. We are currently using these markers to generate data sets from panels of C. immitis that were isolated from clinical and environmental sources from a range of geographical locations spanning the entire distribution of this pathogen. Used together, these data will enable recent epidemiological patterns to be characterized, as well as analysis of deeper evolutionary processes. We would like to thank Ron Talbot for supplying the isolates used in this study, and D. Greene and S. Kroken for helpful discussion and comments. This work was supported by grants from the National Institutes of Health.