基于多组学解析B380鸡黑尾与白尾羽色性状的相关候选基因

doi:10.3864/j.issn.0578-1752.2026.16.014

摘要/Abstract

摘要：

【目的】探究Babcock B380 黑白尾羽颜色形成机制，发掘与尾羽颜色相关关键候选基因及位点。为家禽育种和羽毛颜色相关性状的分子育种提供理论依据。【方法】以80只B380黑尾和白尾母鸡组成的自然群体为材料，通过组织切片（光学显微镜）和透射电镜观察，分析毛囊黑素小体的形态与分布；利用转录组数据分析该材料的差异表达基因，并通过加权基因共表达网络分析（weighted gene co-expression network analysis，WGCNA）探究与尾羽颜色相关的共表达模块，识别模块核心基因，实时荧光定量 PCR（quantitative real-time PCR，qRT-PCR）方法对差异表达基因进行验证。结合自然群体基因型分析结果，利用全基因组关联分析（genome-wide association study，GWAS）方法，挖掘与Babcock B380黑/白尾羽颜色相关的遗传位点，并对其进行群体验证及整合分析，同时分析PMEL mRNA密码子偏好性。【结果】显微镜观察发现，黑素小体主要沉积于鸡毛囊羽轴区域，黑尾鸡黑素小体面积显著高于白尾鸡。转录组分析筛选到373个差异表达基因（differentially expressed genes，DEGs），通路富集到黑色素合成过程，PMEL、TYR、TYRP1和SLC45A2在黑尾毛囊高表达。WGCNA富集得到25个模块，MEmagenta模块与黑素小体合成密切相关，网络连通性与蛋白互作网络分析挖掘到10个潜在调控核心基因，包括：PMEL、PIAS2、NDEL1、MSRA、HDLBP、GCNT4、FMR1NB、TBLX1、ST3GAL5、RAB11A。qRT-PCR结果显示4个DEGs表达趋势与RNA-seq结果一致。GWAS分析共筛选出968个显著SNP位点，分别位于鸡1、5和34号染色体上，注释到221个基因，7个PMEL SNP与表型显著关联；rs316665588位点与黑白尾羽性状显著关联，密码子偏好性指数（codon bias index，CBI）显示该位点突变导致稀有密码子UGU变为常见密码子UGC。【结论】 Babcock B380 黑尾鸡毛囊中黑素小体分布面积显著高于白尾鸡。转录组筛选出4个羽色关键DEGs，鉴定到10个潜在调控核心基因。PMEL为B380黑尾白尾性状候选基因，rs316665588 T/C 同义突变是Babcock B380黑/白尾形成的重要候选位点。

关键词: B380鸡, 全基因组关联分析, 转录组分析, 密码子偏好性, PMEL

Abstract:

