Individual Macquarie perch genotypes for sex-linked WGS loci on scaffold 633
Examination of individual genotypes for 50 females and 50 males for the 5 Kb region of scaffold 633 (90000-95000; Supplementary Material S4) revealed 22 loci with male-specific alleles, including eight XY gametologous ones. All females were homozygous at the X-linked alleles and all males were heterozygous at two or more of the 22 loci (Fig. 1). For the first 13 loci (region 90152-92914), 16% of males were homozygous, whilst the remaining males were heterozygous for two (52%), ten (14%) or four loci (2%). Alternative alleles for these loci were present in four distinct linkage patterns, suggesting that multiple Y- haplotypes are segregating in Macquarie perch (Fig. 1). The next seven loci (93182-93327) comprised the 146-bp sexing region, which had a male-specific deletion and insertion in addition to five SNPs detected with the CMH test, all inherited in a fashion consistent with XY-gametology (Table 3). All females were homozygous for X-alleles and 35/50 males were heterozygous for these and Y-alleles at all seven loci (Fig. 1). Of the remaining males, 14 were either homozygous for the Y-allele or heterozygous at most of these loci, and one (MP_CBR72) was homozygous for the X-allele for all seven loci. The 146-bp sexing region was followed by a SNP with male-specific allele (93879; numbered 21 on Fig. 1) occurring in 37 heterozygous and one homozygous males, and by another XY-gametologous SNP (94017; numbered 22) for which all males were heterozygous, except one homozygous for the Y-allele. Notably, the male MP_CBR72 noted above as displaying a female-like genotype for the 146-bp sexing region, was heterozygous at this locus, as were most other males, suggesting it was not simply a mis-sexed individual or sample mixed-up. The pattern of male heterozygosity for this region suggests a working hypothesis of sex determination in which presence of Y-alleles at one or more of these 22 loci triggers the development of a male phenotype. A sexing assay screening all these 22 loci is expected to achieve ~100% reliability in the Dartmouth and Yarra populations. Occurrence of homozygotes for X- alleles for up to seven of eight XY-gametologous loci in four (of 50) males suggests rare past or ongoing recombination between X and Y haplotypes.
Visual examination of mapped mate-pair reads for the 146-bp sexing region in males confirmed physical linkage of male-specific SNPs (a Y-haplotype). It also revealed a strong drop of read depth compared to the genome average in this region, in both sexes, including zero coverage around position 93400 for 59% of individuals regardless of their overall sequence depth (Fig. 2). This lower depth might be linked to difficulty of sequencing through a low-complexity region, including the (GT)n microsatellite that ends around 93193 (Figure C1). The drop in depth did not result from failed mapping due to repetitive regions, because neither the 146-bp sexing region nor the 41-bp region 93380-93420 spanning the lowest depth were highly similar to any other region in the Macquarie perch genome. Despite this drop in depth, comparison of male and female read depth for loci with male-specific alleles suggested presence of segregating deletions on X chromosomes. In region 90152-92914 average depth was the same in both sexes, approaching genome-wide average (not shown), but sex-differences were apparent in the region containing the 146-bp sexing region (Fig. 2). Average depth for females was consistently lower than that for males from ~93250 to ~93600, containing the majority of the 146-bp sexing region, suggesting that some or most females could be hemizygous for this region (through deletion on one of the X chromosomes, thus the region being X0). Lower depth of reference alleles compared to alternative alleles in males for sex-linked SNPs 93229, 93299, 93315 and 93327 supports the region being haploid in some males too (i.e. Y0; Fig. 3). In contrast, the SNP locus 93879 with a male-specific allele was associated with a drop of male depth compared to that of females (Fig. 2), with male read depth for the alternative allele being just a half of that for the reference allele, suggesting deletion of the Y-allele in some males (Fig. 3). The read depth for the last XY-gametologous SNP locus in this region, 94017, was close to the genome-wide average for both sexes.