
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00797-1
10.1016/j.psj.2024.104218
104218
GENETICS AND MOLECULAR BIOLOGY
A selection breeding pattern for sexually dimorphic breast plumage color in Guangxi Yao chickens
Li Jianbo *†1
Wu Rifu *1
Wang Yan *
Ma Jie *‡
Peng Zhi ‡
Luo Wei *
Liu Tianfei *
Shu Dingming *
Qu Hao quhao@gdaas.cn
*2
⁎ State Key Laboratory of Swine and Poultry Breeding Industry, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China
† State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China
‡ Guangdong Wiz Agricultural Science & Technology Co. Ltd, Guangzhou, 510640, China
2 Corresponding author: quhao@gdaas.cn
1 These authors contributed equally to this work.

14 8 2024
11 2024
14 8 2024
103 11 10421815 5 2024
9 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The breast plumage color of Guangxi Yao chickens shows obvious sexual dimorphism, with roosters showing black and black with red, and hens displaying partridge and red. Black plumage in roosters is considered a sign of quality, necessitating the purification of plumage color. Here, we developed an effective method based on genetic variations within MC1R and plumage characteristics. We clarified the distribution of 5 single nucleotide polymorphisms (SNP) and 3 haplotypes (H1, H2, and H3) of MC1R gene, and revealed potential associations between haplotype H1 and black breast plumage in the F2 resource population derived from a backcross between Guangxi Yao and Yellow chickens. Subsequently, using H1/H1 diplotype roosters and hens to construct families (n = 1,244) notably increased the proportion of offspring with black plumage. Further analysis suggested that red plumage in hens may be the putative phenotype of black plumage in roosters, driven by haplotype H1 of the MC1R gene, as verified by genotype and phenotype analysis. As expected, we found that almost all male offspring of hens with red breast plumage showed black plumage. In short, we established a selection pattern based on the combination of black-plumage roosters and red-plumage hens can significantly purify the sexually dimorphic plumage color and improve the efficiency of breeding programs in Guangxi Yao chickens. Our findings provide a novel technical framework to accelerate the breeding process for plumage trait in poultry.

Key words

Guangxi Yao chicken
plumage color
sexual dimorphism
haplotype
selection model
==== Body
pmcINTRODUCTION

Plumage color is a vital quality trait in poultry, distinguishing breeds and significantly impacting consumer preferences for high-quality live poultry (Ng and Li, 2018). Given its crucial role as a selection criterion in breeding, producing chickens with plumage color variations that align with market preferences is economically significant. The Guangxi Yao chicken is highly sought due to its ability to withstand rough feeding conditions, strong resistance to stress, and exceptional meat quality (Yang et al., 2020; Sun et al., 2021). Notably, it showed distinct sexual dimorphism in plumage color. Male chickens typically display black breast plumage, with some showing black with red breasts, while females usually have red breast plumage, with some individuals having partridge- and yellow-colored breasts. Black feathers on roosters are often regarded as indicators of superior quality. However, the offspring exhibit disorganized plumage color patterns, directly influencing consumer choices and farmers' economic gains. Purifying plumage color has become a pressing issue during the breeding of Guangxi Yao chickens. The conventional approach for breeding inheritance has advanced at a slow pace, with limited enhancement in the proportion of black plumage traits among offspring. Consequently, there is a critical need to devise effective methods for controlling the sexual dimorphism in plumage color among Guangxi Yao chickens.