【Objective】 To investigate the molecular mechanisms underlying black-tailed and white-tailed feather coloration in Babcock B380 and to identify key candidate genes and loci associated with tail feather color. It provides a theoretical basis for poultry breeding and molecular breeding of feather color related traits.【Method】 A natural population consisting of 80 Babcock B380 hens with either black-tailed or white-tailed phenotypes was used in this study. The morphology and distribution of melanosomes within tail feather follicles were analyzed using histological sections observed under light microscopy and transmission electron microscopy. Transcriptome data were utilized to identify differentially expressed genes (DEGs), and weighted gene co-expression network analysis (WGCNA) was performed to identify co-expression modules associated with tail feather color and to screen hub genes within key modules. The expression levels of selected DEGs were subsequently validated by quantitative real-time PCR (qRT-PCR). Furthermore, based on genotypic data from the natural population, a genome-wide association study (GWAS) was conducted to identify genetic loci associated with black-tailed and white-tailed feather coloration in Babcock B380 chickens. The identified loci were further validated at the population level and subjected to integrative analysis. In addition, codon usage bias of PMEL mRNA was analyzed.【Result】 Microscopic observations revealed that melanosomes were predominantly deposited in the feather shaft region of chicken feather follicles, with the melanosome area being significantly greater in black-tailed chickens than in white-tailed chickens. Transcriptome analysis identified 373 DEGs, which were significantly enriched in melanin biosynthesis- related pathways. Key pigmentation-related genes, including PMEL, TYR, TYRP1, and SLC45A2, were highly expressed in black-tailed feather follicles. Weighted gene co-expression network analysis (WGCNA) identified 25 co-expression modules, among which the MEmagenta module was closely associated with melanosome biosynthesis. Integrated analyses of network connectivity and protein-protein interaction networks identified ten potential hub regulatory genes: PMEL, PIAS2, NDEL1, MSRA, HDLBP, GCNT4, FMR1NB, TBLX1, ST3GAL5, and RAB11A. qRT-PCR validation showed that the expression patterns of 4 DEGs were consistent with the RNA-seq results. GWAS identified a total of 968 significant SNPs located on chicken chromosomes 1, 5, and 34, which were annotated to 221 genes. Among these, 7 SNPs within the PMEL gene showed significant associations with tail feather color. Notably, the rs316665588 locus was significantly associated with black-tailed and white-tailed feather phenotypes. Codon bias index (CBI) analysis indicated that the mutation at this locus resulted in a transition from the rare codon UGU to the more frequently used codon UGC.【Conclusion】 The melanosome distribution area in feather follicles was significantly larger in B380 black-tailed chickens than in their white-tailed counterparts. Transcriptome analysis identified 4 key DEGs associated with plumage coloration and revealed 10 potential hub regulatory genes. PMEL was identified as a candidate gene underlying the black-tailed and white-tailed phenotypes in B380 chickens, and the synonymous T/C mutation at rs316665588 represents a key candidate locus contributing to the formation of black/white tail plumage.

Key words: B380 chicken, genome-wide association study (GWAS), transcriptome analysis, codon usage bias, PMEL

任敏鹏, 张夕霏, 葛华梁, 耿立英, 李祥龙, 禹保军, 王坤杨, 张传生. 基于多组学解析B380鸡黑尾与白尾羽色性状的相关候选基因[J]. 中国农业科学, 2026, 59(16): 3673-3686.

REN MinPeng, ZHANG XiFei, GE HuaLiang, GENG LiYing, LI XiangLong, YU BaoJun, WANG KunYang, ZHANG ChuanSheng. Identification of Candidate Genes Associated with Black-Tailed and White-Tailed Plumage Traits in B380 Chickens Based on Multi- Omics Analysis[J]. Scientia Agricultura Sinica, 2026, 59(16): 3673-3686.

