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.