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.