Investigation into the performance characteristics and genomic variations associated with the rumpless phenotype of the indigenous Cay Cum chickens (Gallus gallus L.)

Thi Thom Bui¹, Duc Quan Nguyen², Van Tung Nguyen², Ngoc Lan Nguyen², Thanh Hien Nguyen² and Huy Hoang Nguyen^2,3

¹ Thai Nguyen University of Agriculture and Forestry, Tan Thinh Ward, Thai Nguyen City 50000, Vietnam
nhhoang@igr.ac.vn
² Institute of Genome Research - Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, 10000, Vietnam
³ Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, Vietnam

Abstract

Cay Cum chicken, an indigenous breed in the northern regions of Vietnam, is known for its distinct rumpless phenotype, adaptability to the highland climate and resistance to common poultry diseases. Despite the fact that Cay Cum is a unique breed, little research has been done on its growth, productivity, and the mechanisms underlying the rumpless phenotype. In this study, our results revealed a substantial difference in the reproductive performance between the domestic Ri and Cay Cum chickens, with the latter breed having a lower egg-laying rate, hatchability, and egg size. On the basis of whole genome sequencing analysis, four genomic variants were identified in four genes involved in the development and axial patterning of the vertebral column, including PAX1, PAX9, WNT5B and WNT16. Our findings suggest that the structural and functional disruption of proteins encoded by the four candidate genes may be associated with the development of the rumpless phenotype in the indigenous Cay Cum chickens.

Keywords: Cay Cum chicken, growth performance, reproductive performance, rumpless phenotype, whole genome sequencing

Introduction

Cay Cum is an indigenous chicken breed in the northern provinces of Vietnam. Cay Cum chickens have been reared in the local households for decades because of their ability to adapt to the mountainous terrain and their resistance to common poultry diseases. Cay Cum chickens have a similar appearance to domestic chickens in terms of size and plumage color, with the exception of the rumpless phenotype, i.e., the lack of a pointy rump, preen gland, and the long, colorful sickle feathers. At anatomical level, the rumpless phenotype is characterized by the truncation or absence of the free caudal vertebrae (FCV) and pygostyle (P) structures (Dunn and Landauer 1934; Noorai et al 2012).

The rumpless phenotype has been reported to be caused by improper development and elongation of the vertebral column, including mouse, chicken, frog, zebrafish (Andre et al 2015; Heisenberg et al 2000; Tada and Smith 2000; Wang et al 2014); the biological processes that are regulated by a wide range of gene families, including WNTs (Karner and Long 2017; Qu et al 2021; Tada and Smith 2000), paired-box PAXs (Chen et al 2014; Sivakamasundari et al 2018), SRY-box SOX (Lefebvre 2019), collagen-type-2-alpha-1 COL2A1 (Rolvien et al 2020), aggrecan ACCAN and homeobox HOX genes (Davidson and Zon 2006; Marom et al 1997).

WNT genes encode a large family of highly conserved cysteine-rich glycoproteins (Andre et al 2015; Karner and Long 2017; Qu et al 2021). Embryological investigations in mouse and zebrafish have revealed that WNTs act as key regulators of many vertebrate developmental processes. WNT5A, WNT5B and WNT11 have been found to play a role in regulating the non-canonical WNT signaling/planar cell polarity (PCP) pathway, a process which is responsible for the posterior extension and maintenance of the notochord – a template for the vertebrate body plan (Navajas Acedo et al 2019; Ukita et al 2009; Wang et al 2014; Ward et al 2018). WNT4, WNT6, WNT9A, and WNT16 are responsible for maintaining homeostasis in the development of bones (cortical and trabecular bone mass, thickness, and strength) and joints (Qu et al 2021; Teufel et al 2018; Wang et al 2014). While WNT7, WNT10, and WNT11 induce the formation of large bone-forming cells during osteoblastogenesis (Andre et al 2015; Navajas Acedo et al 2019; Wang et al 2014).

