The spindlin gene as a universal marker for molecular sex identification in different poultry species

Abstract

Due to the lack of pronounced sexually dimorphic traits in many poultry species, or the presence of such traits only in adulthood, visual sexing becomes a challenging task, which can result in considerable difficulties in breeding and conservation management. This study performed laboratory sex identification analysis of eleven poultry species based on the sexing gene Spindlin (SPIN). Genomic DNA was extracted from blood or feather samples, encompassing both males and females. PCR was performed using primers SPIN319F and SPIN472R, which flanked intron 2 of SPIN. The outcomes were visualized using agarose gel electrophoresis and capillary electrophoresis plots. The length polymorphism between SPIN-W and SPIN-Z homologous genes allows for clear sex discrimination in chicken, Japanese quail, pheasant, turkey, guinea fowl, chukar partridge, mallard duck, Muscovy duck, mule duck, swan goose, and rock pigeon. The result demonstrated that SPIN was relatively conserved within the same species. We found strong support for the monophyletic grouping of all W sequences from three avian orders in one clade and of all Z sequences in the other. The research indicates that the SPIN marker is effective for genetic sex identification in the poultry species tested in this study.

Introduction

Sex identification is important for reliable estimation of sex ratios within the poultry industry, as it directly influences production efficiency and economic value. In the large-scale egg-laying chicken farming industry, the sex of chicks is determined on the first day after hatching. All female chicks are retained, while male chicks are culled, as they neither lay eggs nor convert feed into meat efficiently as broilers. According to incomplete statistics, nearly 6 to 7 billion male chicks are culled globally each year, leading to both significant economic losses and serious ethical concerns (Krautwald-Junghanns et al., 2018). Research on sex identification mechanisms in poultry can facilitate the development of early sex selection methods. Utilizing genetic engineering techniques, such as gene editing, to produce chicks of a single sex can enhance breeding efficiency, intensify selection pressures, and ultimately accelerate genetic progress (Xie et al., 2020). A few species can be auto sexed based on sex-linked phenotypic features, such as plumage color and pattern, feathering rate, and other similar characteristics (Magdalena et al., 2014). Most methods of sex identification based on morphological features are unreliable in poultry, particularly in juvenile individuals (Ravindran et al., 2018). Therefore, sex identification requires specialized techniques such as behavioral observations (Catherine and Keith, 2001), differences in morphometric traits (Dechaume-Moncharmont et al., 2011), acoustic sexing (Volodin et al., 2015), or genetic analyses (Mazzoleni et al., 2021; Petrou et al., 2024). While these methods can often be used to identify the sex of sexually mature adults that exhibit differences in secondary sex characteristics or behavior, molecular methods are typically the only reliable approach for accurately sexing juveniles and embryos (Morinha et al., 2012). Sex chromosomes in birds (ZW system) are different from mammals (XY system). Male birds are homogametic (ZZ), whereas females are heterogametic (ZW) (Turcu et al., 2023a), allowing for sex identification through molecular methods based on sequence differences between the W and Z chromosomes (Ellegren, 1996). Due to the fact that the size and gene content of the W chromosome are variable across different lineages (Zhou et al., 2014), no single locus has been identified that can reliably sex all birds (Morinha et al., 2012; Dawson et al., 2016).

