Sex-specific ecology has management implications, but rapid sex-chromosome turnover in fishes hinders development of markers to sex monomorphic species. Here, we use annotated genomes and reduced-representation sequencing data for two Australian percichthyids, the Macquarie perch Macquaria australasica and the golden perch M. ambigua, and whole genome resequencing data for 50 Macquarie perch of each sex, to detect sex-linked loci, identify a candidate sex-determining gene and develop an affordable sexing assay. In-silico pool-seq tests of 1,492,004 Macquarie perch SNP loci revealed that a 275-Kb scaffold, containing the transcription factor SOX1b gene, was enriched for gametologous loci. Within this scaffold, 22 loci were sex-linked in a predominantly XY system, with females being homozygous at all 22, and males being heterozygous at two or more. Seven XY-gametologous loci were within a 146-bp region. Being ~38 Kb upstream of SOX1b, it might act as an enhancer controlling SOX1b transcription in the bipotential gonad that drives gonad differentiation. A PCR-RFLP sexing assay, targeting one of the Y-linked SNPs, tested in 66 known-sex Macquarie perch and two individuals of each sex of three confamilial species, and amplicon sequencing of 400 bp encompassing the 146-bp region, revealed that the few sex-linked positions differ between species and between Macquarie perch populations. This indicates sex-chromosome lability in Percichthyidae, also supported by non-homologous scaffolds containing sex-linked loci for Macquarie- and golden perches. The resources developed here will facilitate genomic research in Percichthyidae. Sex-linked markers will be useful for determining genetic sex in some populations and studying sex chromosome turnover.

Understanding sex-specific biology can aid conservation management. But understanding genomic sex differences of monomorphic fish species and developing molecular sexing assays is challenged by their diverse sex-determination systems. To facilitate research on Percichthyid fish, predominant in the Australian freshwater biota, we report whole genome sequences and annotations of the endangered Macquarie perch Macquaria australasica and its sister species, the golden perch M. ambigua. To identify sex-linked loci, we conducted whole genome resequencing on 100 known-sex Macquarie perch. In-silico pool-seq comparisons revealed few sex differences, but a 275-Kb SOX-containing scaffold was enriched for gametologous loci- homozygous in females, heterozygous in males. Within this scaffold we reconstructed X- and Y-linked 146-bp haplotypes containing 5 sex-linked SNPs, ~38 Kb upstream of SOX, and developed a PCR-RFLP sexing assay targeting the Y-linked allele of one SNP. We tested this assay in a panel of known-sex Macquarie perch, and smaller panels of three other confamilial species. Amplicon sequencing of 400 bp encompassing the 146-bp region revealed that the few sex-linked positions differ interspecifically, and within Macquarie perch such that its sexing test approached 100% reliability only for the populations used in assay development. Similarly, Macquarie- and golden perch genome-wide DArTseq SNPs revealed different sex-linked loci across non-homologous scaffolds. Overall, we identified 22 sex-linked SNPs in Macquarie perch in a predominantly XX/XY system in which females are homozygous at all 22, and males are heterozygous at 2 or more. The resources here will facilitate multi-locus sexing assays for both species and research on Percichthyid biology.