Previous study showed that variations in plumage color primarily result from differences in the distribution and ratio of 2 melanin types: eumelanin, causing black or brown plumages, and pheomelanin, resulting in red or yellow plumages (Moreiras et al., 2021). Melanin synthesis occurs primarily in specialized melanocytes, governed by various signaling pathways, such as the α-MSH/MC1R/cAMP, the Wnt/β-catenin, and the MAPK pathway (Liu et al., 2018; Zou et al., 2021; Singh et al., 2024). The MC1R gene, encoding a G protein-coupled receptor situated on the plasma membrane of melanocytes, stands out as a pivotal candidate gene for coat and plumage color across different vertebrates (Dürig et al., 2018; Jin et al., 2020; Jiang et al., 2021; Hauser et al., 2022; Liu et al., 2023; Cumer et al., 2024; Liu et al., 2024a). In addition, the MC1R gene, part of the melanocortin receptor (MCR) family, acts as a crucial switch governing the generation and transformation of eumelanin and pheomelanin (Liu et al., 2023). MC1R can combine with α-MSH to initiate a series of reactions, thereby regulating tyrosinase activity, which in turn affects the formation and distribution of melanin, thereby producing a range of skin, fur and feather colors (Guida et al., 2022; Liu et al., 2023). Generally, MC1R is thought to be involved in the formation of primary patterns of plumage color in chickens.

Marker-assisted selection (MAS) has been extensively in selecting and breeding plumage color traits in poultry (Huang et al., 2020; Mastrangelo et al., 2020; Zhang et al., 2020b; Xue et al., 2023). Single nucleotide polymorphism (SNP), a prevalent form of genetic variation, are commonly employed to investigate the genetic basis of diverse phenotypes and traits (Yang et al., 2017; Mastrangelo et al., 2020; Nam et al., 2021; Wang et al., 2022; Xue et al., 2023). Several single nucleotide sites in the coding sequence (CDS) region of the MC1R gene have been shown to be significantly associated with different feather colors in chickens (Zhang et al., 2017; Deng et al., 2020; Nam et al., 2021; Fan et al., 2022). Notably, the loci of c.212T>C, c.274G>A, and c.644A>C have been identified as genetic markers for black plumages in Chinese native chickens (Deng et al., 2020). Considering that traits based on genetic markers can be rapidly improved (Khan et al., 2024), identification the genetic variants responsible for the sex-specific development of the black feather trait will help establish methods to purify sexually dimorphic feather colors.

To establish an effective selection pattern and improve the proportion of black breast plumage color in Guangxi Yao chickens, we investigated the potential relevance between 3 haplotypes of MC1R gene and various plumage traits. We employed roosters with diplotype (H1/H1) and 2 types of hens (partridge and red) to form families, and found that the proportion of male individuals with black plumage was significantly increased, accompanied by a rising ratio of females with red plumage. Further, genotypic and phenotypic analyses confirmed that the red plumage in hens is a potential manifestation of the sexually dimorphic black plumage in roosters, revealing haplotype H1 as the key driver of this phenomenon. Remarkably, the selection pattern of black-plumage roosters and red-plumage hens effectively improve the number and proportion of male offspring with black plumage and enhance the efficiency of breeding process in Guangxi Yao chickens. In summary, our findings provide a novel perspective to accelerate breeding process and genetic improvement in Chinese native poultry.

MATERIALS AND METHODS

Ethics Statement

The experiments were carried out in accordance with the Guide for the Animal Care and Use Committee of Institute of Animal Science, Guangdong Academy of Agricultural Sciences.

Chicken Population Generation

Chickens were housed in climate-controlled (22°C) facilities with a 16-h light/8-h dark (16 L:8 D) cycle and were provided with a standard diet and water ad libitum. F1 individuals were generated through crossbreeding between the male Guangxi Yao chicken line (n = 48) and the female indigenous specialized yellow chicken S line (n = 100), both high-quality chicken lines from Guangdong Wiz Agricultural Science & Technology Co. Ltd. (Guangzhou, China). The F2 population (n = 241) was established by backcrossing Guangxi Yao chicken roosters with F1 generation hens. At 10 wk of age, the color of the breast and back plumage of chickens (n = 389) is evaluated.

