Results
The proportion of reads mapping to each of the autosomes was highly
consistent between samples (Fig. 1). Further, autosomal read-dosage
appeared to be positively correlated with scaffold length, as expected
if restriction motifs are randomly distributed. We tested this
correlation by performing a linear regression in RStudio (proportion of
reads ~ scaffold length), which resulted in a slope
coefficient of 3.833e-10 (adjusted
R2 = 0.7, p < 2e-16).
Unlike the autosomes, values for the proportion of reads mapping to the
X chromosome formed two clusters, putatively representing females (with
higher read-dosage values) and males (with lower read-dosage values).
However, the mean proportion of reads mapping to the X chromosome
(length = 171,031,299 bp) for the putatively female samples (0.0308) was
substantially lower than the expectation (0.0656) based on the
relationship between the proportion of reads mapped and scaffold length
inferred from the autosomes. This difference may represent an inherent
bias against sex-linked loci in the DArT pre-sequencing workflow or a
depletion in the restriction motif on the X chromosome relative to the
autosomes.
The read-dosage sex-assignment program (sexassign ) allowed us to
successfully assign all individuals in the dataset as either male
(heterogametic, XY; X read-dosage = ~0.5X) or female
(homogametic, XX; X read-dosage = ~1X, Fig. 2, Table 1).
Of the 60 individuals sequenced, 33 were determined to be female and 27
to be male, consistent with the typical sex ratio in rodent populations
under normal conditions (Labov et al., 1986; Rosenfeld et al., 2003).
Genetic sex determination had a ~94% concurrence rate
with field determined sex, a typical human error margin considering the
lack of obvious sexual dimorphism within the species and the difficulty
of accurately sexing rodents in the field, particularly during
non-reproductive periods (Hoffmann et al., 2010; Jacques et al., 2015).