Molecular sexing offers advantages in terms of accuracy and precision. The relatively recent methods have improved our ability to determine sex using non-invasive tissue samples such as feathers (Jia et al., 2023) or oral swabs (Turcu et al., 2023a). The chromodomain helicase DNA-binding (CHD) was the first proposed as a valid sex-linked marker for sex differentiation (Ellegren, 1996). CHD has two homologous alleles, CHD-Z and CHD-W, which locate on the Z and W chromosome, respectively. The intron sequence lengths of the two alleles are different and can be used for sex identification in several bird species (Griffiths et al., 1998). Nevertheless, accumulating evidence has demonstrated that CHD-based methods are not universally reliable. Several studies have reported amplification failure, weak band separation, or ambiguous banding patterns in diverse avian taxa (Vucicevic et al., 2013; Kroczak et al., 2021). In some cases, CHD primers require species-specific primer redesign or the use of multiple markers to avoid misidentification (Kroczak et al., 2021). In some poultry species, the length difference is too small to be scored by using standard agarose gels (Gruszczyńska and Grzegrzółka, 2021). Several alternative molecular methods, such as single strand conformation polymorphism (SSCP), microsatellites, oligonucleotide microarrays, capillary electrophoresis, real-time PCR analysis and other probe-based methods, are available. Apart from CHD, other genes have been used for detecting the sex of birds, such as the Spindlin (SPIN) (de Kloet and de Kloet, 2003, 2005; Dawson et al., 2016; Wancham et al., 2024), the Nipped-B homolog (NIPBL) (Suh et al., 2011), the RAS p21 protein activator 1 (RASA1) (Li et al., 2012), and the ubiquitin-associated protein 2 (UBAP2) (Romanov et al., 2019; Jia et al., 2024). The avian SPIN gene is among the limited number of genes that have copies present on both the W and Z chromosomes. SPIN evolves relatively slowly and is composed of six exons and five introns. Due to the lack of recombination on the W chromosome, the sequences of the gene copies located on the W and Z chromosomes have become increasingly distinct over time (de Kloet and de Kloet, 2003; Handley et al., 2004). The exons have remained highly conserved across at least 130 million years, while the intron sequences have diverged significantly. This contrast has made SPIN as valuable marker for determining the sex of most bird species. The SPIN primer system successfully distinguished male and female individuals in a wide range of non-ratite birds, including many species for which CHD assays are problematic (Dawson et al., 2016). The size difference between SPIN alleles on the Z and W chromosomes produces a clear and easily distinguishable separation on standard agarose gel electrophoresis. In contrast, the smaller size difference in CHD amplification products frequently requires higher-resolution techniques for reliable interpretation (Vucicevic et al., 2013). SPIN may serve as a solution for species where CHD amplification is inconsistent or unreliable.

Poultry is conventionally defined as domesticated birds raised for commercial purposes, primarily the production of meat and eggs. Key species are predominantly from the orders Galliformes (e.g., chicken, turkey, Japanese quail, pheasant, guinea fowl, chukar partridge) and Anseriformes (e.g., mallard duck, Muscovy duck, swan goose), with additional important species such as the rock pigeon. Given the economic importance of these species and the challenges associated with sex identification, this study aimed to assess the viability of SPIN as a molecular marker for poultry sex identification.

Materials and methods

Study samples and genomic DNA extraction

Feather or blood samples were collected from multiple individuals of known sex and unknown sex for each of the eleven poultry species (Table 1). All samples were collected from commercial farms. The genomic DNA was extracted from each feather using the commercial kit (Genomic DNA kit; Tiangen Bioscience, Beijing, China) following the manufacturer’s protocol. After DNA extraction, the integrity was verified by 1 % agarose gel electrophoresis. A single bright band suggested intact genomic DNA, while degradation appeared as diffuse smearing.

Table 1.

The eleven poultry species investigated in this study.

Family	Species	Type	Individuals (known/unknown sex)
Phasianidae	chicken (Gallus gallus)	Chinese native chicken	8/24
	turkey (Meleagris gallopavo)	American bronze turkey	10/17
	Japanese quail (Coturnix japonica)	Japanese quail	8/17
	chukar partridge (Alectoris chukar)	American Chukar	10/17
	pheasant (Phasianus colchicus)	Chinese pheasant	10/17
Anatidae	mallard duck (Anas platyrhynchos)	Chinese native duck	10/16
	Muscovy duck (Cairina moschata)	Chinese Muscovy duck	10/17
	mule duck (Anas platyrhynchos x Cairina moschata)	Chinese native hybrid duck	6/8
	swan goose (Anser cygnoides)	Chinese native goose	10/17
Numididae	guinea fowl (Numida meleagris)	African guinea fowl	12/17
Columbidae	rock pigeon (Columba livia)	Chinese and American pigeon	8/17

All animal experiments conducted in this study received approval from the Animal Care and Use Committee at the Jiangsu Institute of Poultry Science. The approval date is 23 October 2022 (Approval ID S20221023, Yangzhou, China). The procedures adhered to the relevant guidelines and regulations established by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China.

