Assigning the sex-specific markers via genotyping-by-sequencing onto the Y chromosome for a torrent frog Amolops mantzorum

We use a genotyping-by-sequencing (GBS) approach to identify sex-linked markers in a torrent frog (Amolops mantzorum) using wild-caught individuals of 21 males and 19 females from the same population. A total of 141 putatively sex-linked markers were screened from 1,015,964 GBS tags through three approaches, respectively based on sex differences in allele frequencies, sex difference in heterozygosity, and sex-limited occurrence. With validations, 69 sex-linked markers were confirmed, all of which point to male heterogamety. The male specificity of eight sex markers was further verified by PCR amplifications with a large number of additional individuals covering the whole geographic distribution of the species. Y chromosome (No. 5) was microdissected under a light microscope, amplified by whole-genome amplification, and assembled a draft Y genome. 55 out of 69 sex-linked markers could be mapped to the Y chromosome assembly (i.e 79.7 %). Thus the chromosome 5 could be added as candidate chromosomes that particularly favored to recruit for sex determination than others among frogs. Three sex-linked markers that mapped on Y chromosome were aligned to three different promoter regions of Rana rugosa CYP19A1 gene, which might be considered as a candidate gene to trigger sex determination in A. mantzorum.