酉州乌羊背黑线性状全基因组关联分析

doi:10.3864/j.issn.0578-1752.2026.17.015

摘要/Abstract

摘要：

【目的】背黑线性状在哺乳动物中普遍存在，对品种鉴定、遗传育种和生物进化的研究具有重要的意义。通过筛选与酉州乌羊背黑线性状相关联的分子标记及候选基因，揭示该性状的遗传基础，为地方遗传资源的保护与利用提供科学依据。【方法】选取137只健康成年酉州乌羊作为研究对象，根据背黑线性状进行表型分类。静脉采血后提取基因组DNA，进行全基因组重测序（whole-genome resequencing，WGS），获得原始测序数据。数据经质量评估后与山羊参考基因组ARS1（GCF_001704415.1）比对，获取全基因组单核苷酸多态性（single nucleotide polymorphism，SNP）位点。利用Plink软件对SNP进行严格质控，获得高质量位点后，进行主成分分析（principal component analysis，PCA）与群体结构分析（Admixture），评估群体遗传背景；采用Pair-wise算法估算连锁不平衡（linkage disequilibrium，LD）水平。运用GEMMA软件开展全基因组关联分析（genome-wide association study，GWAS），筛选与背黑线性状显著相关的SNP，设定显著性阈值为7.57×10^-7，并通过Bedtools对显著位点进行基因注释，挖掘标记位点附近的候选基因。同时，基于VCFtools以50 kb窗口、10 kb步长进行全基因组选择信号扫描，计算群体遗传分化指数（Fst）与核苷酸多样性比值（π-ratio），取二者共同的前5%区域作为受选择区域，注释候选基因并进行KEGG通路富集分析。此外，针对18号染色体（NC_030825.1）上12403900—17000000的关键区域，提取SNP并利用ANNOVAR进行变异注释，分析该区域内基因的变异分布。【结果】表型统计显示，137只酉州乌羊中，60只为有背黑线表型，77只为无背黑线表型。WGS共产生4.04 Tb有效数据，平均有效率为99.31%，Q30为96.48%，平均测序深度为10.49×，质控后共保留13 211 396个高质量SNP用于后续分析。群体结构分析表明，有背黑线与无背黑线群体未形成明显遗传分化。GWAS研究发现，3个SNP位点与山羊背黑线性状显著相关，其中SNP1和SNP2注释到过氧化物酶体生物合成因子5样基因（peroxisomal biogenesis factor 5 like，PEX5L）内含子上，SNP3注释到遗传抑制元件1基因（genetic suppressor element 1, GSE1）内含子上。卡方检验表明，3个位点的基因型分布在有背黑线与无背黑线群体间存在极显著差异（P<0.001），其中SNP1的GG基因型和SNP2的TT基因型主要存在于有背黑线群体中，SNP3的CC基因型主要存在于无背黑线群体中。选择信号分析发现，共筛选到4 373个选择信号区域，注释到1 544个候选基因，其中包含PEX5L和GSE1；受选择基因显著富集到18条信号通路（P<0.05），包括cAMP信号通路、间隙连接、粘着连接等。针对18号染色体关键区域的深入分析表明，GSE1变异最为丰富，共检出1 277个SNP（外显子18个，内含子1 259个），其变异数量显著高于该区域内其他基因。【结论】本研究基于WGS数据，整合GWAS与选择信号分析，筛选出PEX5L和GSE1作为酉州乌羊背黑线性状的关键候选基因，为解析山羊背黑线性状的分子遗传机制提供了理论依据，亦有助于该性状在育种实践中的标记辅助选择及地方品种遗传资源的可持续利用。

关键词: 全基因组关联分析(GWAS), 酉州乌羊, 背黑线性状, 选择信号分析, 全基因组重测序(WGS)

Abstract:

