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Identification of Y chromosome markers in the eastern three-lined skink (Bassiana duperreyi) using in silico whole genome subtraction

Citation

Dissanayake, Duminda; Holleley, Clare; Georges, Arthur (2020), Identification of Y chromosome markers in the eastern three-lined skink (Bassiana duperreyi) using in silico whole genome subtraction, Dryad, Dataset, https://doi.org/10.5061/dryad.pvmcvdnj1

Abstract

Background: Homologous sex chromosomes can differentiate over time because recombination is suppressed in the region of the sex determining locus, leading to the accumulation of repeats, progressive loss of genes that lack differential influence on the sexes and sequence divergence on the hemizygous homolog. Divergence in the non-recombining regions leads to the accumulation of Y or W specific sequence useful for developing sex-linked markers. Here we use in silico whole-genome subtraction to identify putative sex-linked sequences in the scincid lizard Bassiana duperreyi which has heteromorphic XY sex chromosomes.

Results: We generated  96.7 x 109 150 bp paired-end genomic sequence reads from a XY male and 81.4  x 109 paired-end reads from an XX female for in silico whole genome subtraction to yield Y enriched contigs. We identified 7 reliable markers which were validated as Y chromosome specific by polymerase chain reaction (PCR) against a panel of 20 males and 20 females.

Conclusions: The sex of B. duperreyi can be reversed by low temperatures (XX genotype reversed to a male phenotype). We have developed sex-specific markers to identify the underlying genotypic sex and its concordance or discordance with phenotypic sex in wild populations of B. duperreyi. Our pipeline can be applied to isolate Y or W chromosome-specific sequences of any organism and is not restricted to sequence residing within single-copy genes. This study greatly improves our knowledge of the Y chromosome in B. duperreyi and will enhance future studies of reptile sex determination and sex chromosome evolution.

Methods

Reads from a focal male and a focal female were analysed independently as follows. First, reads were decomposed into k-mers of 27 bp using Jellyfish 2.0. Unique k-mers were counted, again using Jellyfish 2.0 and k-mers in common between the male and female sets were removed from the male set. This yielded a (subtracted) k-mer set that was enriched for Y chromosome sequence. The Y enriched k-mers were then reassembled into contigs using an inchworm assembler (kassemble.cgi uploaded here) with stringent extension criteria. Briefly, the assembler initially took a focal k-mer at random and searched for other k-mers that matched exactly k-1 bp of the focal k-mer. If this second k-mer was unique, then the focal k-mer was extended by one bp, and the process was repeated. If the k-mer was not unique, then the extension process was terminated. The extension occurred to both the left and the right, yielding relatively short contigs (up to ca 1400 bp) that contain sequence unique to the male individual.

The resultant contigs were selected on length and used to develop primers for screening individuals of known sex. PCR validation on 20 males and 20 females was the basis for selecting a final set of primer pairs for a definitive sex test.

Usage Notes

Refer to the README file.

Funding

Australian Research Council, Award: DP110104377

Australian Research Council, Award: DP170101147