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.