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参考文献 38

[1]	李东华, 付亚伟, 张晨曦, 曹艳芳, 李文婷, 李转见, 康相涛, 孙桂荣. 淅川乌骨鸡全基因组转座子的鉴定与分析[J]. 中国农业科学, 2020, 53(7): 1491-1500. DOI: 10.3864/j.issn.0578-1752.2020.07.017.
	Li D H, Fu Y W, Zhang C X, Cao Y F, Li W T, Li Z J, Kang X T, Sun G R. Genome-wide identification and characterization of transposable elements in Xichuan black-bone chicken[J]. Scientia Agricultura Sinica, 2020, 53(7): 1491-1500. DOI: 10.3864/j.issn.0578-1752.2020.07.017. (in Chinese)
[2]	D’Alba L, Van Hemert C, Spencer K A, Heidinger B J, Gill L, Evans N P, Monaghan P, Handel C M, Shawkey M D. Melanin-based color of plumage: Role of condition and of feathers' microstructure[J]. Integrative and Comparative Biology, 2014, 54(4): 633-644. doi: 10.1093/icb/icu094 pmid: 24987010
[3]	Ito S, Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin in humans, mice, and other animals: A comparative review[J]. Pigment Cell Research, 2003, 16(5): 523-531. pmid: 12950732
[4]	Cao W, Zhou X H, McCallum N C, Hu Z Y, Ni Q Z, Kapoor U, Heil C M, Cay K S, Zand T, Mantanona A J, Jayaraman A, Dhinojwala A, Deheyn D D, Shawkey M D, Burkart M D, Rinehart J D, Gianneschi N C. Unraveling the structure and function of melanin through synthesis[J]. Journal of the American Chemical Society, 2021, 143(7): 2622-2637. doi: 10.1021/jacs.0c12322 pmid: 33560127
[5]	赵兵令, 李亚楠, 陈天直, 刘颖, 常露程, 范瑞文, 薛霖莉, 王海东, 董常生. GPNMB通过调控MITF下游色素相关基因表达影响黑色素细胞色素的生成[J]. 中国农业科学, 2017, 50(7): 1334-1342. DOI: 10.3864/j.issn.0578-1752.2017.07.016.
	Zhao B L, Li Y N, Chen T Z, Liu Y, Chang L C, Fan R W, Xue L L, Wang H D, Dong C S. GPNMB affects melanin synthesis in the melanocytes via MITF to regulate the downstream pigmental genes[J]. Scientia Agricultura Sinica, 2017, 50(7): 1334-1342. DOI: 10.3864/j.issn.0578-1752.2017.07.016. (in Chinese)
[6]	Mastrangelo S, Cendron F, Sottile G, Niero G, Portolano B, Biscarini F, Cassandro M. Genome-wide analyses identifies known and new markers responsible of chicken plumage color[J]. Animals, 2020, 10(3): 493. doi: 10.3390/ani10030493
[7]	Wang X L, Li D H, Song S F, Zhang Y H, Li Y F, Wang X N, Liu D L, Zhang C X, Cao Y F, Fu Y W, Han R L, Li W T, Liu X J, Sun G R, Li G X, Tian Y D, Li Z J, Kang X T. Combined transcriptomics and proteomics forecast analysis for potential genes regulating the Columbian plumage color in chickens[J]. PLoS ONE, 2019, 14(11): e0210850. doi: 10.1371/journal.pone.0210850
[8]	Zheng X T, Zhang B, Zhang Y W, Zhong H A, Nie R X, Li J Y, Zhang H, Wu C X. Transcriptome analysis of feather follicles reveals candidate genes and pathways associated with pheomelanin pigmentation in chickens[J]. Scientific Reports, 2020, 10: 12088. doi: 10.1038/s41598-020-68931-1 pmid: 32694523
[9]	Peng S C, Zhang L, Ali S, Lin S D, Liao Z G, Peng Q Q, Xie P P, Zhang Z H. Whole genome resequencing uncovers candidate genes related to plumage color in Yuexi frizzled feather chicken[J]. Poultry Science, 2025, 104(11): 105680. doi: 10.1016/j.psj.2025.105680
[10]	Yang L J, Zhao W, Chen S Y, Xue L, Tian J L, Xu H R, Zhang H B, Wang H, Gu Y L, Zhang J. Whole genome resequencing reveals genetic markers for plumage colour in Jingyuan Chicken[J]. Poultry Science, 2025, 104(11): 105666. doi: 10.1016/j.psj.2025.105666
[11]	Nie C S, Qu L, Li X H, Jiang Z H, Wang K H, Li H Y, Wang H E, Qu C Q, Qu L J, Ning Z H. Genomic regions related to white/black tail feather color in dwarf chickens identified using a genome-wide association study[J]. Frontiers in Genetics, 2021, 12: 566047. doi: 10.3389/fgene.2021.566047
