Discussion
We have proposed a general method for sexing wild birds using sex-linked SNP markers based on sequencing unique loci mapping outside the pseudoautosomal region where sexual chromosomes exchange genetic material. We hypothesized heterozygote genotypes for the Z-linked SNP would refer to males as the homogametic sex in birds. In contrast, the detection of W-linked SNP variants would occur only in females. We demonstrate the accuracy and reliability of the method in the monomorphic Western Jackdaw. Below, we provide further insights into the use of our molecular technique to sex wild birds, highlighting its benefits and limitations compared to other available tools for molecular sexing and the broader implications of the use of sex-linked SNPs on the study of ecology and evolutionary biology.
The proposed technique is expected to be particularly useful in studies aimed at genotyping large number of individuals within species and populations. On one hand, the method only requires sampling feathers. Feathers provide sufficient quality and quantity of DNA for molecular analyses like ours (Horváth et al. 2005). Indeed, we found that in feathers where the concentration of the DNA extracted was considered low (<50 ng/μL), genotyping was still successful in 86.55% of cases. On the other hand, our technique can be integrated into multiple genetic analysis based on SNP data without the need of additional PCR amplification and electrophoresis. Combined with the use of high-throughput sequencing method of OpenArray® genotyping based on real-time PCR technology (Broccanello et al. 2020), which results in a significant reduction on the sequencing time and costs required per sample and locus (Hudson 2008), the technique represents a cost-effective method for SNP genotyping in studies that require genotyping thousands of individuals (Jenkins and Gibson 2002).
Despite the capabilities of OpenArray® genotyping, its implementation is not exempt of difficulties. In our case, the genotyping variants of one locus included in the array was undefined for the totality of the samples. Because the estimated error rate of automated high-throughput methods for genotyping with SNPs is fewer than 1 in 2000 genotypes (Ranade et al. 2001), we can safely discard standard sequencing errors as the main source of the observed result. Instead, the allelic variants identified in individuals from the Sweden population for this locus in particular might not be present in Lleida’s population. Furthermore, candidate loci meeting the screening criteria are more challenging to identify from sequencing artifacts in small sample sizes (N=3 males and N=1 females). In practice, multiple individuals of each sex would be required to avoid falsely identifying rare SNP variants as sex‐linked contigs (Palmer et al. 2019).
The capabilities of OpenArray® genotyping are also limited by the quality of DNA samples. Although genotyping proved to be successful in poor-quality samples, a 3.03% of the samples (29 of the whole batch of 957) did not amplify for any loci due to low DNA concentrations. From the remaining 928 sequenced samples, the Z-allele could not be identified in 9 of them, leading to a total of 919 genotyped samples. The low mean DNA concentration (47.27 ng/μL) of these samples indicated, again, problems of sample quality rather than sequencing errors. Therefore, increasing the quality threshold should reduce the number of not genotyped samples.
More difficult to understand is the observation of five samples (5 of the 919 genotyped samples) that could not be sexed with certainty because showed two variants in the Z-chromosome and a third in the W-chromosome. A likely alternative is a differential specificity of the F5C and F7C primers for the W-unique loci. Indeed, in 2.4% of the cases where genotypes were defined, samples showed a mismatch between the genotypic variants amplified with the W-linked F5C and F7C SNPs. The existence of differential specificity of the F5C and F7C primers is further supported by a blast analysis, which shows that specificity was higher for F7C. Although in our sexing method we employed F7C, the fact that we still detected samples with two variants in the Z-chromosome may indicate that this may still be insufficient to fully resolve the specificity issue. Alternatively, the observation of ZZW may indicate possible cross-contamination events (mixing samples from male and female individuals) and/or sequencing errors (misassignment of genotype calls). We should also consider the possibility of trisomies which, although rare, have been documented in some bird species like Gallus domesticus (Lin et al. 1995), Charadrius alexandrines (Küpper et al. 2012), Ara ararauna (Tiersch et al. 1991) andAcrocephalus arundinaceus (Arlt et al. 2004).
Regardless of the limitations, the high accuracy (100% of individuals correctly sexed) and reliability (over 99% of samples correctly processed) of the proposed approach make it an efficient way to sex birds. Although there are other powerful approaches for sexing individuals (e.g. Griffiths et al. 1998; Lois-Milevicich et al. 2021), the use of sex-specific SNP markers located in unique fragments of Z- and W-chromosomes, as we propose here, will contribute to reliably sex individuals while genotyping samples for other purposes. While the approach requires reference sequences to identify and annotate sex chromosomes, this information is becoming increasingly available in non‐model species through fast and affordable next generation (NGS) and whole-genome sequencing (WGS). Even in species for which only one reference sex chromosome is available, as in our case, sex-linked scaffolds can still be identified based on sex differences in genomic coverage (Palmer et al. 2019).
Currently, SNPs are widely used in a variety of research programs like human forensics, crop improvement, aquaculture, drug discovery and wildlife research (Garvin et al. 2010). In ecology and evolution, SNPs are increasingly used for population genetic analysis, pedigree reconstruction and phenotype mapping (Garvin et al. 2010). Given that males and females often differ in habitat preferences, feeding specializations, parental investment and dispersion, among many other ecological roles, these studies heavily rely on the accurate identification of the sex of individuals. Thus, we expect that the sex-linked SNPs protocol we present here will be useful to a broad range of fields because it will allow genotyping and sexing a high number of individuals in parallel and with independence of their life cycle stage.