10.5061/DRYAD.PVMCVDNJ1
Dissanayake, Duminda
Institute for Applied Ecology
Holleley, Clare
0000-0002-5257-0019
Commonwealth Scientific and Industrial Research Organisation
Georges, Arthur
0000-0003-2428-0361
Institute for Applied Ecology
Identification of Y chromosome markers in the eastern three-lined skink
(Bassiana duperreyi) using in silico whole genome subtraction
Dryad
dataset
2020
Australian Research Council
https://ror.org/05mmh0f86
DP110104377
Australian Research Council
https://ror.org/05mmh0f86
DP170101147
2020-09-11T00:00:00Z
2020-09-11T00:00:00Z
en
2728146 bytes
1
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
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.
Refer to the README file.