【Objective】The dorsal black stripe trait is widespread among mammals and holds significant importance for breed identification, genetic breeding, and evolutionary studies. This research aimed to reveal the genetic basis of this trait by screening molecular markers and candidate genes associated with the dorsal black stripe trait in Youzhou dark goats, thereby providing scientific support for the conservation and utilization of local genetic resources.【Method】A total of 137 healthy adult Youzhou dark goats were selected as subjects and phenotypically classified based on the presence or absence of the dorsal black stripe trait. Genomic DNA was extracted from venous blood samples and subjected to whole-genome resequencing (WGS) to generate raw sequencing data. After quality assessment, the data were aligned to the goat reference genome ARS1 (GCF_001704415.1) to obtain genome-wide single nucleotide polymorphism (SNP) markers. High-quality SNPs were retained after strict quality control using Plink software, followed by principal component analysis (PCA) and population structure analysis (Admixture) to assess genetic background, and pairwise algorithm-based linkage disequilibrium (LD) estimation. Genome-wide association study (GWAS) was performed using GEMMA to identify SNPs significantly associated with the dorsal black stripe trait (threshold = 7.57×10^-7), and significant loci were annotated using Bedtools to mine nearby candidate genes. Meanwhile, genome-wide selection scans were conducted with VCFtools using a 50 kb sliding window and 10 kb step size to compute population genetic differentiation index (Fst) and nucleotide diversity ratio (π‑ratio). Regions falling within the top 5% of both metrics were defined as positively selected regions, from which candidate genes were annotated and subjected to KEGG pathway enrichment analysis. Additionally, SNPs within a key region on chromosome 18 (NC_030825.1: 12403900-17000000) were extracted and annotated using ANNOVAR to analyze variant distribution among genes in this region.【Result】Phenotypic statistics showed that among the 137 Youzhou dark goats, 60 individuals exhibited the dorsal black stripe trait, while 77 did not. WGS generated a total of 4.04 Tb of valid data, with an average efficiency rate of 99.31%, Q30 of 96.48%, and an average sequencing depth of 10.49×. After quality control, a total of 13 211 396 high-quality SNPs were retained for subsequent analysis. Population structure analysis indicated no clear genetic differentiation between goats with the have and non-have dorsal black stripe. GWAS identified three SNPs significantly associated with the dorsal black stripe trait in goats. Among these, SNP1 and SNP2 were annotated within introns of the peroxisomal biogenesis factor 5‑like gene (PEX5L), while SNP3 was located in an intron of the genetic suppressor element 1 gene (GSE1). The chi-square test showed that the genotype distributions of the three SNPs differed significantly between the groups with have and non-have dorsal black stripe (P < 0.001). Specifically, the GG genotype of SNP1 and the TT genotype of SNP2 were primarily observed in the group with have dorsal black stripe, while the CC genotype of SNP3 was mainly found in the group with non-have dorsal black stripe. Selection signature analysis detected 4 373 candidate selective sweep regions, corresponding to 1 544 candidate genes, including PEX5L and GSE1. These selected genes were significantly enriched in 18 KEGG pathways (P < 0.05), such as the cAMP signaling pathway, gap junction, and adherents junction. Further analysis of the key region on chromosome 18 demonstrated that GSE1 exhibited the highest genetic variability, with a total of 1 277 SNPs (18 in exons, 1 259 in introns), significantly exceeding the variant counts of other genes in this region.【Conclusion】Integrating WGS‑based GWAS and selection signature analyses, this study identified PEX5L and GSE1 as key candidate genes for the dorsal black stripe trait in Youzhou dark goats. The findings provide a theoretical foundation for elucidating the molecular genetic mechanisms underlying this trait in goats and would facilitate marker‑assisted selection in breeding programs as well as sustainable utilization of local genetic resources.

Key words: genome-wide association study (GWAS), Youzhou dark goat, dorsal black stripe trait, selection signature analysis, whole-genome sequencing (WGS)

蒋婧, 王高富, 孙晓燕, 李杰, 陈灿灿, 刘良佳, 刘鸿宇, 黎年富, 任航行, 吕士鹏. 酉州乌羊背黑线性状全基因组关联分析[J]. 中国农业科学, 2026, 59(17): 3920-3932.

JIANG Jing, WANG GaoFu, SUN XiaoYan, LI Jie, CHEN CanCan, LIU LiangJia, LIU HongYu, LI NianFu, REN HangXing, LÜ ShiPeng. Genome-Wide Association Analysis of Dorsal Black Stripe Trait in Youzhou Dark Goat[J]. Scientia Agricultura Sinica, 2026, 59(17): 3920-3932.

