Developing PCR-RFLP molecular sexing assay for Macquarie perch and testing it in four percichthyids
To develop the rapid and affordable PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) molecular sexing assay, we targeted a male-specific allele of an XY-gametologous SNP in the Macquarie perch sexing region. Conserved PCR primers (F1, F2, R2 and R1; Table C1) were designed based on the Geneious Pro alignment of the 600-bp region of Macquarie perch scaffold 633 containing the sexing region (above) and the homologous region of the golden perch genome (scaffold VMKM01003747 bases 16365-16988; Appendix C, Figure C1). SMS online tool (https://www.bioinformatics.org/sms2/rest_map.html) was used to select Ase I restriction endonuclease, which recognizes a male-specific sequence ATTAAT and cuts it at Y-linked SNP 93229. After digestion of PCR products with Ase I, this assay was expected to yield, during visualization on an agarose gel, 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. After initial tests on two individuals of each sex for four percichthyids, the final assay using F1-R1 primers was tested on 45 hatchery-sexed Macquarie perch from Dartmouth, Yarra and Abercrombie, 21 field-sexed Macquarie perch from King Parrot Creek and Holland’s Creek, and 41 Macquarie perch of unknown sex from eight other populations (two inland and six coastal; Appendix C; list of samples in Supplementary Material S1).