Genomic DNA Extraction and PCR Amplification

Blood samples were collected from the aforementioned chickens and stored at −20°C. Genomic DNA was isolated from the blood samples using phenol-chloroform methods, according to the manufacturer's instructions. The concentration and optical density (OD) values at 260/280 of DNA samples were measured using a Nanodrop 2000c spectrophotometer (Thermo, Waltham, MA). A 1,308 bp DNA fragment comprising the 945 bp coding sequence of MC1R gene was amplified using the PrimeSTAR® HS DNA Polymerase with GC Buffer kit (Takara, Japan). The specific primers (forward-5′- CTTCCCCATCCTTGTGCCTG-3′, reverse-5′- CCTTTATTTGGGAGCGCGAG-3′) of MC1R gene were designed by DNAMAN version 6.0 software. The PCR thermal cycling program was as follows: 98°C for 30 s, 35 cycles of 98°C for 5 s, 62°C for 5 s, 72°C for 1min, followed by a further 1 cycle of 72°C for 5min. The PCR amplified products were sequencing by Sangon Biotech Co. Ltd. (Shanghai, China).

Polymorphism, Genotyping and Haplotype Analysis

Each experimental group consisted of at least 100 individuals, with 3 biological replicates for each PCR amplification. The screen of polymorphism of MC1R gene was performed in F2 resource population, containing 118 male chickens and 123 females. The PCR products in each individual were analyzed by the DNAMAN and Chromas software to determine the SNPs. The MC1R haplotypes were reconstructed by PHASE2.1 software (http://www.bioinf.man.ac.uk/resources/phase/). SNP, genotype, and haplotype frequency differences were analyzed using Microsoft Excel (Microsoft Corporation, Redmond, WA) and SPSS 22.0 (SPSS, Chicago, IL). Linkage disequilibrium analysis (LD) between SNPs was performed using SHEsis software (http://analysis.bio-x.cn/myAnalysis.php). Chi-square tests were used to compare the frequencies of haplotypes between different plumage color groups. Sort and classify breast plumage colors. The generalized linear model within the R package haplo.stats (https://CRAN.R-project.org/package=haplo.stats) was utilized to detect the correlation between different phenotypes and 3 haplotypes, returning a P value that indicates the significance of the correlation. P-values less than 0.05 were regarded as significant. The size and sign (positive or negative) of the haplotype effect score represent the magnitude and direction of the haplotype's impact on the trait.

RESULTS

Generation of F2 Resource Population

We employed backcross methods between Guangxi Yao chicken and Chinese native yellow chicken (F0) to generate an F2 resource population, which showed clear segregation of plumage colors (Figure 1 and Supplementary Figure S1). The F2 population was divided into 9 categories based on sex and breast and dorsum plumage colors (Table 1). The F2 male chickens (n = 118) were divided into 4 types (Table 1), including: (1) black breast & black with red dorsum (B&BR, n = 32), (2) black with red breast & black with partridge dorsum (BR&BP, n = 25), 3) red with black breast & red with partridge dorsum (RB&RP, n = 40), and 4) red breast & red with partridge dorsum (R&RP, n = 21). In the female F2 population (n = 123) (Table 1), 5 types of plumage color were observed: (1) partridge breast & brown with partridge dorsum (P&BrP, n = 32), (2) red breast & brown with partridge dorsum (R&BrP, n = 39), (3) red breast & partridge with brown dorsum (R&PBr, n = 18), (4) yellow breast & partridge with yellow dorsum (Y&PY, n = 22), and (5) yellow breast & yellow with partridge dorsum (Y&YP, n = 12).Figure 1 Schematic representation of generation of Guangxi Yao chicken population (F0 and F2, F0 represents Guangxi Yao and Chinese native Yellow chicken, F2 represents offspring of Guangxi Yao chicken based on backcross).

Figure 1

Table 1 The description of different plumage colors in F0 (Yao and Yellow chicken) and F2 population.

Table 1Population	Sex	Day	Number	Plumage	
Breast	Dorsum	Breast&Dorsum	
Yao chicken	Male	189	48	B	BP	B&BP	
Yellow chicken	Female	230	100	R	R	R&R	
F2	Male	88	32	B	BR	B&BR	
25	BR	BP	BR&BP	
40	RB	RP	RB&RP	
21	R	RP	R&RP	
Female	88	32	P	BrP	P&BrP	
22	R	PBr	R&PBr	
39	R	BrP	R&BrP	
18	Y	PY	Y&PY	
12	Y	YP	Y&YP	
Abbreviations: B, black; BP, black with partridge; BR, black with red; Br, brown; BrP, brown with partridge; P, partridge; PBr, partridge with brown; PY, partridge with yellow; R, red; RP, red with partridge; Y, yellow; YP, yellow with partridge.

