鸡线粒体ND1基因变异/异质性及其与性状的关联分析

doi:10.3864/j.issn.0578-1752.2019.16.013

摘要/Abstract

摘要：

【目的】探究鸡线粒体ND1基因的异质性,并在此基础上进行线粒体异质性在不同品种间和固始鸡资源群中的分布研究及其对重要经济性状的效应分析。【方法】选用固始鸡、卢氏鸡、来航鸡、白洛克鸡、白羽丝毛乌骨鸡、藏鸡、肉鸡以及河南斗鸡8种各具特色鸡种的血样DNA,利用PCR-RFLP技术进行mt.A4589G变异/异质性在自然群体的分布研究。此外,利用实验室先前构建的固始鸡资源群的血样DNA,通过PCR-RFLP技术进行mt.A4589G变异/异质性在固始鸡资源群的分布研究及其与固始鸡资源群F₂代性状的关联分析,包括腹脂、腿肌率、盲肠长度等屠体性状指标,胫长、胸深、体斜长等生长性状指标,肌纤维直径、肌肉pH、剪切力等肉质性状指标以及葡萄糖、淀粉酶、甘油三酯等血液生化指标,进而掌握鸡线粒体ND1基因异质性及其潜在效应。【结果】（1）在8个鸡品种中,从线粒体ND1基因全长检测到一个同义突变mt.A4589G,其分布具有明显的品种特性：外来品种如肉鸡、白洛克和白来航鸡的优势等位基因为A,而国内地方品种如丝羽乌骨鸡、河南斗鸡、卢氏鸡、固始鸡和藏鸡均为G;（2）通过PCR-RFLP检测到mt.A4589G在不同品种以及固始鸡资源群中皆存在异质性。在8个不同品种中,仅从固始鸡、河南斗鸡和肉鸡中检测到5个AG异质性个体,异质性发生的频率为0.063,表明mt.A4589G异质性的分布具有一定的品种特性。而在固始鸡资源群中,mt.A4589G存在较不同品种间更为广泛的异质性,在F₀、F₁和F₂代的异质性发生频率分别为0.81、0.41和0.70,表明mt.A4589G异质性具有普遍性;（3）mt.A4589G异质性与固始鸡资源群F₂代性状的关联分析表明,mt.A4589G异质性对腹脂、皮脂厚度、8周胫长、腿肌纤维直径、葡萄糖和乳酸脱氢酶等的影响都达到差异显著水平(P<0.05);（4）mt.A4589G不同的异质性等级与固始鸡资源群F₂代性状的关联分析表明,mt.A4589G异质性等级除与腹脂、腹脂率和腿肌率等屠体性状显著相关（P<0.05）外,还与生长性状（8周胫长等）、血清生化指标（葡萄糖等）均产生显著的关联（P<0.05）。【结论】mt.A4589G是一个异质性变异,其异质性的分布具有明显的品种特性,在自然群体的异质性发生频率较低,而mt.A4589G在固始鸡资源群中存在广泛的异质性。通过mt.A4589G异质性及其不同异质性等级与固始鸡资源群F₂代性状的关联分析发现,mt.A4589G变异/异质性与鸡的腹脂、血液中葡萄糖水平等性状均产生了显著的关联（P<0.05）。

关键词: 鸡, ND1基因, PCR-RFLP, 异质性, 关联分析

Abstract:

