Sex-linked markers by genome-wide RAD sequencing to identify XX/XY Sex Chromosomes in the spiny frog (Quasipaa boulengeri)

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.

Methods
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.