[12]	Zheng X T, Chen J F, Nie R X, Miao H H, Chen Z W, He J H, Xie Y K, Zhang H. Differential expression of ASIP transcripts reveals genetic mechanism underpinning black-tail independence from body plumage in yellow-bodied chickens[J]. Animal Genetics, 2024, 55(2): 249-256. doi: 10.1111/age.v55.2
[13]	Zhou X, Stephens M. Genome-wide efficient mixed-model analysis for association studies[J]. Nature Genetics, 2012, 44(7): 821-824. doi: 10.1038/ng.2310 pmid: 22706312
[14]	Otasek D, Morris J H, Bouças J, Pico A R, Demchak B. Cytoscape Automation: Empowering workflow-based network analysis[J]. Genome Biology, 2019, 20(1): 185. doi: 10.1186/s13059-019-1758-4 pmid: 31477170
[15]	Chen C J, Wu Y, Li J W, Wang X, Zeng Z H, Xu J, Liu Y L, Feng J T, Chen H, He Y H, Xia R. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining[J]. Molecular Plant, 2023, 16(11): 1733-1742. doi: 10.1016/j.molp.2023.09.010
[16]	Soliman S A. Morphological and histochemical description of quail feather development[J]. The Anatomical Record, 2020, 303(7): 1865-1883. doi: 10.1002/ar.v303.7
[17]	Eliason C M, Shawkey M D, Clarke J A. Evolutionary shifts in the melanin-based color system of birds[J]. International Journal of Organic Evolution, 2016, 70(2): 445-455.
[18]	Rubenstein D R, Corvelo A, MacManes M D, Maia R, Narzisi G, Rousaki A, Vandenabeele P, Shawkey M D, Solomon J. Feather gene expression elucidates the developmental basis of plumage iridescence in African starlings[J]. Journal of Heredity, 2021, 112(5): 417-429. doi: 10.1093/jhered/esab014 pmid: 33885791
[19]	叶宇恒. 固原乌鸡黑色素分布沉积特征及酪氨酸对其组织黑色素含量与黑色素合成相关基因表达量的影响[D]. 银川: 宁夏大学, 2023.
	Ye Y H. Distribution and deposition characteristics of melanin in Guyuan black-bone chicken and the effect of tyrosine on melanin content and expression of genes related to melanin synthesis in its tissues[D]. Yinchuan: Ningxia University, 2023. (in Chinese)
[20]	McGraw K J, Safran R J, Wakamatsu K. How feather colour reflects its melanin content[J]. Functional Ecology, 2005, 19(5): 816-821. doi: 10.1111/fec.2005.19.issue-5
[21]	Nakamura H, Fukuda M. Establishment of a synchronized tyrosinase transport system revealed a role of Tyrp1 in efficient melanogenesis by promoting tyrosinase targeting to melanosomes[J]. Scientific Reports, 2024, 14: 2529. doi: 10.1038/s41598-024-53072-6 pmid: 38291221
[22]	Wang F F, Ma W J, Fan D J, Hu J, An X H, Wang Z D. The biochemistry of melanogenesis: An insight into the function and mechanism of melanogenesis-related proteins[J]. Frontiers in Molecular Biosciences, 2024, 11: 1440187. doi: 10.3389/fmolb.2024.1440187
[23]	Liu X H, Zhou R Y, Peng Y D, Zhang C S, Li L H, Lu C X, Li X L. Feather follicles transcriptome profiles in Bashang long-tailed chickens with different plumage colors[J]. Genes & Genomics, 2019, 41(11): 1357-1367.
[24]	Khalil S, Enbody E D, Frankl-Vilches C, Welklin J F, Koch R E, Toomey M B, Sin S Y W, Edwards S V, Gahr M, Schwabl H, Webster M S, Karubian J. Testosterone coordinates gene expression across different tissues to produce carotenoid-based red ornamentation[J]. Molecular Biology and Evolution, 2023, 40(4): msad056.
[25]	Pan R, Hua T, Ding Y F, Bai H, Jiang Y, Wang Z X, Hu M, Chen G H, Wu X S, Chang G B. Study on changing disciplinarian of beak colors in ducks and the regulation network based on transcriptome sequencing[J]. Poultry Science, 2024, 103(2): 103266. doi: 10.1016/j.psj.2023.103266