Genotype Frequency Distribution of SNP

We examined SNPs in MC1R gene from F0 (n = 148) and F2 (n = 241) populations via sequence alignment. Only 5 SNPs were detected in the CDS region of MC1R gene, including one synonymous mutation (c.69C>T) and 4 missense mutations (c.212T>C, c.274G>A, c.644A>C, and c.919C>G) (Figure 2). In total, 389 chicken genomic DNA templates were genotyped, and the genotype frequencies of the 5 SNPs were shown in Table 2. The CC, TT, GG, and AA genotypes of these SNPs (c.69C>T, c.212T>C, c.274G>A, and c.644A>C), which serve as genetic markers for black plumage in Chinese chicken, displayed the same frequency in the consistent chicken populations (Guangxi Yao (F = 1.00); F2 male (B&BP, F = 0.94); and F2 female (R&BrP, F = 0.90)) (Table 2). Oppositely, TT, CC, AA, and CC genotypes of these SNP were predominant in Chinese native Yellow chicken (F = 0.83) (Table 2). Using the SHEsis software, we analyzed the LD pattern among the 5 SNPs (Figure 3). The LD map was generated based on r2. The r2 among 4 SNPs (c.69C>T, c.212T>C, c.274G>A, and c.644A>C) are all 1.00 (Figure 3), indicating that the 4 SNPs are in complete linkage disequilibrium. We employed the chi-squared test to compare genotype frequencies of 5 SNPs in the MC1R gene across different plumage color groups. The genotype distribution of these SNPs (c.69C>T, c.212T>C, c.274G>A, and c.644A>C) in the F2 chicken populations with different plumage colors was significant (P < 0.01, Table 2). However, the distribution of SNP (c.644A>C) significantly differs (P < 0.05) only in the F2 female population with different plumage colors.Figure 2 The sequencing results of the CDS in the MC1R gene in F0 and F2 populations revealed 5 SNPs: c.69C>T, c.212T>C, c.274G>A, c.644A>C, and c.919C>G.

Figure 2

Table 2 The genotype frequency and diversity parameters in F0 (Yao and Yellow chicken) and F2 population.

Table 2SNP	Genotype	Yao chicken	Yellow chicken	F2 (Male)	F2 (Female)	
B&BP	R&R	B&BR	BR&BP	RB&RP	R&RP	χ2	P value	P&BrP	R&BrP	R&PBr	Y&PY	Y&YP	χ2	P-value	
C69T	CC	1	0.01	0.94	0.4	0.5	0.38	26.43	<0.01	0	0.9	0.55	0.67	0.42	61.52	<0.01	
CT	0	0.16	0.06	0.56	0.45	0.62	0.94	0.1	0.46	0.33	0.58	
TT	0	0.83	0	0.04	0.05	0	0.06	0	0	0	0	
T212C	TT	1	0.01	0.94	0.4	0.5	0.38	26.43	<0.01	0	0.9	0.55	0.67	0.42	61.52	<0.01	
TC	0	0.16	0.06	0.56	0.45	0.62	0.94	0.1	0.46	0.33	0.58	
CC	0	0.83	0	0.04	0.05	0	0.06	0	0	0	0	
G274A	GG	1	0.01	0.94	0.4	0.5	0.38	26.43	<0.01	0	0.9	0.55	0.67	0.42	61.52	<0.01	
GA	0	0.16	0.06	0.56	0.45	0.62	0.94	0.1	0.46	0.33	0.58	
AA	0	0.83	0	0.04	0.05	0	0.06	0	0	0	0	
A644C	AA	1	0.01	0.94	0.4	0.5	0.38	26.43	<0.01	0	0.9	0.55	0.67	0.42	61.52	<0.01	
AC	0	0.16	0.06	0.56	0.45	0.62	0.94	0.1	0.46	0.33	0.58	
CC	0	0.83	0	0.04	0.05	0	0.06	0	0	0	0	
C919G	CC	0.21	1	0.38	0.52	0.4	0.48	3.1	0.798	0.59	0.41	0.23	0.44	0.25	16.68	<0.05	
CG	0.54	0	0.44	0.4	0.5	0.38	0.41	0.33	0.59	0.39	0.42	
GG	0.25	0	0.19	0.08	0.1	0.14	0	0.26	0.18	0.17	0.33	

Figure 3 Linkage disequilibrium analysis of SNPs in the MC1R gene was conducted based on r2.