PAX genes are members of a gene family that encodes highly conserved paired-box transcriptional activators and play an important role in the formation of the vertebral column, a load-bearing structure that is designed to support the body’s stability and mobility, in many vertebrates (Chen et al 2014; Sivakamasundari et al 2018). To date, PAX1 and PAX9 are the only two PAX genes known to regulate the differentiation of somites into sclerotomes, the cells that eventually organize to form the vertebral column and rib cartilage of chickens (Scaal 2016). The activation of PAX1 and PAX9 is induced by the Sonic hedgehog (Shh) and Noggin (Nog) signals secreted by the notochord (Sivakamasundari et al 2018). Furthermore, PAX1 and PAX9 have also been reported to control the condensation of intervertebral disc, tail and limb cartilage tissues via direct regulation of the expression of a group of genes, such as SOX5, SOX9 (Lefebvre 2019), COL2A1 (Rolvien et al 2020), ACCAN (Rashid et al 2017) and bone morphogenetic protein 4 (Bmp4) (Salazar et al 2016).

Genome wide association studies and transcriptomic analyses have shown that changes in the expression of WNTs and PAXs either by overexpression or knock out/down processes adversely disrupt the normal embryonic development of vertebrates. The consequences include increased mortality, reduced reproductive performance (Noorai et al 2012), defective vertebral column, kinked tail phenotype (Chen et al 2014; Dietrich and Gruss 1995; Navajas Acedo et al 2019; Peters et al 1999), deformed limbs, ribs and caudal vertebrae (Peters et al 1998; Qu et al 2021; Rolvien et al 2020; Sivakamasundari et al 2018) and/or shortened the anterior – posterior (A-P) axis (Davidson and Zon 2006; Lefebvre 2019; Qu et al 2021; Teufel et al 2018). These findings clearly imply that the construction of the correct body plan requires tight control and precise regulation of genes involved in the development of the vertebral column and elongation of the A-P axis.

In this study, we investigated the performance characteristics and molecular mechanisms underlying the formation of the rumpless phenotype by whole genome sequencing (WGS) analysis. Our results revealed that the growth performance of evaluated Cay Cum chickens was comparable to that of Ri chickens, with Cay Cum roosters and hens weighing roughly 2.4 and 1.9 kg at 30 weeks of age, respectively. However, Cay Cum chickens showed considerably worse reproductive performance (egg laying rate, egg weight, and hatchability) than their counterparts. Using WGS analysis, four genomic variants, c.778G>A (p.Gly260Arg), c.965C>T (p.Ala322Val), c.122G>C (p.Cys41Ser), and c.412C>T (p.Pro138Ser) were identified in WNT5B, WNT16, PAX1, and PAX9, respectively, and; they were bioinformatically predicted to be responsible for the formation of the rumpless phenotype in Cay Cum chickens.

Material and methods

Animals

The domestic, Ri, and Cay Cum chickens (Gallus gallus L.) used in this study were raised with standard daily water and food supplies at Thai Nguyen University of Agriculture and Forestry, Vietnam. All chickens were vaccinated to prevent common poultry diseases. Ri chickens with pointy tail shapes and preen glands (normal phenotype), and Cay Cum chickens with rounded rumps and a lack of preen glands (rumpless phenotype) were kept apart to avoid crossbreeding.

Observation of the appearance and tailbone structure

Twelve Cay Cum roosters and hens were chosen for examination of the size and color of their combs, beaks, feathers, shanks, and toes. Three roosters and hens of Ri (control) and Cay Cum were slaughtered at a local butcher according to the slaughtering method demonstrated by Zaman et al (2017). The FCV and P structures were photographed for visual assessment.

Analysis of body weight and reproductive performance

The body weight of twelve randomly selected Ri and Cay Cum chickens was measured at 15, 25, and 30 weeks of age.

Reproductive performances, such as the number of eggs laid by a hen in a month (monthly average), egg-laying rate, and egg weight, were conducted on three groups of six Ri and Cay Cum hens at 25 weeks of age for three months. The total number of eggs laid and the total weight of eggs were recorded.