PCR and electrophoresis

The design of PCR primers was referenced from (Handley et al., 2004), which flanked intron 2 of SPIN. The exon-intron structure of SPIN-Z/SPIN-W was inferred by comparing the Z-linked chicken cDNA sequence (AB047853) to the sequence of human SPIN (NT023935.13). Genomic DNA was tested for the presence of SPIN-W and SPIN-Z genes by standard PCR, using the primers SPIN319F (5′-TATGGACTAGAACTGCACAAAG-3′) and SPIN472R (5′-AGACCATCCCCCTCCATTCATC-3′) (Sangon Biotechnology Co., Ltd., Shanghai, China). The PCR was conducted in a total volume of 25 μL containing 12.5 μL of 2 × Rapid Taq Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 0.5 μL of each primer (10 μM), 1 μL of template, and 10.5 μL of sterile ultrapure water. The PCR was performed under the following conditions: 95°C for 5 min for initial incubation, followed by 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and 72°C for 10 min. A 1.2 % agarose gel electrophoresis was performed at 90 V for 30 min to visualize the products. Additionally, the amplification of SPIN-W and SPIN-Z were also analyzed in capillary and detected using an ABI 3100 DNA analyzer (Applied Biosystems), allowed for mutual confirmation of the correctness of the individual sex identification. LIZ1200 (Applied Biosystems) was used as an internal standard. A GeneMarker v3.0.0 (Soft Genetics LLC, State College, PA, USA) was used to determine the size of the amplified fragments.

TA cloning and sanger sequencing

To get male and female sequences for each species, PCR products were sequenced using the TA cloning and Sanger sequencing methods (Sangon Biotechnology Co., Ltd., Shanghai, China). If only one band was present in the gel, the amplicon was directly purified by Monarch® PCR & DNA Cleanup Kit. In the case of female samples with two bands in the gel, each DNA fragment was initially extracted from the gel, followed by DNA purification using the Monarch® DNA Gel Extraction Kit. SEQme Ltd., Czechia, sequenced the purified products. The obtained sequences were identified by the Basic Local Alignment Search Tool (BLAST) in NCBI (https://blast.ncbi.nlm.nih.gov/BlastAlign.cgi). Before the experiment, we conducted a BLAST search on the selected PCR primers, which are introns located in SPIN. After verification, the amplicon sequences used for poultry species sex identification have Z and W chromosome specific copies, and their lengths are polymorphic, allowing for differentiation between male and female.

Phylogenetic analysis

Sequences were aligned using MAFFT (Katoh et al., 2002). Nucleotide divergence between Z and W introns in the eleven species were calculated with Jukes-Cantor correction, and 95 % confidence intervals were derived from standard error estimates based on 1000 bootstrap replicates in MEGA11 (Tamura et al., 2021). Maximum likelihood was used to construct unrooted phylogenetic trees and support was obtained from 1000 bootstrap replicates in IQ-TREE (Minh et al., 2020).

Results

Electrophoretic analysis of sex identification

The SPIN319F and SPIN472R sexing method was successful for eleven tested species (Table 1). The length polymorphism between SPIN-W and SPIN-Z homologous genes allows for apparent sex discrimination by agarose gel electrophoresis in chicken, Japanese quail, pheasant, turkey, guinea fowl, chukar partridge, mallard duck, Muscovy duck, mule duck, swan goose, and rock pigeon. PCR amplification of SPIN provided two bands in females, corresponding to the SPIN-W and SPIN-Z genes, and a single band in males, corresponding only to the SPIN-Z gene (Figure 1). Results from additional samples can be found in the Supplementary material.