Figure 3

Association of Haplotypes With Plumage Color Traits

To further investigate the relationship between the MC1R gene and different plumage color phenotypes, we conducted haplotype construction and analysis based on 5 SNP. We identified 3 haplotypes, including H1 (CTGAC), H2 (CTGAG), and H3 (TCACC), in F0 and F2 chicken populations (Table 3). H1 and H2 were dominant haplotypes in F0 (Guangxi Yao chickens) with distribution frequencies of 0.48 and 0.52 (Table 3), respectively. In addition, H1 and H2 were advantage haplotypes in F2 male population with black breast and black with red dorsum (H1 = 0.56 and H2 = 0.41) and F2 female population with red breast and brown with partridge dorsum (H1 = 0.50 and H2 = 0.45) (Table 3). Notably, H3 was the advantage haplotype in Chinese native Yellow chicken, with a distribution frequency of 0.91 (Table 3).Table 3 The haplotype frequency in F0 (Yao and Yellow chicken) and F2 population.

Table 3Haplotype	SNP	Yao chicken	Yellow chicken	F2 male	F2 female	
69	212	274	644	919	B&BP	R&R	B&BR	BR&BP	RB&RP	R&RP	Total	P&BrP	R&BrP	R&PBr	Y&PY	Y&YP	Total	
H1	C	T	G	A	C	0.48	0.09	0.56	0.44	0.38	0.36	0.436	0.27	0.5	0.3	0.47	0.17	0.366	
H2	C	T	G	A	G	0.52	0	0.41	0.24	0.35	0.33	0.339	0.2	0.45	0.48	0.36	0.54	0.386	
H3	T	C	A	C	C	0	0.91	0.03	0.32	0.28	0.31	0.225	0.53	0.05	0.23	0.17	0.29	0.248	

Given that breast plumage purification in Guangxi Yao chicken populations is the breeding goal, our subsequent research will focus on this trait. To determine the key factor controlling breast plumage color on the MC1R gene, we performed association analysis between different haplotypes and plumage traits in the F2 population. Breast plumage color was categorized by depth of color (Table 4). We found that the haplotype H1 (Effect < 0, P < 0.05) and H3 (Effect > 0, P < 0.05) were significantly associated with black and red breast plumage color in F2 male chickens, respectively (Table 5). Furthermore, the haplotype H2 (Effect > 0, P < 0.05) and H3 (Effect < 0, P < 0.05) were significantly associated with partridge and yellow breast plumage color in F2 female chickens, respectively. In light of the association analysis result, we hypothesized that the haplotype H1 might be the core driver of black breast plumage phenomenon in male Guangxi Yao chickens.Table 4 The sorted list of breast plumage in the F2 population.

Table 4Chicken	Breast	Number	
F2 male	B	32	
BR	25	
RB	40	
R	21	
F2 female	P	32	
R	61	
Y	30	

Table 5 Association analysis between haplotypes and breast plumage in the F2 population.

Table 5F2	Haplotype	Frequency	Effect	P	
Male	H1	0.44	−2.63	0.01	
H2	0.34	−0.47	0.64	
H3	0.23	3.55	0.00	
Female	H1	0.37	0.95	0.34	
H2	0.39	2.52	0.01	
H3	0.25	−5.05	0.00	

Application of Haplotype for Plumage Color Purification

In the Guangxi Yao chicken purebred population, we counted the number of various breast plumage colors. We found that 62.14% of roosters had black plumage and 37.86% had black with red plumage (Table 6). Among hens, 33.56% had partridge plumage and 66.44% had red plumage (Table 6). To confirm the regulatory role of haplotype H1, we selected H1/H1 diplotype roosters with black breast plumage trait and hens with partridge (H1/H1, H1/H2, H2/H2, H1/H3, and H2/H3) and red (H1/H1, H1/H2, and H2/H2) breast plumage colors to construct family pedigrees (n = 1,244) (Table 7). As expected, the proportion of males with black breast plumage phenomenon increased to 89.57%, and the proportion of hens with red breast plumage color rose to 90.66% (Table 7), significantly higher than before selection (62.14% and 66.44%, respectively) (Table 6).Table 6 The proportion of different plumage colors in Guangxi Yao chickens.

