Individual Macquarie perch genotypes for sex-linked WGS-derived loci on scaffold 633 region 90152-94017
a. Complex patterns of inheritance of Y-specific polymorphisms suggests multiple Y-linked haplotypes
We explored individual strelka genotypes for 50 females and 50 males for a 5 Kb region of scaffold 633 (90,000-95,000; Supplementary Material S4), focussing on loci with sex-specific alleles in the region 90152-94017 containing the nine most significantly differentiated loci (CMH test p<1e-20; Fig. C1). The region 90152-92914 contained 13 SNP-loci bearing recent-Y-specific polymorphism (Fig. C1). Of these, two (90152 and 91773) were heterozygous in >54% of males (34 and 27, respectively) and 11 were heterozygous in 14-32% of males, with the two sets of loci generally present in different sets of males (Fig. C1). The other males and all females were homozygous for the reference allele at these loci. The 146-bp sexing region 93182-93327 had five SNPs and two indels (a male-specific deletion at 93184 and insertion at 93278) inherited in a fashion consistent with XY-gametology (Table 4; Supplementary Material S4; Fig. C1). All females were homozygous for reference (X-) alleles and 35/50 males were heterozygous for this and alternative (Y-) alleles at all seven loci. The remaining 15 males included 11 homozygous for the Y-allele for one or more locus and heterozygous for the remaining ones, three homozygous for the Y-allele at two loci and homozygous for the X-allele for one locus and heterozygous for three loci, and one (MP_CBR72) homozygous for the X allele for all seven loci. For the SNP-locus 93879 bearing recent-Y-specific polymorphism, 37 males were heterozygous, one homozygous for the alternative allele, and the other 12 males and all females were homozygous for the reference allele. Lastly, SNP-locus 94017 was consistent with being XY-gametologous: all females were homozygous for the reference X-allele and all males were heterozygous, except one male (MP_CBR101) homozygous for the Y-allele. Notably, 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. Overall, females were homozygous for the reference allele at all 22 loci with sex-specific alleles, and males were heterozygous at two or more of these, indicating presence of multiple Y-haplotypes.
b. Depressed read-depth coverage indicates short-read Illumina sequencing issues
Examination of mapped mate-pair reads for the 146-bp sexing region of scaffold 633 (93182- 93327) in heterozygous 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 (Fig. 1). Near position 93250, average read-depth coverage (calculated for each base of the region 93000-94200 and normalized for each individual by average genome-wide depth before being averaged across the four sex-by-population samples) was close to the genome average, but dropped to almost zero at ~93400 (zero reads in 31 females and 28 males), then gradually returned to the genome-wide average by ~93800, except that the depth recovered earlier (at ~93650) for Dartmouth males.
c. Sex-differences in read depth coverage suggests segregating sex-linked deletions
For the loci bearing recent-Y-specific polymorphism 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. 1). Average depth for females was consistently lower than that for males from ~93250 to ~93600, containing 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. 2). In contrast, the SNP locus 93879 (bearing a recent-Y-specific polymorphism) was associated with a drop of male depth compared to that of females (Fig. 1), with male read depth for the alternative allele being just a half of that for the reference allele, suggesting deletion of Y-allele in some males (Fig. 2). The last XY-gametologous SNP locus in this region, 94017, was in a region where depth was close to the genome-wide average for both sexes (Table 4; Fig. 1).