Amplicon-based sequencing to test for sex-specific variation at
the Macquarie perch sex-linked region in other species
We investigated whether the Macquarie perch sex-linked region is also
sex-linked in three other species of Percichthyids. The 600-bp
sex-linked fragment of Macquarie perch scaffold 633 (SEMN01000633;
93182-93327) had 91% identity (6% gaps) with a corresponding part of
golden perch scaffold 3747 (VMKM01003747; 16365-16989) and 88% identity
(7% gaps) with a section of Murray cod scaffold 2546 (LKNJ01002546;
82756-83361). An alignment of scaffolds of all three species revealed
that one of the Macquarie perch sex-linked SNPs (scaffold 633 base
93181) was located in a microsatellite, part of which was missing from
the Murray cod sequence (Fig. B1). Positions corresponding to the other
four putatively sex-linked Macquarie perch SNPs were present in all
three genomes (Fig. B1).
Initial tests of the PCR-RFLP sexing assay (above) on two females and
two males of the golden perch, Murray cod and trout cod did not reveal
any sex-specific variation in length of the amplified fragments or
presence of a male-specific cut site (Appendix B, Fig. B7). We then
amplified a ~400 bp region using primers conservative
across the three genomes (F2 and R3, Table B1) and sequenced it on an
Illumina ISeq100 system by GeneSEQ Sdn Bhd (Malaysia) using the
unidirectional read configuration of 1 x 300 bp (details in Appendix A).
Two males and two females of each of the four species were sequenced,
along with two coastal Macquarie perch individuals of unknown sex but
appearing to be one of each sex based on the PCR-RFLP sexing assay (list
of samples in Supplementary Material S1). Reads in the fastq files were
trimmed of poly-G sequences (a sequencing artefact in the newer
NextSeq500/NovaSEQ6000/ISeq100 Illumina system) using cutadapt(Martin, 2011) via the option –nextseq-trim . Each individual
was then genotyped for the 400-bp amplicon region using best-practice
GATK v4.1.0 protocol (Van der Auwera et al., 2013), containerized and
saved on the docker repository with tag:trust1/gatk:version0.3.1 . The ~600 bp region was
used as a reference (LKNJ01002546 bases 82756- 83361 for Murray cod and
trout cod, VMKM01003747 bases 16365-16988 for golden perch and
SEMN01000633 bases 93001-93600 for Macquarie perch). Raw reads were also
examined in Geneious Pro for the presence of sex-specific haplotypes.