A fast molecular tool for sexing Gibber Italicus, song thrush, and redwing birds

Abstract

Gibber Italicus, song thrush (Turdus philomelus), and redwing (Turdus iliacus) are 3 bird species in which sexual dimorphism is not readily apparent. Therefore, molecular sexing is a valuable tool for breeding, selection, and conservation purposes. We compared DNA extraction by commercial kit and an alkaline method from feathers, then developed a molecular method for sexing these species using the P2/P8 and CHD1F/CHD1R primer pairs. Both protocols were successful in extracting DNA; the alkaline method is more cost-effective, whereas the commercial kit protocol is easier to standardize. All DNA samples were successfully amplified using both the P2/P8 and CHD1F/1R primer pairs. The use of the CHD1F/1R primer pair is notably advantageous as it produces fragments of different lengths of the sex chromosomes, making them easily distinguishable via electrophoresis. The sequence analysis of the amplicons obtained with the CHD1F/1R primer pair revealed specific single-nucleotide polymorphisms for song thrush and redwing, which could be used as markers to differentiate between the 2 species if required.

Gibber Italicus or Gibber, song thrush (Turdus philomelus), and redwing (Turdus Iliacus) are 3 bird species highly favored by amateur bird keepers. The selection for Gibber Italicus, an Italian breed of Serinus canaria, began in the 1930s, originating from Curled or Dutch of the South and Belgian Bossu breeds. It was officially recognized as a breed by the World Ornithological Confederation in 1951.

To date, the song thrush and redwing, both species of the Turdus genus, are under observation regarding their conservation status by the International Union for the Conservation of Nature (IUCN). Although the population of the song thrush appears to be experiencing a slight increase, climate change and human-induced habitat alterations in many areas could lead to a rapid decline in the coming years (https://www.iucnredlist.org/species/22708822/166346863). The redwing is already experiencing a population reduction due to the aforementioned factors, leading to its inclusion on the IUCN Red List (https://www.iucnredlist.org/species/22708819/110990927).

Molecular sexing of bird species, when sexual dimorphism is not evident from birth, is a valuable tool for breeding, selection, and conservation. This method enables males to be differentiated from females at a very early age without stressing the animals. Furthermore, it facilitates the verification of consistency between phenotypic, genetic, and chromosomal sex, which is crucial in studies concerning sexual differentiation^2,7,12 and disorders of sexual development.^1,8,13,12

Since the elucidation of the avian chromodomain helicase DNA binding 1 (CHD1) gene, ⁴ most non-ratite bird species have been sexed using the variability gathered from the 2 copies located on the Z (CHD1Z) and W (CHD1W) sex chromosomes. Despite its high conservation, CHD1 has accumulated various mutations during evolution, given its location between the non-recombining regions of the 2 sex chromosomes. Consequently, 2 forms are commonly recognized: CHD1W and CHD1Z. Most of these mutations occur in noncoding regions, thereby avoiding alterations in gene function. However, they serve as useful markers to distinguish individuals carrying 2 identical sex chromosomes (males = Z/Z) from those carrying 2 different sex chromosomes (females = Z/W).

CHD1 is a protein-coding gene that potentially modulates gene expression by altering access of the transcriptional machinery to its chromosomal DNA template, thus affecting chromatin structure. ¹⁰ In the chicken (Gallus gallus), it comprises 39 exons, with CHD1W being longer than CHD1Z. Notably, the proteins encoded by both genes exhibit no discernible differences, leaving the precise involvement of CHD1 in avian sex differentiation unclear and unconfirmed.

The substantial differences in the length of certain intronic regions of the 2 CHD1 genes ¹¹ have been utilized to distinguish the 2 sex chromosomes and thus for molecular sexing purposes. Several primer pairs have been developed for amplifying these regions, with the most common being CHD1F/CHD1R, spanning the 18th intron of the gene (NC_052571), ⁹ and P2/P8, spanning the 24th and 25th exons of the gene (NC_052571). ⁵

There is a lack of specific protocols for sexing Gibber Italicus, song thrush, and redwing. To address this gap, we developed an efficient and cost-effective molecular method based on PCR and agarose gel electrophoresis, which are widely recognized as simple to perform, fast, reproducible, reliable, and sensitive. Using primers P2/P8 and CHD1F/1R, known for their high success rates in the order Passeriformes, we tested feathers from 2 known males and 2 females of each species, with DNA from a male and a female canary (Serinus canaria) as positive controls. Our optimized approach provides a valuable tool for breeders to successfully deal with sexing of the studied species.

