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.