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.