Identification of nine candidate sexual SNP loci
The exploration of candidate sexual SNP loci was carried out through the
compilation of individual sequences using our own data (ten birds from
Lleida) and data kindly provided by Weissensteiner et al. (2020) (four
whole-genome sequenced birds from Sweden) (see Section S2 and Table S3
in Supplementary Information). We first located the sexual chromosomes
and the PAR region within them based on the four whole-genome sequenced
Jackdaws from Sweden. To do so, we assembled the sequences of the four
individuals and assumed that the homologous regions between
non-autosomal chromosomes should map within the PAR region. We annotated
and identified the CHD gene (chromo-helicase-DNA-binding), traditionally
used for sex identification in birds (Griffiths et al. 1998), to locate
this region. Similar to Palmer et al. (2019), we used the amount of
reads mapping to this gene to distinguish between males and females.
Individuals J01, J02 and J08, with a high number of reads mapped, were
classified as males while individual J03 was classified as a female
based on the low number of reads mapped (see Section S2 in Supplementary
Information). Since reads mapped heterogeneously in multiple regions
along the chromosome Z, with variable coverage ranging from 4 to 30 and
a median of 12, this approach did not allow us to identify the starting
and ending positions of the PAR region. We located the chromosome W
based on a sequence deposited in NCBI from a female of a close-related
species, the New Caledonia Crow Corvus moneduloides (accession
number CM018842.1). Reads not mapping to Z nor to any other part of the
genome were assumed to be unique to W.
Once we identified the PAR region and the W chromosome, we used the
sequences of the individuals from Lleida and Sweden to identify SNPs
located in sexual chromosomes and outside the PAR region to assess their
utility for sexing. From the W-chromosome, we selected four SNPs from
different regions far from CHD and potentially unique from W. From the
Z-chromosome, we selected one SNP previously tested in the studied
population (F9A, see Section S2 in Supplementary Information) and four
additional SNPs. These four SNPs presumably presented fixed sex-specific
allele variants at least within the analysed individuals from both
populations, and were located in regions close to the CHD gene, assumed
to be within the PAR region. Therefore, we used a total of nine SNPs
located in sexual chromosomes to evaluate for their usefulness for
sexing.
SNP genotyping with OpenArray Real-Time PCR
The OpenArray® technology is an advanced real-time PCR method that
enables a broad range of applications, including SNP genotyping and gene
expression analysis (Schleinitz et al. 2011; Broccanello et al. 2020).
The thermocycler measures FAM and VIC fluorescence signals of the
amplified product (SNPs sequences, in our case) that is generated during
the reaction. Such fluorescence signals are expressed when the threshold
cycle (CT) value is reached, which is the number of PCR
cycles until the genotype calling groups are specified. The fact that
the detection of the DNA amplification occurs while the reaction is
proceeding —instead of at the end-point— makes the technology faster
and more precise and accurate than other PCR technologies (Valasek and
Repa 2005). Excluding 3 blanks and 29 samples without amplified genotype
variants of 960 samples, we analysed the genotyping results of 928
samples from 927 individuals.