We tested 306 birds, comprising 201 Gibber Italicus, 58 song thrush, and 47 redwing individuals, which were submitted to the Veterinary Hospital Genetic Diagnostic Service (University of Naples Federico II, Naples, Italy) for molecular sexing. We analyzed all samples within 48 h of collection. We divided each sample into 2 subsamples of 5–6 feathers each. We cut the calamus and harvested it in 2 separate 1.5-mL tubes. Subsequently, we performed DNA extraction using 1 of 2 protocols:

• Protocol 1 (Extracta DNA Prep for PCR–Tissue kit, cat. 95091; Quantabio). We added 70 µL of extraction solution to each tube that was then incubated at 95°C for 50 min; later we added 70 µL of stabilizing solution at room temperature.

• Protocol 2 employed the alkaline method. ⁶ We added 20 µL of a 0.2 M NaOH solution to each tube, incubated at 72°C for 50 min, and then added 180 µL of 0.04 M Tris–HCl at room temperature.

We evaluated DNA quality with electrophoresis on 0.8% agarose gels and carried out the PCR reaction in 25 μL of total volume containing 12.5 µL of master mix (GoTaq green; Promega), 0.4 µL of primers, DNA template (~100 ng), and nuclease-free water up to the final volume. The PCR amplification protocol for P2/P8 uses an initial denaturation step at 94°C for 5 min, followed by 40 cycles at 94°C for 40 s, 48°C for 40 s, and an extension at 72°C for 60 s. A final step was performed at 72°C for 10 min. For CHD1F/1R, the PCR protocol was an initial denaturation step at 94°C for 5 min, followed by 37 cycles of 3-steps each: 94°C for 30 s, 49°C for 45 s, and an extension at 72°C for 45 s. A final step was performed at 72°C for 5 min.

We visualized amplicons from all PCRs using electrophoresis on 2.0% agarose gels, thus establishing the length of each fragment. Subsequently, we extracted PCR amplicons for 3 males and 3 females of each species obtained using both primer pairs (total of 54 amplicons) from the gel (Clean-Easy agarose purification kit; Canvax), following the user manual instructions, and then performed Sanger sequencing. We identified sequences with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and analyzed them with BioEdit (v.7.7, https://thalljiscience.github.io/), Mega X (v.10.0.13, https://www.megasoftware.net/), and T-Coffee. ³

The electrophoresis of freshly extracted DNA had a clear, thin band with a slight smear for the extraction carried out with both protocols, indicating that both methods were successful in extracting DNA for molecular sexing, although they resulted in slight degradation. The average concentration was 117 ± 59 and 60 ± 19 ng/µL, and the A260/280 ratio was 1.52 ± 0.24 and 1.60 ± 0.27 for protocols 1 and 2, respectively.

We successfully amplified all DNA samples using both P2/P8 and CHD1F/1R primer pairs. The P2/P8 primer pair generates 2 fragments that differ in length by a few bps in females and a single fragment in males for the 3 investigated species (Table 1; Fig. 1). The CHD1F/1R primer pair generates 2 fragments of different lengths in females of all 3 species, which are easily distinguishable by electrophoresis (Table 1; Fig. 2). We preferred the protocol with the CHD1F/1R primer pair for the remaining DNA samples to be tested.

Table 1.

Length of the amplicons obtained with P2/P8 and CHD1F/CHD1R primer pairs.

Primer pair	Length of the amplicons, bp
	Gibber Italicus		Song thrush		Redwing
	CHD1Z	CHD1W	CHD1Z	CHD1W	CHD1Z	CHD1W
P2/P8	340	380	340	370	340	370
CHD1F/1R	520	325	550	335	550	335

Figures 1, 2.