Table 6Sex	Plumage	Number	Proportion	
Male	B	1,699	62.14%	
BR	1,035	37.86%	
Female	P	1,431	33.56%	
R	2,833	66.44%	
Total		6,998		

Table 7 The number and proportion of haplotypes and plumage colors in Guangxi Yao chickens.

Table 7Sex	Plumage	Number	Proportion	Diplotype	Number	Proportion	
Male	B	481	89.57%	H1/H1	239	49.49%	
H1/H2	242	50.31%	
H1/H3	0	0.00%	
BR	56	10.43%	H1/H1	7	12.50%	
H1/H2	6	10.71%	
H1/H3	43	76.79%	
Female	P	66	9.34%	H1/H1	9	13.64%	
H1/H2	6	9.09%	
H1/H3	51	77.27%	
R	641	90.66%	H1/H1	338	52.73%	
H1/H2	303	47.27%	
H1/H3	0	0.00%	
Total		1,244			1,244		

To get a better understanding of the phenomenon, we detected the genotype of MC1R gene in the whole offspring of Guangxi Yao chickens. In total, 3 diplotypes (H1/H1, H1/H2 and H1/H3) were present in the chicken populations (Table 7). H1/H3 showed a higher proportion in male chicken with black with red breast plumage (76.79%) and in females with partridge breast plumage (77.27%) (Table 7). Interestingly, H1/H1 and H1/H2 were the dominant diplotypes in male chicken with black breast plumage (49.49% and 50.31%) and in females with red breast plumage (52.73% and 47.27%) (Table 7). Based on these observations, we hypothesized that red plumage in hens may be a potential manifestation of sexually dimorphic black plumage in roosters.

In order to substantiate this hypothesis, we calculated the number and proportion of various plumage colors in the offspring of hens with partridge and red breast plumage. Interestingly, a notable observation was found in the plumage color variants of the offspring. In the offspring (n = 477) of hens with partridge breast plumage color, the proportion of black breast plumage phenomenon of male individuals and red breast plumages of females increased to 76.73% and 77.09%, respectively (Table 8). Notably, in the offspring (n = 767) of hens with red breast plumage color, the proportion rose to 97.31% and 99.31%, respectively (Table 8). These results indicate that phenotype selection of hens with red plumage can effectively improve the ratio of male offspring with black breast plumage color traits in Guangxi Yao chickens.Table 8 The proportion of different plumage colors in offspring of Guangxi Yao chickens.

Table 8Parent	Offspring	Number	Propotion	
Male	Female	Male	Female	
B	P	B		155	76.73%	
BR		47	23.27%	
	P	63	22.91%	
	R	212	77.09%	
R	B		326	97.31%	
BR		9	2.69%	
	P	3	0.69%	
	R	429	99.31%	
Total				1,244		

DISCUSSION

Plumage color serves as a breed and gender identifier in poultry, with significant economic implications in production (Ng and Li, 2018; Xu et al., 2022). Previous studies showed that plumage color was controlled by the MC1R gene, with variations closely linked to the black feather trait (Zhang et al., 2017; Deng et al., 2020; Kabir et al., 2020; Zhang et al., 2020b; Fan et al., 2022). In this study, we characterized SNPs in the MC1R gene of the Guangxi Yao chicken backcross population, clarified the functional relationship between haplotype and male black breast plumage color trait, and developed an effective method for purifying plumage color. The current work provides a new research framework for purifying and breeding sex-specific feather colors in native chickens.

