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.

populations.sumstats.gz

Stacks2.41 populations output provides summary statistics for each locus containing SNPs. To identify sex markers, we split  samples into male and female :  males were assigned to Pop M, females were assigned to Pop F.

catalog.tags.tsv, catalog.fa.gz

Stacks2.41 denovo_map.pl pipeline was used to construct a catalog of loci from GBS reads of S samples.  We specified a minimum depth of coverage (m) of two, a maximum number of mismatches between stacks (alleles) within an individual (M) of two, and a maximum number of mismatches between individuals (n) of three to build the catalog. M and n allow for assembly of polymorphic loci within individuals and fixed differences among individuals at a locus, respectively. 

matches.gz

Samples' stacks are matched to the catalog to create a matches.tsv file for each sample. We counted the matches to the catalog to find the depth of each locus in each individual sample. 

Amolops_mantzorum_Sex_linked_marker.gz

Our goal was to isolate single nucleotide polymorphisms (SNP) and presence-absence (PA) markers that are sex-linked in A. mantzorum. Three approaches were used to identify sex-linked markers for both male (XY) and female (ZW) heterogametic systems.

Y_chromosome_assembly.gz

We completed Y chromosome isolation and DNA amplification.  We also performed a de novo assembly using SOAPdenovo2 for the trimmed reads.

Sequence.gz

After amplification in additional samples (12 male,12 female) from three populations, the PCR products were sequenced at Sangon Sequencing Center (Shanghai, China). Except for some individuals were not successfully sequenced, all male individuals and female individuals shown heterozygosity and homozygosity at the polymorphic sites, respectively.