Agarose gel (2%) electrophoresis of PCR products with: Figure 1. P2/P8 in male (L2) and female (L3) redwing; in male (L4) and female (L5) song thrush; in male (L6) and female (L7) Gibber; male (L8) and female (L9) Serinus canaria. Molecular marker 100 bp (L1); negative control (L10); Figure 2. CHD1F/1R in male (L2) and female (L3) redwing; in male (L4) and female (L5) song thrush; in male (L6) and female (L7) Gibber; male (L8) and female (L9) Serinus canaria. Molecular marker 100 bp (L1); negative control (L10).

Molecular analyses revealed that among the 201 Gibbers Italicus samples, 121 were females and 77 were males; 3 samples did not amplify. Of the 58 song thrush samples, 23 were females and 35 were males. Of the 57 redwing samples, 32 were females and 25 were males. The sex of all of the sexed birds was later confirmed when adults, demonstrating the reliability of the test.

We sequenced the amplicons for all species (54 in total) and subsequently analyzed them using nucleotide BLAST to check for correspondence with known CHD1W/1Z sequences in related species. In all 3 species, the sequences most similar to those input into BLAST belonged to birds of the order Passeriformes and matched sequences within the CHD1W/1Z gene. This confirms that P2/P8 and CHD1F/1R primer pairs amplify fragments of these genes in Gibber Italicus, song thrush, and redwing.

We aligned the sequences obtained for 3 females and 3 males of each species with each other to examine the presence of single-nucleotide polymorphisms (SNPs) and potential SNPs responsible for differences in length between CHD1W and CHD1Z in the 3 species. In Gibber Italicus, we observed no differences when aligning CHD1W/1Z sequences amplified with the P2/P8 primer pair in all studied birds. Similarly, we observed no differences when aligning the short and long fragments obtained with the CHD1F/1R primer pair. However, when aligning the CHD1Z fragments with CHD1W fragments, only a short region of the 2 sequences had perfect identity (Suppl. Figs. 1, 2). This suggests that the differences in length in both regions (the one amplified with P2/P8 and the one amplified with CHD1F/1R) are likely due to several mutations accumulated in both regions.

Sequence analyses revealed that the alignment of the fragments from the P2/P8 primer pair in song thrush had a 40-bp insertion in the sequence of the longest fragment (CHD1W), along with a different nucleotide sequence extending for 152 bp (Suppl. Fig. 3). When aligning the sequences of the shorter amplicon with the longer one generated by the CHD1F/1R primer pair, we observed that they had only 47% similarity (Suppl. Fig. 1). We obtained similar results when analyzing the fragments obtained for both primer pairs in the redwing (Suppl. Figs. 2, 3). Moreover, because song thrush and redwing are 2 species in the same genus, we conducted an alignment between their sequences revealing some species-specific polymorphisms (Fig. 3).

Figure 3.

Alignment of CHD1 sequences on CHDW and CHDZ obtained with A, B. P2/P8 and C, D. CHD1F/1R in song thrush and redwing, respectively.

Use of the commercial kit (Extracta DNA Prep for PCR-Tissue), rather than the alkaline method ⁶ for DNA extraction from feathers, yielded similar results. The DNA obtained worked well with both PCR protocols, indicating no preference for one method over the other in terms of performance. However, the cost is lower for the alkaline method, while the commercial kit offers easier protocol standardization, as the reagents are supplied ready to use and with quality control certification.

Using the CHD1F/1R primer pair was certainly more convenient for all 3 species. This primer pair generates a fragment that is longer on the W chromosome than on the Z chromosome, which is distinguishable in agarose gel electrophoresis. This allows for rapid, economical, and unequivocal identification of the sex of the analyzed birds. Furthermore, the comparison of CHD1F/1R sequences revealed specific SNPs between song thrush and redwing, allowing for their differentiation by sequencing this region. These SNPs could be used to distinguish the 2 species if needed.