Fig. 1.

The electrophoresis results of PCR products for eleven poultry species on a 1.2 % agarose gel using primers set SPIN319F/SPIN472R. The maker is DL2000, M =Male, F = Female.

To improve the resolution of the results and accurately determine the lengths of these amplification products, the SPIN-W and SPIN-Z amplicons were analyzed by capillary electrophoresis (Figure 2). We found that the females displayed two visible peaks, while only a single peak was observed in males. In most cases, the female SPIN-Z gene yields the larger product, while the opposite is shown in the pigeon. Except for the pigeon, the amplification products of SPIN-Z ranged from 973 bp to 1157 bp, which were longer than those of SPIN-W, whose lengths varied between 791 bp and 841 bp (Table 2). The size difference between the two products ranged from 132 bp to 351 bp. Notably, the pigeon deviated from this pattern, as female exhibit a longer W-linked allele (1165 bp) compared to the Z-linked copy (940 bp). PCR amplification of the SPIN-Z gene in male mule duck produced two distinct bands corresponding to the SPIN-Z gene in duck and Muscovy duck (1151 bp and 1157 bp).

Fig. 2.

The capillary electrophoresis of the Spindlin (SPIN) gene PCR products across eleven poultry species.

Table 2.

Capillary electrophoresis fragment length (bp) of the Spindlin (SPIN) gene for Z and W chromosomes across eleven poultry species.

Species	Male (SPIN-Z/SPIN-Z)	Female (SPIN-Z/SPIN-W)
chicken	985/985	985/798
Japanese quail	1089/1089	1089/791
pheasant	1151/1151	1151/800
turkey	1115/1115	1115/792
guinea fowl	990/990	990/797
chukar partridge	973/973	973/841
mallard duck	1151/1151	1151/841
Muscovy duck	1157/1157	1157/831
mule duck	1151/1157	1157/841
swan goose	1147/1147	1147/832
rock pigeon	940/940	940/1165

Sequencing analysis of SPIN

To determine the fragment length more accurately, the SPIN-W and SPIN-Z amplicons were analyzed by TA cloning and sequencing. The sequencing results of the two fragments indicated that the exon of SPIN is relatively conservative, whereas the intron displays considerable variability. Sequence analysis revealed multiple mutation sites and insertion/deletions between SPIN-Z and SPIN-W amplicons across poultry species.

Intronic nucleotide divergence was estimated for ten species from SPIN (Table.3). The sequence length obtained through TA clone sequencing aligns closely matched those from capillary electrophoresis, with minor discrepancies potentially attributable to differences in experimental techniques. The divergence among ten poultry species ranged from 0.402 to 0.542, with quail exhibiting the highest divergence and pigeon demonstrating the lowest. To assess sequence variation, the analysis of mutations was conducted on a minimum of three sequences per sex for each species (GenBank accession number PX654925-PX655065). Sequencing of the Z chromosome was consistently more straightforward than that of the W chromosome. The result demonstrated that intron 2 of SPIN was relatively conserved within the same species, with no mutations observed among the majority of sequences from the same sex chromosome within a species, thus the impact of intronic variation on subsequent phylogenetic analysis was considered minimal. The maximum number of SNPs (n = 10) was found in a female rock pigeon, which might be attributed to the genetic distance between the Chinese and American pigeon breeds. Insertion-deletions were only detected as 1 bp gap in pheasant, Muscovy duck, and rock pigeon, with the maximum of 4 bp found in turkey. These mutations were not found to affect the reliability or practical applicability of the SPIN primer set.

Table 3.

Intron length and nucleotide divergence of the Spindlin (SPIN) gene between Z and W chromosomes in poultry species.