To test for the rate of hatchability, twenty-six to thirty fertilized eggs from Ri and Cay Cum chickens were incubated in an electric Laminar 020 egg incubator (Mitsubishi, Japan) for 21 days at 37.5 ± 0.5^oC and 55% humidity. The experiment was repeated for three months.

Whole genome sequencing and variants screening

Blood samples were collected from the wing veins of five Cay Cum chickens at 30 weeks of age. Blood collection practices were approved by Thai Nguyen University with the protocol number of DHNLTN 2019-106. Genomic DNA was isolated from blood samples using QIAamp DNA Blood Kits (Qiagen, Germany) according to the manufacturer’s instructions with some minor modifications. The DNA concentration and quality were determined using a NanoDrop™ 1000 spectrophotometer (ThermoFisher Scientific, USA) and via standard electrophoresis on a 1.0% agarose gel. Good quality DNA samples, which had an A260/A280 ratio of 1.8–2.0, were chosen for whole genome sequencing.

DNA samples for WGS were prepared by pooling 1 µg of DNA from each of the five chickens. All the sequencing data in this study were obtained from this DNA sample. Library preparation and WGS were performed using the TruSeq Nano DNA Kit (Illumina, USA) and the Illumina HiSeq 2500 platform (USA), respectively. Post the completion of sequencing, the high quality read pairs were filtered and extracted with the Trimmomatic (version 0.38; RRID: SCR_011848) and FastQC (version 0.11.8; RRID: SCR_014583) tools. The high quality data was aligned to the chicken reference genome ( Gallus_gallus-6.0; http://ftp.ensembl.org/pub/release-104/fasta/gallus_gallus/dna) using the Burrows-Wheeler Aligner (version 0.7.17; RRID: SCR_010910). The duplicated reads from the BAM and SAM files were removed by the Sambamba tool (version 0.6.8; http://lomereiter.github.io/sambamba/) using the CIGAR string. Finally, the SnpEff tool (version 4.3t; RRID: SCR_005191) was used to identify single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) variants.

Potential variants causing rumplessness of Cay Cum chickens were screened by searching for genes involved in development of the vertebral column and elongation of the A-P axis. All the identified intronic and synonymous variants were excluded. The identified variants in the Cay Cum genome sequencing data were also investigated by the variant effect predictor tool on the Ensembl genome database (RRID: SCR_002344).

Confirmation of the identified variants

Polymerase chain amplification reaction (PCR) was performed using the primer sets listed in Table S1, and the obtained PCR products were then sequenced. Sequencing results were compared to the reference sequences of PAX1 (gene ID: 100859138), PAX9 (395740), WNT5B (418154), and WNT16 (427856), obtained from the NCBI database using the Geneious software (RRID: SCR_010519).

The existence of the four variants at the indicated locations in PAX1, PAX9, WNT5B, and WNT16 was confirmed by Sanger sequencing (Figure S1). These results also clearly validate the accuracy and reliability of the obtained WGS data.

Gene ontology and pathway enrichment analyses

The identified variants were subjected to Gene Ontology (GO) annotation (RRID: SCR_002811) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (RRID: SCR_012773) with the default analysis settings to determine their biological function, cellular component, biological process, and metabolic pathway.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Results and discussion

Appearance and tailbone structure of Cay Cum chicken

Appearance observation of twelve Cay Cum chickens revealed a clear distinction between the roosters and the hens. The plumage of the roosters is more colorful and eye-catching, with the color ranging from bright orange to blackish red (Figure 1A). It is also made up of a thicker set of long and pointed neck and back feathers. The colorful and attractive plumage of the roosters is believed to show their sexual and territory supremacy over other roosters (Jawor and Breitwisch 2003; Roulin 2004). The hens, on the other hand, have feathers that are thinner, shorter, rounder with a dull color scheme of mostly dark yellow and brown coloration (Figure 1D). Cay Cum chickens were found to have different types of comb. The single comb type was found solely in the roosters, whereas the rose comb was only observed in the hens. Cay Cum roosters and hens both have a sharp, pointed yellow beak, yellow-to-brown colored shanks, and long toes (Figure 1).