[26]	Du J Y, Miller A J, Widlund H R, Horstmann M A, Ramaswamy S, Fisher D E. MLANA/MART1 and SILV/PMEL17/GP100 are transcriptionally regulated by MITF in melanocytes and melanoma[J]. The American Journal of Pathology, 2003, 163(1): 333-343. doi: 10.1016/S0002-9440(10)63657-7
[27]	Bai R M, Yin P, Xing Z X, Wu S B, Zhang W, Ma X Y, Gan X Y, Liang Y X, Zang Q J, Lei H, Wei Y, Zhang C N, Dai B L, Zheng Y. Investigation of GPR143 as a promising novel marker for the progression of skin cutaneous melanoma through bioinformatic analyses and cell experiments[J]. Apoptosis, 2024, 29(3): 372-392. doi: 10.1007/s10495-023-01913-6
[28]	骆娜, 安炳星, 魏立民, 文杰, 赵桂苹. 全基因组关联分析筛选文昌鸡体尺性状相关分子标记[J]. 中国农业科学, 2024, 57(10): 2046-2060. DOI: 10.3864/j.issn.0578-1752.2024.10.015.
	Luo N, An B X, Wei L M, Wen J, Zhao G P. Identification of molecular markers associated with body size traits through genome-wide association analysis in Wenchang chickens[J]. Scientia Agricultura Sinica, 2024, 57(10): 2046-2060. DOI: 10.3864/j.issn.0578-1752.2024.10.015. (in Chinese)
[29]	Bissig C, Rochin L, van Niel G. PMEL amyloid fibril formation: The bright steps of pigmentation[J]. International Journal of Molecular Sciences, 2016, 17(9): 1438. doi: 10.3390/ijms17091438
[30]	dos Santos Videira I F, Moura D F L, Magina S. Mechanisms regulating melanogenesis[J]. Anais Brasileiros de Dermatologia, 2013, 88(1): 76-83. doi: S0365-05962013000100076 pmid: 23539007
[31]	Makarova A V, Mitrofanova O V, Vakhrameev A B, Dementeva N V. Molecular-genetic bases of plumage coloring in chicken[J]. Vavilov Journal of Genetics and Breeding, 2019, 23(3): 343-354. doi: 10.18699/VJ19.499
[32]	Hua G Y, Chen J F, Wang J K, Li J Y, Deng X M. Genetic basis of chicken plumage color in artificial population of complex epistasis[J]. Animal Genetics, 2021, 52(5): 656-666. doi: 10.1111/age.13094 pmid: 34224160
[33]	Kerje S, Sharma P, Gunnarsson U, Kim H, Bagchi S, Fredriksson R, Schütz K, Jensen P, von Heijne G, Okimoto R, Andersson L. The dominant white, dun and smoky color variants in chicken are associated with insertion/deletion polymorphisms in the PMEL17 gene[J]. Genetics, 2004, 168(3): 1507-1518. doi: 10.1534/genetics.104.027995 pmid: 15579702
[34]	Palma C, Lai A, Scholz-Romero K, Chittoory H, Van Haeringen B, Carrion F, Handberg A, Lappas M, Lakhani S R, McCart Reed A E, McIntyre H D, Nair S, Salomon C. Differential response of placental cells to high D-glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras-related protein RAB-7A[J]. Journal of Extracellular Biology, 2024, 3(1): e135. doi: 10.1002/jex2.v3.1
[35]	Massacci G, Perfetto L, Sacco F. The Cyclin-dependent kinase 1: More than a cell cycle regulator[J]. British Journal of Cancer, 2023, 129(11): 1707-1716. doi: 10.1038/s41416-023-02468-8 pmid: 37898722
[36]	Liu Y, Yang Q, Zhao F Z. Synonymous but not silent: The codon usage code for gene expression and protein folding[J]. Annual Review of Biochemistry, 2021, 90: 375-401. doi: 10.1146/annurev-biochem-071320-112701 pmid: 33441035
[37]	Bartoszewski R, Króliczewski J, Piotrowski A, Jasiecka A J, Bartoszewska S, Vecchio-Pagan B, Fu L W, Sobolewska A, Matalon S, Cutting G R, Rowe S M, Collawn J F. Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator[J]. Cellular & Molecular Biology Letters, 2016, 21(1): 23.
[38]	Xin T X, Zhang Z, Zhang Y Y, Li X T, Wang S H, Wang G Q, Li H X, Wang B W, Zhang M Z, Li W J, Tian H J, Zhang Z H, Xiao Y L, Tang W X, He C, Ding Y L, Huang S W, Yang X Y. Recessive epistasis of a synonymous mutation confers cucumber domestication through epitranscriptomic regulation[J]. Cell, 2025, 188(17): 4517-4529.e15. doi: 10.1016/j.cell.2025.06.007 pmid: 40602402

