Introduction
The growing development of advanced molecular and bioinformatic tools
have revolutionized the study of ecology and evolution by allowing to
sequence hundreds of samples in parallel at a whole-genome scale (Hudson
2008). Next-generation sequencing (NGS) techniques are sensible,
accurate, low time-consuming, and can be applied to non-model organisms.
Such availability of massive genomic data provides a promising potential
in the field of genomics, transcriptomics, and proteomics, as well as a
wide application for microsatellites and single-nucleotide polymorphisms
(SNP) screening.
SNPs are increasingly used to study wild populations. They are suitable
for population-level genotyping due to their abundance and widespread
distribution along the genome, low genotyping error, high-throughput and
low-cost per locus (Kaiser et al. 2017). The use of SNP markers is
currently widely used in population genetic analysis, pedigree
reconstruction, extra-pair paternity assignments and phenotypic trait
mapping (Garvin et al. 2010), providing crucial information to address
important aspects of evolutionary and conservation ecology.
Sex largely influences a wide array of key ecological and evolutionary
processes, including habitat use, feeding specializations, parental
care, dispersal and migration (Selander 1966; Durell 2000). Thus, it is
not surprising that devising molecular tools for reliable sex
identification has long been in the agenda of wildlife ecologists. Yet,
tools for sexing using SNP markers have only been developed for
commercial animals (Andrews et al. 2016), including a range of
aquaculture fish and crustacean species for which the factors involved
in determining sexes are not identified yet (Palaiokostas et al. 2015;
Shi et al. 2018; Wang et al. 2019; Fang et al. 2020). The use of SNPs
for sexing remains, however, largely unexplored in wild animals, despite
their great potential to reduce costs and improve performance of
multiple genetic analysis from the same multi-locus panel including all
SNPs of interest without the need of additional PCR amplifications and
electrophoresis.
Here, we describe a novel protocol for molecular sexing of birds based
on single nucleotide polymorphisms. Birds are well-known for their
extraordinary diversity of sexually dimorphic characters. The elaborate
breeding performances by colourful males with striking plumage-colours,
bill sizes and shapes in birds-of-paradise (family Paradisaeidae) or the
extreme adaptations in the tail of male peacocks (Pavo cristatus )
are among the most emblematic displays (Beehler 1989; Owens and Hartley
1998). However, the sex of birds cannot always be easily identified by
phenotypic traits. In fact, about half of all avian species are sexually
monomorphic, with males and females showing very similar appearances
(Price and Birch 1996). These include geese, cranes, rails, raptors,
owls, parrots, doves, auks, shearwaters and many passerines (Volodin et
al. 2015). Even in sexually dimorphic species, males and females rarely
show sex-linked morphological differences shortly after hatching, making
it difficult to obtain information on the sex ratio at birth exclusively
from phenotypic measurements.
Our method is based on the identification of two unique loci, one in
each sexual chromosome. Birds have the ZW sex-determination system, in
which males are the homogametic sex (ZZ), while females are
heterogametic (ZW) (Bloom 1974). As for the XY system in mammals, Z and
W chromosomes share homologous sequences of nucleotides in the
Pseudo-Autosomal Region (PAR) (Fridolfsson and Ellegren 1999). In this
region, genes are inherited the same way as any autosomal gene rather
than sex-linked, and both males and females have two copies of this
region. Therefore, SNPs found in the PAR region would distinguish
females and males only in the rare event that each allele variant is
fixed and specific for each sex. For those cases, females would be
heterozygotes whereas males would be homozygotes. Outside the PAR
region, however, discriminating among sexes is expected to be easier
because unique Z-linked SNPs would amplify two SNP alleles in
heterozygotic males and only one in females, while unique W-linked SNPs
would amplify only in females. Only hemizygotic (individuals in which
only one member of a chromosome pair is present) or homozygotic males
would express one Z allele call but not for W. ZZ males are then defined
by either homozygote or heterozygote genotype calls for the
Z-chromosome-linked SNP and the lack of the calling variants in the
W-chromosome-linked SNP. Conversely, ZW females amplify for the
W-chromosome-linked SNP but do not show heterozygosity for
Z-chromosome-linked SNP.
To illustrate the method, we use the Western Jackdaw (Corvus
monedula ) as our study system. Jackdaws are small corvids from the
Palearctic (Madge and De Juana 2019), characterized by a black plumage,
grey nape and distinctive pale-blue irises. The identification of sexes
in the wild is difficult because sexual dimorphism in plumage is absent
and, although males tend to be larger than females, there is
considerable overlap between sexes (Green and Theobald 1989; Henderson
1991; Fletcher and Foster 2010). The traditional molecular method for
bird sexing based on the PCR amplification of the CHD sexual gene
(Griffiths et al. 1998) works well in Jackdaws (e.g. de Kort et al.
2003; Arnold and Griffiths 2003; Salomons et al. 2006; Woods et al.
2018; Aastrup and Hegemann 2021; Hahn et al. 2021). However, the
alternative use of SNPs holds great potential for its use in reliable
high-throughput sexing within broader population genetic studies from
the same multi-locus SNP panel. Despite the limitation of lacking a
W-chromosome reference in the Jackdaw, our protocol made it possible to
locate the Pseudo-Autosomal Region (PAR) in which both sexual
chromosomes share homologous sequences as well as to detect unique
W-linked markers that can be used to reliably sex individuals. Thus, our
approach can easily be extended to other avian species.