Using Sex-Linked Markers via Genotyping-by-Sequencing to Identify XX/XY Sex Chromosomes in the Spiny Frog (Quasipaa boulengeri).

We used genotyping-by-sequencing (GBS) to identify sex-linked markers in 43 wild-collected spiny frog (Quasipaa boulengeri) adults from a single site. We identified a total of 1049 putatively sex-linked GBS-tags, 98% of which indicated an XX/XY system, and finally confirmed 574 XY-type sex-linked loci. The sex specificity of five markers was further validated by PCR amplification using a large number of additional individuals from 26 populations of this species. A total of 27 sex linkage markers matched with the Dmrt1 gene, showing a conserved role in sex determination and differentiation in different organisms from flies and nematodes to mammals. Chromosome 1, which harbors Dmrt1, was considered as the most likely candidate sex chromosome in anurans. Five sex-linked SNP makers indicated sex reversals, which are sparsely present in wild amphibian populations, in three out of the one-hundred and thirty-three explored individuals. The variety of sex-linked markers identified could be used in population genetics analyses requiring information on individual sex or in investigations aimed at drawing inferences about sex determination and sex chromosome evolution.