Carcasses and tailbones analyses revealed a distinct morphology of Cay Cum chickens (Figure 1). The carcasses of Cay Cum chickens display a rounded rump with no preen glands (Figure 1B, C, E, and F). Because of this distinct feature, Cay Cum chickens are often found to be lacking long, colorful sickle feathers. At anatomical level, in contrast to the presence of the fully developed synsacrum, FCV and P structures in the Ri chickens, Cay Cum chickens display a severely truncated FCV (with less than two pieces of FCV bone in all chickens observed) and no P tailbone structures (Figure 1G, H, and I).

Figure 1. Appearance and skeleton analyses of the indigenous Cay Cum chicken. Cay Cum rooster (A) and hen (D) at 30 weeks old. Carcass of Cay Cum rooster (B and C) and hen (E and F) with the rumpless phenotype. (G) Skeleton of the Ri chicken (control) with the presence of synsacrum (S), free caudal vertebrae (FCV) and pygostyle (P) structures. (H) Skeleton of Cay Cum rooster with truncated FCV and no P structures. (I) Skeleton of Cay Cum hen with no FCV and P structures.

Growth and reproductive performance characteristics of Cay Cum chickens

The body weight of Cay Cum chickens was measured at three stages of their development. The 15 weeks of age - the adolescent period of development at which the physical distinctions between male and female birds become more apparent (Ohkubo et al 2000). The 25 and 30 weeks of age – the stages at which Cay Cum chickens reach full sexual maturity and physical development (adult), respectively (Ohkubo et al 2000). Across three groups of age, the roosters were found to be heavier than the hens, weighing roughly 0.29 ± 0.02, 0.34 ± 0.02 and 0.46 ± 0.03 kg (Figure 2). Specifically, Cay Cum roosters weighed 1.34 ± 0.05, 1.75 ± 0.09 and 2.35 ± 0.1 kg, and their counterparts weighed 1.05 ± 0.07, 1.39 ± 0.11 and 1.89 ± 0.13 kg at 15, 25 and 30 weeks old, respectively. Interestingly, there was no significant difference in body weight between Ri and Cay Cum chickens across both sexes and all age groups (Figure 2). The obtained statistical data clearly indicates that the lack of tail and preen glands has no negative impact on the overall growth performance of all Cay Cum chickens evaluated. Our findings were consistent with those of Sandilands et al (2004), Chen et al (2015) and Wang et al (2017), who reported that body weight had no correlation with the sexes as well as the size and weight of preen glands in ducks and chickens.

Figure 2. Body weight of Ri and Cay Cum chickens at 15, 25 and 30 weeks of age

The weight difference is significant between the roosters and hens, but insignificant between the two breeds across all sexes and age groups. Statistical analysis was done using One-way ANOVA with posthoc Tukey. The statistically significant differences are represented by a different letter (p-value < 0.05).

Despite their physical similarities, Cay Cum and the Ri hens showed statistically significant differences in all reproductive performance parameters, including the quantity of produced eggs, egg weight, egg-laying rate, and hatchability (Table 1). Cay Cum chickens lay eggs at approximately half the rate of domestic hens. Specifically, a Cay Cum hen produced 12.3 ± 0.99 eggs per month on average, compared to 20.1 ± 1.2 eggs per month on average for a Ri hen (Table 1). Furthermore, Cay Cum hens were also found to lay eggs that were approximately 14.9% smaller in size (51.5 ± 2.3 g) than their counterparts (64.1 ± 3.1 g). In terms of hatchability, the majority of fertilized eggs produced by the domestic hens were hatched into baby chicks, namely 95.2 ± 0.7%. However, only 78.1 ± 0.7 % of the eggs produced by Cay Cum hens were successfully hatched (Table 1). The lower rate of hatchability is thought to be due to the smaller size of the Cay Cum eggs. Previous research on the hatchability of various chicken breeds, such as Vanaraja and Gramapriya (Patra et al 2016), Venda (Mbajiorgu 2011) and Atak-S chickens (Kamanli et al 2010), showed that small-sized eggs had a higher rate of embryonic mortality due to smaller yolks and an insufficient supply of essential nutrients required for embryonic development. (Patra et al 2016; Ramaphala 2013).

