Molecular sexing in Macquarie perch using PCR-RFLP
The sexing assay (PCR product bounded by F1-R1 primers digested withAse I at the Y-specific scaffold 633 allele T at base 93229) tested in two males and two females with known genotypes (C/C homozygote in females, C/T heterozygote in males based on WGS data) showed a single band in females, consistent with XX genotype, and three bands in males, consistent with uncut X allele and two fragments of Y-allele cut at the Y-specific SNP, as expected (Appendix B; Fig. B2). A larger sample of 10 females and 10 males from Dartmouth/Yarra also showed a single band in females, but only two short cut fragments of Y in males, consistent with Y0 genotype (Fig. B3). The lack of the uncut X-specific band in digested PCR product for some of these males was unexpected, because five males had WGS data and of these three showed C/T heterozygotes for the cut-site (although all five displayed Y0 genotype for >3 of other male-specific loci in the 146 bp sexing region). The PCR-RFLP assay also revealed length variation for all bands, likely reflecting variation at microsatellite repeat number. Despite this variation, and WGS showing a single male out of 50 being homozygous for a putative X-allele at 93229, the sexing assay appears promising in these two populations.
The same assay tested in known-sex Macquarie perch from King Parrot Creek/Holland’s Ck and Abercrombie populations showed that males consistently yielded 2- or 3-band patterns, but so did 20% of King Parrot Creek females and 60% of Abercrombie ones, indicating the presence of the T allele at 93229. The remaining females showed a single band, expected from homozygotes for the X-linked C allele at 93229 (Appendix B, Fig. B4). Abercrombie females with a male-specific allele (i.e. potentially feminized males) appear to have had eggs with lower fertilization rate (Supplementary Material 1). Of four females that produced a single female-like band on the PCR-RFLP assay (out of ten that yielded bands), two produced eggs that were successfully fertilized in hatchery, and two had eggs that failed to be fertilized. Of six females that produced two bands (male-like), four produced eggs that were not fertilized, one showed an eggs fertility rate of <1%, and one had eggs which were successfully fertilized. The same PCR-RFLP assay tested in Macquarie perch of unknown sex from six additional populations yielded a single band in 34% of individuals, and two or three bands in 66% of individuals (Appendix B, Fig. B5).