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).