Plumage color traits in Guangxi Yao chickens show notable diversity: roosters have black and red breast plumage, while hens exhibit red, partridge, and yellow breast plumage. Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentationv (Marathe et al., 2017). Mutations at SOX10, which underlie the dark brown phenotype, affect the balance of black eumelanin and red pheomelanin, shifting towards a more pheomelanistic (reddish) plumage color (Schwochow et al., 2021; Zhu et al., 2022). Given the similarity in plumage color changes caused by SOX10 mutations to those seen in Guangxi Yao chickens, we further examined SOX10 gene. Unexpectedly, we found that all individuals exhibited common sequence characteristics (data not shown), suggesting that the SOX10 gene is not associated with plumage color variants in Guangxi Yao chickens. These findings can contribute to clearly exploring the complex genetic basis of sexually dimorphism plumage color in chickens.

The MC1R gene plays a crucial role in regulating melanin synthesis, transport, and deposition (Guida et al., 2022), significantly impacting plumage and skin color variations (Xi et al., 2020; Ascsillán and Kemény, 2024; Castejón-Griñán et al., 2024; Liu et al., 2024b). Multiple mutation sites in the MC1R gene are associated with chicken feather color characteristics (Zhang et al., 2017; Yang et al., 2019; Zhang et al., 2020b). By characterizing the sequence of the MC1R gene, we identified only 5 SNPs in the CDS region. Three SNPs (c.212T>C, c.274G>A, and c.644A>C) are crucial markers for black feather traits in Chinese indigenous chickens (Deng et al., 2020), aligning with our findings. Further analysis revealed that these SNPs had a high genotype frequency in male Guangxi Yao chickens and F2 males with black breast plumage color traits. Comparison of SNPs and haplotypes of the MC1R gene between 30 Japanese breeds and 8 non-Japanese breeds revealed that the c.274G>A substitution contributes to black plumage coloration (Kabir et al., 2020). A previous study found that c.274G>A activates MC1R gene activity, promote the formation of eumelanin and black feathers (Kabir et al., 2020). In contrast, c.644A>C inhibits eumelanin formation and promotes pheomelanin synthesis. When both sites mutate simultaneously, the mutation at site c.274G>A does not cause black feathers due to the suppressive effect of c.644A>C (Zhang et al., 2017; Deng et al., 2020; Kabir et al., 2020). These findings indicate that the core factor of these SNPs in the MC1R gene for black plumage coloration is not a single genetic locus and requires further investigations.

Although some advances have been made in assessing genetic basis of sexually dimorphism plumage color, the core driver remain largely unknown. SNPs, as the most prevalent genetic variations in a population, generate distinct haplotypes through their linkage relationships. These haplotypes often influence various phenotypes, significantly contributing to trait diversity within the population (Zhang et al., 2020a; Ye et al., 2023). Since the functional relationship between genetic markers and trait phenotypes cannot be effectively captured by a single SNP, we used linkage disequilibrium and haplotype analysis to explore the association between genetic loci and plumage color. We found that 4 SNP sites (c.69C>T, c.212T>C, c.274G>A, and c.644A>C) were in complete linkage disequilibrium, forming 3 haplotypes in the Guangxi Yao chicken backcross population. This suggests that the black breast plumage trait may be jointly regulated by genetic variants of MC1R. Previous studies have demonstrated that the diversity of haplotypes in the MC1R gene may be the key driver of black, grey, and white plumage color in various chickens (Zhang et al., 2017; Yang et al., 2019; Kabir et al., 2020; Zhang et al., 2020b). We found haplotype H1 was significantly associated with black plumage color in F2 male chickens, suggesting that the H1 haplotype might enhance MC1R activity, thereby increasing eumelanin synthesis (Almathen et al., 2018; Kabir et al., 2020; Fan et al., 2022; Liu et al., 2024a). In addition, the H1 haplotype could include specific SNPs that alter the MC1R protein's structure or function. These changes might increase the receptor's affinity for α-MSH or its signaling efficiency (Dall'Olmo et al., 2023; Cavatão et al., 2024), promoting eumelanin production more effectively than other haplotypes.