Species	Length (Z/W)	Divergence	95 % C.I.	Mutations (Z/W)1
chicken	808/623	0.515	0.556-0.474	13/6, 1/0, 1/0
Japanese quail	909/616	0.542	0.585-0.499	5/3, 3/0, 0/0
pheasant	969/625	0.497	0.536-0.458	3/13, 0/1, 1/0
turkey	934/617	0.475	0.513-0.437	7/4, 0/0, 0/4
guinea fowl	811/622	0.468	0.505-0.431	4/5, 3/0, 0/0
chukar partridge	797/666	0.499	0.538-0.460	9/3, 0/0, 0/0
mallard duck	972/667	0.523	0.562-0.484	16/10, 3/0, 0/0
Muscovy duck	979/657	0.480	0.516-0.443	11/3, 3/0, 1/0
swan goose	970/658	0.481	0.519-0.444	13/4, 5/4, 0/0
rock pigeon	762/978	0.402	0.432-0.371	5/4, 0/10, 0/1

¹The format of mutations is Z/W (number of sequences, number of SNPs, length of insertion-deletion).

Phylogenetic analysis of SPIN Z-W

The divergence of Z- and W-linked copies of SPIN-Z/SPIN-W, relative to ordinal divergence, was investigated using a phylogenetic approach. Similar analyses have previously been reported for CHD1-Z/CHD1-W (Fridolfsson and Ellegren, 2000; García-Moreno and Mindell, 2000), ATP5A1-Z/ATP5A1-W (the ATP synthase a-subunit gene) (Ellegren and Carmichael, 2001), and SPIN-Z/SPIN-W (Kroczak et al., 2022).

Exon and intron data were processed separately to investigate potential differences in molecular evolution. For the analysis of intron data, the entire intron sequences were concatenated. The ModelTest module within the IQ-TREE software package was used to determine the optimal nucleotide substitution model for phylogenetic analysis. The best-fit model selected was TIM3+F + I, which incorporates the invariable-sites model (+I), a discrete-rates model that assumes a certain proportion of sites (pinv = 0.075) remains constant across sequences. Phylogenetic tree inference was subsequently performed using the maximum likelihood algorithm as implemented in IQ-TREE, with branch support values calculated through ultrafast bootstrap resampling. Evolutionary convergence was observed between the Z and W chromosome of turkey and pheasant, Japanese quail and chukar partridge within the Galliformes, as well as mallard duck, Muscovy duck and swan goose within the Anseriformes. Chicken was clustered with turkey and pheasant on the W chromosome, but with Japanese quail and chukar partridge on the Z chromosome. The divergence of Z-linked and W-linked copies of SPIN-Z/SPIN-W, relative to ordinal divergence, was investigated using a phylogenetic approach. Similar to the cases of CHD1-Z/CHD1-W and HINT-Z/HINT-W, and in contrast to the cases of ATP5A1-Z/ATP5A1-W and UBAP2-Z/UBAP2-W, the phylogenetic analysis of SPIN-Z/SPIN-W provided strong support for the classification of Z and W copies into a single group across all poultry species (Figure 3) (Ellegren and Carmichael, 2001; Handley et al., 2004). The ATP5A1-Z/ATP5A1-W and UBAP2-Z/UBAP2-W ceased to recombine independently with-in each order after ordinal radiation. The contrasting phylogenetic patterns of CHD1-Z/CHD1-W, HINT-Z/HINT-W, and SPIN-Z/SPIN-W versus UBAP2-Z/UBAP2-W and ATP5A1-Z/ATP5A1-W support the definition of two evolutionary strata on the avian Z chromosome.

Fig. 3.

A maximum-likelihood phylogeny of ten poultry species based on the Spindlin (SPIN) gene.

