DOI: 10.5061/dryad.mkkwh70wb
title: Assigning the sex-specific markers via genotyping-by-sequencing onto the Y chromosome for a torrent frog Amolops mantzorum

description: 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.
description: 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. 
 <strong>matches.gz</strong>
 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. 
 <strong>Amolops_mantzorum_Sex_linked_marker.gz</strong> Our goal was to isolate single nucleotide polymorphisms (SNP) and presence-absence (PA) markers that are sex-linked in <i>A</i>. <i>mantzorum</i>. Three approaches were used to identify sex-linked markers for both male (XY) and female (ZW) heterogametic systems. <strong>Y_chromosome_assembly.gz</strong> We completed Y chromosome isolation and DNA amplification.  We also performed a <i>de novo</i> assembly using SOAPdenovo2 for the trimmed reads.<br> <br> <strong>Sequence.gz</strong> 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.