品种 Breed	缩略词 Abbreviation	样本数量 Sample size	登录号 Accession	表型 Phenotype
矮脚鸡 Aijiao chicken	AJ	10	PRJNA947391	黑尾Black-tailed
怀乡鸡 Huaixiang chicken	HX	10	PRJNA482210	黑尾Black-tailed
和田鸡 Hetian chicken	HT	10	PRJNA482210	黑尾Black-tailed
广西黄鸡 Guangxi yellow chicken	GXH	10	PRJNA482210	黑尾Black-tailed
江汉鸡 Jianghan chicken	JH	10	PRJNA482210	黑尾Black-tailed
彭县黄鸡 Pengxian yellow chicken	PX	10	PRJNA656352	黑尾Black-tailed
黔东南小香鸡 Qiandongnan xiaoxiang chicken	QDN	10	PRJNA947391	黑尾Black-tailed
威宁鸡 Weining chicken	WN	10	PRJNA947391	黑尾Black-tailed
文昌鸡 Wenchang chicken	WC	10	PRJNA800119	黑尾Black-tailed
正阳黄鸡 Zhengyang yellow chicken	ZY	10	PRJNA482210	黑尾Black-tailed

基因 Gene name	描述 Description	F/R	引物序列 Primer sequence (5'-3')	Tm (℃)	产物大小 Production size (bp)
PMEL	前黑素体蛋白 Premelanosome protein	F	TTGTCTACGTGTGGTGGACG	60	173
PMEL	前黑素体蛋白 Premelanosome protein	R	CTGGTCGGTGATGCTGAACT	59	173
TYR	酪氨酸酶 Tyrosinase	F	CACTCTTAGGTGGCTCCAATGTG	60	168
TYR	酪氨酸酶 Tyrosinase	R	CAGTCCCAGTAGGGGATGGTGAA	60	168
TYRP1	酪氨酸酶相关蛋白1 Tyrosinase related protein 1	F	CAGAAGCTCAGTTCCCTCG	59	198
TYRP1	酪氨酸酶相关蛋白1 Tyrosinase related protein 1	R	TGGTTGAAGAAGCGTATGG	60	198
NPY	神经肽Y Neuropeptide Y	F	GAGGCACTACATCAACCTCATCAC	59	142
NPY	神经肽Y Neuropeptide Y	R	TGTTTTCTGTGCTTTCCCTCAA	58	142
β-actin	β-肌动蛋白 Beta-actin	F	TCACCAACTGGGATGATATGGA	60	181
β-actin	β-肌动蛋白 Beta-actin	R	TTGGCTTTGGGGTTCAGG	59	181

项目 Item	白尾 White-tailed	黑尾 Black-tailed
黑素小体H.E染色面积 H.E-stained area of melanosomes (μm²)	1686.52±111.02^**	7384.13±319.67**
黑素小体电镜图像面积 Melanosome area in transmission electron microscopy images (μm²)	0.20±0.012^**	1.03±0.032**
褐黑素数量占比 Proportion of pheomelanin-containing melanosomes (%)	57.14±0.67	59.25±1.17
真黑素数量占比 Proportion of eumelanin-containing melanosomes (%)	42.85±0.67	40.75±1.17

	CC	TC	合计 Total
黑尾 Black-tailed	79	1	80
白尾 White-tailed	0	80	80
合计 Total	79	81	160
Fisher检验 Fisher’s exact test	1.76E^-45**

氨基酸 Amino acid	密码子 Codon	个数 Number	同义密码子相对使用度 Relative synonymous codon usage, RSCU
Cys	UGU	1	0.13
Cys	UGC	14	1.87