In summary, our findings reveal that the growth performance of Cay Cum and Ri chicken breeds was comparable regardless of their tailbone structures. However, Cay Cum chickens were found to have a significantly lower reproductive rate than their counterparts. More studies on Cay Cum chickens are required to determine any possible correlations between the poor reproductive performance and their rumpless phenotype.

Genome sequencing and variant identification

The total number of cleaned reads for Cay Cum genome sequencing was 278.1 million base pairs (bp), with the mean read length of 151 bp and the sequencing depth of 36.3X. The ratio of bases with a Phred quality score of 20 (Q20) had reached 98.0%. The high scores of Q20 indicated that the majority of nucleotide bases identified by the automated sequencing platform were accurately called and suitable for the downstream processes (Leggett et al 2013). The aligning rate of reads to the reference genome of Gallus_gallus-6.0 was also high in all samples assessed, accounting for more than 97.0%. Our obtained WGS data have been shown to possess similar parameters to those reported in research on rumpless American Araucana, rumpless Korean Araucana, Korean domestic, White Leghorn and DongXiang (Freese et al 2014; Noorai et al 2019; Zhao et al 2018). These parameters indicated that the obtained WGS data was of excellent quality and suitable for subsequent analyses.

For variant identification, cleaned reads were analyzed by SAMTools. Excluding the multi-allelic sites, a total of 7,550,208 good quality variants were identified from the WGS data of Cay Cum chicken. In which, homozygous and heterozygous variants accounted for approximately 62.0 and 37.0%, respectively. The intronic, intergenic, and exonic variants accounted for more than 47.0, 28.0, and 0.4% of the total number of identified variants, respectively. The variants were also detected in 16.0% and 5.0% of the Cay Cum genome's upstream and downstream regions, respectively.

Potential variants causing the rumpless phenotype in Cay Cum chickens

In this study, four variants, c.778G>A (p.Gly260Arg), c.965C>T (p.Ala322Val), c.122G>C (p.Cys41Ser), and c.412C>T (p.Pro138Ser), were identified in PAX1, PAX9, WNT5B, and WNT16, respectively (Table 2). The defective potential of the four variants was predicted to be deleterious by the variant effect predictor tool on the Ensembl database (Table 2). The biological functions of the four genes in Cay Cum chickens were then investigated using GO, KEGG enrichment, and multiple alignment analyses. The bioinformatic results revealed that PAX1 and PAX9 are members of a transcriptional activator family that is involved in the development of the vertebral column, whereas WNT5B and WNT16 are signaling molecules that regulate the elongation of the A-P axis in vertebrates (Table 3) (Andre et al 2015; Freese et al 2014; Hardy et al 2008; Sivakamasundari et al 2018). Multiple alignment analysis further revealed that PAX1 and PAX9 in Cay Cum chickens contain the majority of amino acid residues of the two paired DNA-binding subdomains, PAI (H1–H3) and RED (H4–H6), and the linker connecting two subdomains that are highly conserved in PAX proteins in human, mouse and zebrafish (Figure S2). Similarly, WNT5B and WNT16 have been found to contain up to 24 conversed cysteine residues and the signature Frizzled receptor binding site of WNT proteins in human, mouse, and zebrafish (Figure S3). These findings strongly suggest that PAX1, PAX9, WNT5B, and WNT16 are evolutionarily conversed in Cay Cum chickens, and they are probably involved in the formation of the vertebral column and elongation of the A-P axis of Cay Cum chickens (Hikasa and Sokol 2013; Lako et al 1998; Yang and Mlodzik 2015).