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分组 Grouping	比对率 Mapped (%)			覆盖度 Coverage (%)
分组 Grouping	平均值 Mean	标准差 Standard deviation	变异系数 CV (%)	平均值 Mean	标准差 Standard deviation	变异系数 CV (%)
有背黑线组 Have	99.90	0.501	0.502	94.70	0.0048	0.504
无背黑线组 Non-have	99.70	1.350	1.350	94.60	0.0040	0.425

染色体 Chromosome	SNP编号 SNP No.	物理位置 Position	P值 P value	基因型 Genotype	相对基因位置 Location relative to gene	基因注释 Gene symbol	NCBI基因ID号 Entrez gene ID	基因区间 Gene range
1	SNP1	86740693	5.31×10^-9	G/A	内含子 Intron	PEX5L	102187896	86709401:8699066
1	SNP2	86740750	6.11×10^-8	T/C	内含子 Intron	PEX5L	102187896	86709401:8699066
18	SNP3	12798677	7.28×10^-8	C/A	内含子 Intron	GSE1	102183352	12653900:13048675

SNP编号 SNP No.	性状 Trait	基因型频率 Genotype frequency			等位基因频率 Allele frequency		P值 P value	卡方值 χ²
SNP1	有背黑线 Have	GG（38） 0.63	GA（20） 0.33	AA（2） 0.03	G 0.80	A 0.20	P<0.001	30.90
SNP1	无背黑线 Non-have	GG（16） 0.21	GA（34） 0.45	AA（25） 0.33	G 0.44	A 0.56	P<0.001	30.90
SNP2	有背黑线 Have	TT（38） 0.64	TC（18） 0.31	CC（3） 0.05	T 0.80	C 0.20	P<0.001	27.31
SNP2	无背黑线 Non-have	TT（18） 0.24	TC（31） 0.41	CC（26） 0.35	T 0.45	C 0.55	P<0.001	27.31
SNP3	有背黑线 Have	CC（23） 0.39	CA（29） 0.49	AA（7） 0.12	C 0.64	A 0.36	P<0.001	27.30
SNP3	无背黑线 Non-have	CC（63） 0.82	CA（13） 0.17	AA（1） 0.01	C 0.90	A 0.10	P<0.001	27.30

编号 No.	物理位置 Position	基因型 Genotype	基因 Gene	外显子 Exon	碱基突变 Nucleotide mutation	氨基酸突变 Amino acid mutation	Have MAF	Non-have MAF	P值 P value
1	12654282	C/T	GSE1	exon1	c.C380T	p.A127V	0.2119	0.2303	4.84×10^-1
2	13033621	G/A	GSE1	exon9	c.G3901A	p.G1301S	0.0848	0.1104	6.39×10^-1
3	13033781	C/A	GSE1	exon9	c.C4061A	p.P1354Q	0.3250	0.1558	2.28×10^-3
4	13036369	C/A	GSE1	exon10	c.C4458A	p.F1486L	0.0424	0.0844	2.31×10^-1
5	13040017	C/T	GSE1	exon14	c.C5225T	p.T1742M	0.175	0.2078	4.52×10^-1

基因 Gene	总SNP数 Total number of SNPs	外显子SNP数 Exon SNP number	内含子SNP数 Intron SNP number	基因 Gene	总SNP数 Total number of SNPs	外显子SNP数 Exon SNP number	内含子SNP数 Intron SNP number
GSE1	1277	18	1259	GAS8	44	2	42
TOX3	433	6	427	MAP1LC3B	44	0	44
CBFA2T3	289	6	283	SLC7A5	36	0	36
JPH3	269	4	265	ZNF276	34	0	34
ANKRD11	268	12	256	DPEP1	31	2	29
KIAA0513	248	6	242	GALNS	30	2	28
CRISPLD2	238	3	235	C18H16orf95	29	0	29
BANP	210	3	207	EMC8	28	1	27
PIEZO1	198	16	182	CDK10	25	1	24
ZFPM1	153	6	147	RNF166	25	0	25
ZCCHC14	149	4	145	FAM92B	23	2	21
GINS2	148	0	148	DEF8	19	1	18
KLHDC4	137	4	133	CPNE7	19	0	19
C18H16orf74	137	3	134	VPS9D1	14	2	12
FBXO31	129	3	126	SNAI3	13	1	12
ZC3H18	124	4	120	SLC22A31	11	4	7
ZNF469	117	26	91	TRAPPC2L	10	1	9
SPG7	106	4	102	DBNDD1	10	0	10
CA5A	106	3	103	FOXF1	10	0	10
ACSF3	85	2	83	MVD	9	1	8
ZDHHC7	81	1	80	RPL13	9	0	9
FANCA	80	7	73	CDT1	8	3	5
SPIRE2	74	1	73	IL17C	8	3	5
IRF8	64	4	60	CHMP1A	7	0	7
CTU2	56	3	53	APRT	4	2	2
TCF25	53	0	53	SPATA2L	4	0	4
CDH15	48	5	43	FOXL1	3	3	0
MTHFSD	48	1	47	PABPN1L	3	1	2