Discussion

Sex identification is essential in commercial poultry due to its direct effects on economics, animal welfare, and flock management. For egg-production systems, accurate early sexing prevents the unnecessary rearing and frequent culling of day-old male layer chicks (Jia et al., 2023). Furthermore, separating flocks by sex improves uniformity and feed efficiency, while in breeding programs, it ensures optimal mating ratios, hatchability, and fertility (England et al., 2021). The process of sex identification in poultry is regulated by sex chromosomes (ZZ/ZW). In the early stages of poultry embryo development, genes located on one or both of these sex chromosomes control sexual differentiation. In males, both left and right gonads develop into testes, whereas females show asymmetrical gonadal development, with only the left gonad develops into functional ovary and the right one regressing. Two major hypotheses have been proposed regarding the mechanism of sex identification in birds (Lin et al., 1995). The first is the Z-chromosome dosage hypothesis, which posits that individuals with a single Z chromosome develop as female, whereas those with two Z chromosomes develop as male. This hypothesis suggests that a dosage-dependent mechanism in which the Z chromosome plays a central role in the foundation for sex identification. The second is the W-chromosome dominant effect hypothesis, which proposes that the W chromosome carries dominant, female-determining genes that directly regulate the development of female characteristics during sex identification (Sun et al., 2023).

In an attempt to find a universal method, several molecular genetic techniques for identifying the sex of birds—as well as many PCR markers based on CHD (Fridolfsson and Ellegren, 1999; Turcu et al., 2023b), ATP5A1 (Fridolfsson et al., 1998), W-linked gene for the altered form of protein kinase C-interacting protein (Wpkci) (O'Neill et al., 2000), NIPBL, SPIN, or RASA1 genes—have been tested (Li et al., 2012). These genes are used to identify differences between the homologous regions of the Z and W chromosomes based on variations in the length polymorphism of introns located in these regions.

In the current research, using SPIN319F and SPIN472R primers, PCR yielded two bands in females corresponding to SPIN-W and SPIN-Z, and a single band in males corresponding only to SPIN-Z, indicating that the SPIN gene could be amplified by conventional PCR in those poultry species. The SPIN-Z amplicon was approximately 1092 bp, while the SPIN-W amplicon was approximately 816 bp, except for the pigeon. We speculated that the SPIN-W amplification product in pigeon (1165 bp) exhibited greater length compared to SPIN-Z (940 bp), which may indicate potential gene duplication or structural divergence. A similar problem was reported in other avian species, although previous studies indicated that such polymorphism always occurred on the Z chromosome (Handley et al., 2004). The SPIN-W amplicon is about 275 bp shorter than SPIN-Z, providing a clear distinction that makes it easier to distinguish fragments on the agarose gel. There are two possible reasons that might explain the inability of universal primers to sex certain species of birds. First, PCR amplification of the SPIN-W fragment may fail due to mutations in the primer annealing site. Second, the amplified SPIN-W and SPIN-Z fragments may be of similar or long sizes, preventing their separation by standard agarose gel electrophoresis. Such problems can be avoided by using the specific primers that give PCR products of varying length.

This study aimed to test the performance of SPIN for the sex identification in different poultry species. We successfully developed a pair of specific primers for sexing poultry species, which can be easily to detected using agarose gel electrophoresis. For SPIN-Z/SPIN-W we found very strong support for the monophyletic grouping of all W sequences from three avian orders in one clade and of all Z sequences in the other. Therefore, we conclude that this method is suitable for routine sex identification in poultry.

Funding

This work was supported by the National Key Research and Development Program of China (grant number 2021YFD1200302), National Natural Science Foundation of China (grant number No. 32372869), the Natural Science Foundation of Jiangsu Province (grant number BK20221412), the Open Projects of Key Laboratory for Poultry Genetics and Breeding of Jiangsu 483 Province (grant number JQLAB-ZZ-202303), and “JBGS” Project of Seed Industry Revitalization in Jiangsu Province (grant number No.JBGS(2021)029).

CRediT authorship contribution statement

Wanqiang Chen: Writing – review & editing, Writing – original draft, Software, Data curation. Yushi Gao: Writing – review & editing. Xiujun Tang: Writing – review & editing. Xiaoxu Jia: Writing – review & editing, Writing – original draft, Project administration, Conceptualization.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.