During embryonic development, PAX1 and PAX9 are the only two transcriptional activators that are co-expressed in the ventromedial compartments of the somites to regulate the differentiation of the somites into sclerotomes and the formation of the vertebral column (Peters et al 1999; Sivakamasundari et al 2018). Genetic disruption of PAX1 and PAX9 has been shown to have adverse effects on the formation of bones in many vertebrates (Chen et al 2014; Peters et al 1998; Peters et al 1999; Sivakamasundari et al 2018). In zebrafish, knocking down PAX1 and PAX9 expression resulted in severe malformation of fin buds (Chen et al 2014). In mouse, pax1 mutant, undulated, possessed a short and kinked tail; the phenotype that was revealed to be the consequence of the lumbar vertebrae truncation and the caudal vertebrae misalignment (Dietrich and Gruss 1995; Peters et al 1999). Pax9 mutant mice suffered from defects in limb formation, such as pre-axial polydactyly of the fore- and hind-limbs (Peters et al 1998; Sivakamasundari et al 2018). Loss-of-function PAX1 and PAX9 mice showed even more severe vertebral column defects, with the most defective phenotype displaying a total loss and/or malformation of the vertebral body, intervertebral discs, caudal vertebrae, and ribs (Sivakamasundari et al 2018).

Skeletal abnormalities in vertebrates have also been reported to be associated with the changes in expression of numerous members of theWNT family (Andre et al 2015; Qu et al 2021; Wang et al 2014). WNT5 is involved in the non-canonical WNT signaling/PCP pathway to regulate the development of the vertebral column, elongation of the A-P axis, and differentiation of osteoblasts (Table 3) (Andre et al 2015; Hardy et al 2008). Blocking of the non-canonical WNT signaling pathway can lead to the disruption of osteoblastogensis and the development of a normal body plan (Andre et al 2015; Heisenberg et al 2000). For example, the wnt5 zebrafish (pipetail) and mouse mutants have been shown to exhibit defective elongation at the posterior of the embryo (Andre et al 2015; Heisenberg et al 2000). Similar results were also observed when the expression of WNT16 was knocked out (Qu et al 2021). WNT16 encodes a signaling molecule that is known to be involved in non-canonical WNT signaling/PCP, mTOR, FoxO, and VEGF pathways to control bone formation and resorption via regulating osteoblast and osteoclast activities, respectively, as well as cartilage formation at limb and tail buds (Table 3). As a result of the loss of WNT16, mouse and zebrafish mutants exhibit various abnormalities in the structure and mineral density of their heads, vertebral columns, and tail bones (Ohlsson et al 2018; Qu et al 2021; Wang et al 2014).

Conclusion

• Our study provides fundamental insights into the performance characteristics and the mechanisms underlying the rumpless phenotype of the indigenous Cay Cum chickens. Interestingly, the rumplessness has been found to have no detrimental effect on growth performance, but it does appear to have a negative impact on the reproductive performance of Cay Cum chickens, as evidenced by the poor egg-laying and hatchability rates.
• The WGS analysis also identified four deleterious variants in PAX1, PAX9, WNT5B, and WNT16 genes.
• The findings of our study provide one potential explanation for the malformation of vertebral column development and defective elongation of the A-P axis; the problems that are leading to the formation of the rumpless phenotype in Cay Cum chickens. However, further molecular studies are still required to confirm the link between the disruption of these four gene and the rumplessness in Cay Cum chickens.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Acknowledgement

The authors would like to sincerely thank the Vietnam Academy of Science and Technology for funding our study.

This research was funded by the Vietnam Academy of Science and Technology, grant number NCVCC40.01/22-23.

TTB and HHN conceived the study. TTB, DQN, VTN, NLN, THN and HHN conducted the experiments and analyzed the data. DQN prepared the manuscript. All authors read and approved the final version of the manuscript.

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