We use genotyping by sequencing as an approach to identify sex-linked markers in the spiny frog Quasipaa boulengeri with 43 wild-collected adults from a single site. The GBS methodology identified 2 loci on sex differences in allele frequencies, 50 loci on sex differences in heterozygosity, and 523 loci on male-limited occurrence, altogether associated with males heterogamety, indicating an XX-XY system. The sex specificity of five markers was further validated by PCR amplification with a large number of additional individuals from 26 various populations in this species. A total of 27 sex linkage markers were matched to Dmrt1 gene, a ubiquitous role in sex determination and differentiation from flies and nematodes to mammals. Chromosome 1, that harboring Dmrt1, has further been assigned to a highly potential candidate sex chromosome in anurans. Five sex-linked SNP makers explored 3 sex reversals out of 133 individuals here, sparsely showing sex reversal detected in wild amphibian populations.

Genomic DNA of the 43 samples from a single site (DYYEC) was subjected to GBS by the Novogene. Clean Illumina reads were demultiplexed, trimmed, and filtered by the process_radtags function in STACKS [v 2.4]. e used RStudio 1.2.1335 to generate candidate alleles for each individual. 

catalog.fa.gz, catalog.tags.tsv.gz

The files generated by the Stacks operation, by changing the parameters M (M controls the number of mismatches allowed between the two alleles of a heterozygote sample), m (m controls the number of identical reads required to initiate a new putative allele) and n (n controls the number of mismatches allowed between any two alleles of the population). Depending on the data set, gapped alignments may provide more loci for the analysis. 

matches.zip

Stacks will generate a matches file for each sample through operation comparison. By analyzing these matches files, you can find the depth of each site.

populations.sumstats.tsv

The populations program outputs a summary file for each site of the population. Can be grouped according to needs (such as male and female).

Quasipaa_ boulengeri_Sex_linked_marker

Sex-linked markers were screened with three approaches followed by Brelsford et al. (2017), respectively based on (i) sex differences in allele frequencies, (ii) sex differences in heterozygosity, and (iii) sex-limited occurrence. We used RStudio 1.2.1335 to generate candidate alleles for each individual.

sequences.zip

The primer design method was adopted for four sex-linked sites. After amplification in additional samples (10♀10♂) respectively from three populations, the PCR products were sequenced at Sangon Sequencing Center (Shanghai, China).