Marker development
We studied putatively sex-linked sequences that were identified by RADseq from a sample of 40 agile frogs from a single clutch in Switzerland (Jeffries et al., 2018). The 92 bp long RAD tags were mapped to a genome assembly of the common frog (unpublished data, D.L. Jeffries), a species closely related to the agile frog (Pyron & Wiens, 2011), using Magic-BLAST 1.3 (Boratyn, Thierry-Mieg, Thierry-Mieg, Busby, & Madden, 2019). We concentrated on those tags that hit to the agile frog’s sex chromosomes (Jeffries et al., 2018) uniquely or had an e-value of at least 5 orders of magnitude lower than the next best hit. First, we aimed to check if the putative sex-linked loci carried sex-linked single nucleotide polymorphisms (SNPs) in our study populations as well. Using the NCBI Primer-BLAST tool (Ye et al., 2012), we designed primers for a total of 14 loci based on common frog genome with aim for sequencing agile frog DNA around the sex-linked RAD tags so we could sequence DNA fragments of about 220-1100 bp length. PCRs were performed with these primers on DNA samples of morphologically sexed adult agile frogs from Hungary in the laboratory of the Conservation Genetics Group, Department of Ecology, University of Veterinary Medicine Budapest (for PCR primers used for sequencing and detailed conditions see Table S2; PCR programs are described in Table S3). Clear PCR products in the expected length range were cut and purified from 2% agarose gel, using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), and ran on a 3130xl Genetic Analyzer (Thermo Fisher Scientific) at BIOMI, Gödöllő, Hungary. Sequencer output files were analysed by the STADEN package (Bonfield, Smith, & Staden, 1995; downloaded from https://sourceforge.net/projects/staden/files) and sequences were checked manually. In total, primers designed for 11 loci produced strong PCR products close to the expected fragment sizes which were also suitable for cutting from agarose gel, and 7 of these yielded unambiguous DNA sequences of the target loci (Table S2). Three out of these 7 loci contained sex-linked SNPs based on sequences from 10 agile frogs (5 males and 5 females) from Hungary (Table S2), and we denominated these Rds1, Rds2 and Rds3, according to their order on the common frog’s chromosome 4 (that is corresponding to the agile frog’s sex chromosome). Segregation of SNPs at all three loci matched expectations for an XX/XY sex-determination system as found in Jeffries et al. (2018). Because there is no sex chromosome sequence assembly available for the agile frog, we estimated the distances between the sex-linked SNPs based on the corresponding chromosome 4 of the common frog (unpublished data of D. L. Jeffries; reported as chromosome 5 in Jeffries et al. (2018).
We designed sexing primers for these three putatively sex-linked loci, so that for each locus two fragments could be amplified in a single PCR: one fragment amplified from both chromosomes X and Y, and a shorter fragment amplified only from Y (i.e. if a Y-specific SNP was present). The shorter amplicon is part of the X/Y-universal fragment (see Figure S1). Using this method, successful amplification of the X/Y-universal product means that the target locus is amplifiable in the investigated DNA sample (i.e. positive control). If the Y-specific fragment is amplified as well, that proves the presence of the Y-specific SNP (male genotype). We designed primers specific for the Y-SNPs so that the SNP was present at their 3’ end. To increase allelic specificity, a mismatching base was artificially introduced at the 3rd position closest to the 3’ end of these primers (replacing the original base in the sequence; following Liu et al. (2012). On Rds2 two sex-linked SNPs were situated 11 nucleotides apart, therefore the Y-specific primer binding to both of these SNPs did not require the introduction of any artificial mismatch. PCR conditions for each pool of sexing primers were optimized based on individuals with known DNA sequence at the concerned locus. Specificity of the primers was tested by agarose gel electrophoresis of PCR products from individuals with known DNA sequence. Sexing PCRs were carried out in a final volume of 16 µl containing 1.6 µl DreamTaq green buffer (10x, Thermo Fisher Scientific), 0.65 µl dNTP (2 mM, Thermo Fisher Scientific), primers of varying amount (Table 2), 0.065 µl DreamTaq DNA polymerase (5U/µl, Thermo Fisher Scientific) and 20-100 ng genomic DNA. PCRs were carried out on a Bioer Life ECO gene amplification instrument (TC-96/G/H(b)C). Optimized sexing PCR profiles are described in Table S3.
Because PCR optimization by the above method was insufficient for Rds3, we developed an HRM-based (high-resolution melting) method for sex-linked SNP-identification at this locus in the laboratory of the Ruminant Genome Biology Research Group, NARIC Agricultural Biotechnology Institute, Gödöllő, Hungary. Total HRM reaction volume was 15 µl, containing 3 µl 5x HOT FirePol EvaGreen qPCR Mix Plus (ROX, Solis BioDyne), 1 µl forward and 1 µl reverse primer (10 µM each; Table 2) and 80-100 ng genomic DNA. Reactions were performed in a Roche Light Cycler 96 Instrument (as described in Table S3) and the results were analysed with the Light Cycler 96 v. 1.1.0.1320 software (Roche Diagnostics International LTD). Detailed guidance for HRM-based sexing is available in Figure S2. HRM allows us to differentiate not only between individuals carrying and not carrying Y-SNP but it provides information on further differences between individual genotypes as well (i.e. presence of additional SNPs can be detected: Figure S2). Genotyping with this method was validated by comparing the assumed genotype based on HRM to DNA sequence data of 42 individuals. While PCR-based sexing allowed us to detect the presence or absence of a Y-SNP (Figure S1), the HRM method gave information on the presence of both the Y-SNP and the X-SNP (Figure S2).