PCR-RFLP molecular sexing assay for Macquarie perch
To develop the molecular sexing assay, we targeted a 146-bp region on Macquarie perch scaffold 633 (bases 93182-93327), which contained 5 sex-linked SNP loci homozygous for the (X-linked) reference allele in all sequenced females, with the alternative (Y-linked) allele present in most sequenced males. To develop conservative PCR primers intended to amplify in all Macquarie perch populations and possibly other species, we first used Blastn to search the golden perch genome for a region homologous to 600 bp of Macquarie perch sequence encompassing the 146-bp region of scaffold 633 (SEMN01000633, bases 93001-93600). This search yielded a 624-bp region on golden perch scaffold VMKM01003747 (bases 16365-16988), which we aligned to putative X- and Y-linked Macquarie perch haplotypes in Geneious Pro (Appendix B, Fig. B1). We designed two forward and two reverse primers in regions identical in the two species (Table B1, primers F1, F2, R2 and R1, with the 5’-end starting on scaffold 633 bases 93027, 93107, 93380, and 93474, respectively) and then used the SMS online tool (https://www.bioinformatics.org/sms2/rest_map.html) to find positions of restriction endonuclease cut sites targeting Macquarie perch SNPs with Y-specific alleles. Ase I was chosen because it recognizes ATTAAT that occurs only in the male-specific sequence (T base at 93229).
All four primer combinations were trialled initially in two female and two male Macquarie perch (see Appendix B for details). This assay was expected to produce one long band (of length dependent on primer combination) for XX females (always homozygous) and three bands (including a long X band and two shorter Y ones cut by the enzyme) for XY males. For the final assay on a larger sample, we ran PCRs with F1-R1 primers followed by Ase I digestion (Appendix B; list of samples in Supplementary Material S1). This assay was tested using 45 Macquarie perch hatchery-sexed individuals from Dartmouth, Yarra and Abercrombie, 21 field-sexed individuals from King Parrot Creek and Holland’s Creek, and 41 Macquarie perch of unknown sex from eight other populations (two inland and six coastal).