Association analysis confirmed a significant correlation between haplotype H1 and black breast plumage color in male F2 individuals. Given its domination role in plumage coloration, we selected roosters within the diplotype (H1/H1) and hens with 2 types of breast plumage (various diplotypes) to establish families within Guangxi Yao chickens. Among their offspring, the ratio of male individuals with black breast plumage has significantly increased, indicating that molecular selection can effectively promote uniform plumage color traits. However, the exact methodology for selecting hens remains unclear due to sexual dimorphism in breast plumage patterns. Notably, there was a significant increase in hens with red breast plumage. Further analysis showed similar genotype categories and frequencies, suggesting that red plumage in hens may be a manifestation of the black plumage phenotype in roosters. To verify this hypothesis, we conducted a statistical analysis of the offspring phenotypes from different hens. Phenomenon examination revealed that nearly all male offspring of hens with red breast plumage exhibited black breast plumage, compared to those from hens with partridge breast plumage. Similarly, female offspring of hens with red breast plumage showed consistent alterations in plumage characteristics. Therefore, screening and selecting specific haplotypes associated with desired plumage colors can achieve more consistent and predictable plumage across the poultry population. For instance, selecting haplotype H1 ensure hens exhibit the desired red plumage while roosters display the preferred black plumage, aligning with breeding goals for sex-specific traits. Notably, implementing haplotypes and plumage co-selection for roosters and hens can effectively enhance the feather color uniformity and facilitate the efficiency of breeding programs in the Guangxi Yao chicken population. By using genetic markers and plumage traits to co-select ideal characteristics early, the number of generations needed to reach breeding goals is reduced, saving both time and costs associated with breeding programs.

The differences in plumage color between males and females are a prominent example of sexual dimorphism. Despite we have established selection systems to purify plumage color in the Guangxi Yao chicken population, the genetic regulatory network underlying sexual dimorphism in plumage color remains unknown. Therefore, future work should focus on identifying the spatiotemporal distribution patterns of candidate genes, involved in melanin synthesis, for sex-specific plumage color formation; methods such as single cell RNA-sequencing is ideally for capturing the high-resolution transcriptomes of candidate genes in hair follicles at different developmental stages. Explore how epigenetic modifications and hormone levels interact with genetic to influence plumage color will enhance our understanding of the regulatory landscape underlying sexual dimorphism in plumage. Future research should also validate these findings in other chicken populations with different genetic backgrounds. These research directions will deepen our understanding of the genetic mechanisms behind sex-specific plumage color differences and enhance corresponding breeding programs.

This study has several limitations. First, the sample size (n = 389) used for genotyping in this study, despite providing an ideal gene frequency distribution, may limit statistical power. Second, co-select pattern of black-plumage roosters and red-plumage hens can effectively improve the uniformity of feather color in Guangxi Yao chickens, but the validation, such as purification of sexually dimorphic plumage, has not yet been verified in other similar chicken species. Future studies should include larger sample sizes and multiple populations with different genetic backgrounds to ensure and determine the broader applicability of the results.

CONCLUSIONS

In summary, we revealed that haplotype H1 of the MC1R gene is the key factor driving the formation of black plumage in roosters, while red plumage in hens may be the corresponding phenotype. This study established a novel method for co-selecting roosters and hens based on haplotypes and phenotypic characteristics to purify plumage color traits in Guangxi Yao chickens, and showcased the power of this technical framework in accelerating the efficiency of breeding process of sex-specific plumage color traits in poultry.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This work was supported by STI2030-Major Projects (2023ZD04064 ), the Key Realm R&D Program of Guangdong Province (2022B0202100002 ) and the sub project of “Discipline Construction of Swine and Poultry Breeding Industry” of Special Project on Science and Technology Innovation Strategy (ZX202401-05 ).

Author contributions: HQ and DS conceived and designed the project. RW, YW, JM, ZP, WL and TL performed the experiments and collected samples. JL and RW analyzed the data. JL wrote the manuscript. HQ revised the manuscript. All authors read and approved the final draft.

DISCLOSURES

The authors declare no competing interests.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104218.
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