【Objective】 The purpose of this study was to investigate the heteroplasmy of mitochondrial ND1 gene in chickens and to study the distribution of mitochondrial heteroplasmy among different breeds and Gushi chicken resource population, and furthermore, to analysis the effects of mitochondrial heteroplasmy on valuable economic traits. 【Method】 DNA extracted from blood samples of eight chicken breeds including Gushi chicken, Arbor Acres broiler chicken, Henan Cockfighting chicken, Lushi chicken, White Leghorn chicken, White Plymouth Rock chicken, Silkie chicken and Tibetan chicken, were used to study the distribution of mt.A4589G mutation/ heteroplasmy in natural populations by PCR-RFLP. In addition, DNA extracted from blood samples of Gushi chicken resource population constructed previously were used to study the distribution of mt.A4589G mutation/ heteroplasmy in Gushi chicken resource population by PCR-RFLP and to study the association analysis between mt.A4589G mutation/ heteroplasmy and F₂ traits of Gushi chicken resource population, including carcass traits, growth traits, meat quality indicators and blood biochemical indicators, thereby grasping the heteroplasmy of chicken mitochondrial ND1 gene and its potential effects; 【Result】1) Among eight breeds, one synonymous mutation mt.A4589G was detected from the full length of mitochondrial ND1 gene, and its distribution showed obvious variety characteristics. The dominant alleles of exotic breeds such as broiler chicken, White Plymouth Rock chicken and White Leghorn chicken were A, while domestic local breeds such as Silky chicken, Henan Cockfighting chicken and Lushi chicken were G. (2) The heteroplasmy of mt.A4589G in different breeds and Gushi chicken resource population was detected by PCR-RFLP. Among eight breeds, only five AG heterogeneous individuals were detected in Gushi chicken, Henan Cockfighting chicken and broiler chicken. In the Gushi chicken resource population, mt.A4589G had a wider heteroplasmy and the frequencies of heteroplasmy in F₀, F₁ and F₂ generations were 0.81, 0.41 and 0.70, respectively. (3) The correlation analysis between mt.A4589G heteroplasmy and F₂ traits of Gushi chicken resource population showed that mt.A4589G heteroplasmy was significantly correlated with abdominal fat, sebum thickness eight-week-old shank length, diameter of leg muscle fiber and glucose levels in the blood (P<0.05). (4) The correlation analysis between the levels of heteroplasmy of mt.A4589G and F₂ traits of Gushi chicken resource population indicated that the levels of heteroplasmy was significantly correlated with carcass traits, growth traits and serum biochemical indicators (P<0.05). 【Conclusion】 mt.A4589G is a heteroplasmic variation, and its heteroplasmic distribution exhibits obvious variety characteristics. The mt.A4589G heteroplasmy of the natural population occurs less frequently, while mt.A4589G has a wider heteroplasmy in the Gushi chicken resource population. Furthermore, through the correlation analysis between the heteroplasmy of mt.A4589G and the F₂ traits of Gushi chicken resource population, it was found that the mt.A4589G mutation/heterogeneity was significantly correlated with the abdominal fat and the glucose level in the blood (P<0.05).

Key words: chicken, ND1 gene, PCR-RFLP, heteroplasmy, association analysis

徐媛媛,侯玲灵,姬杰菲,王焕杰,李思露,陈文,康相涛,黄艳群. 鸡线粒体ND1基因变异/异质性及其与性状的关联分析[J]. 中国农业科学, 2019, 52(16): 2880-2890.

XU YuanYuan,HOU LingLing,JI JieFei,WANG HuanJie,LI SiLu,CHEN Wen,KANG XiangTao,HUANG YanQun. The Variation/Heteroplasmy of Chicken Mitochondrial ND1 Gene and Its Association with Traits[J]. Scientia Agricultura Sinica, 2019, 52(16): 2880-2890.

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引物名称 Primer name	引物序列（5′-3′） Primer sequence(5′-3′)	长度 Length（bp）	退火温度 Tm（℃）	所用内切酶 Endonuclease
ND1-1	F:ATGACCCTGCCCACCCTAA R:TTTTGTTCAGGCACGCTTCC	995	57	--
ND1-2	F:TAGCCATCATCCTGTTATCC R:GAGGGTTTTTGTAGCGAGTG	356	55	Bsp1286I

基因型 Genotype	频率Frequency	品种（样本数）Breed ¹⁾（sample number）
基因型 Genotype	频率Frequency	GS (10)	BC (12)	HC (8)	LS (10)	WL (10)	WP (10)	SK (10)	TB (10)
AA	0.375	0.0（0）	0.75（9）	0.0（0）	0.1（1）	0.7（7）	1.0（10）	0.0（0）	0.3（3）
AG ²⁾	0.063	0.2（2）	0.08（1）	0.25（2）	0.0（0）	0.0（0）	0.0（0）	0.0（0）	0.0（0）
GG	0.562	0.8（8）	0.17（2）	0.75（6）	0.9（9）	0.3（3）	0.0（0）	1.0（10）	0.7（7）

基因型 Genotype	F₀		F₁		F₂		总分布 Total
基因型 Genotype	样本数 No.	频率 Frequency	样本数 No.	频率 Frequency	样本数 No.	频率 Frequency	样本数 No.	频率 Frequency
AA	6	0.19	9	0.16	66	0.09	81	0.09
AG	25	0.81	23	0.41	540	0.70	588	0.69
GG	0	0.00	24	0. 43	165	0.21	189	0.22

性状Traits	AA型 AA genotype（n=63）	AG型 AG genotype（n=69）	P值P-value
出生重 BW(g)	29.495±0.200	30.180±0.194	0.0232
腿肌纤维直径 FDL (cm)	37.831±0.925	35.047±0.827	0.0388
8周胸深 CD8(cm)	6.251±0.109	6.644±0.105	0.0159
8周胸骨长 BBL8(cm)	8.718±0.079	8.983±0.077	0.0259
8周胫长 SL8(cm)	7.749±0.078	8.012±0.077	0.0266
8周胫围 SG8(cm)	3.347±0.027	3.421±0.025	0.0644
胸肌pH BpH	6.189±0.038	6.080±0.036	0.0553
盲肠长度 CL(cm)	16.453±0.262	15.689±0.256	0.0538
肌胃重率 GR(%)	2.149±0.036	2.052±0.035	0.0685

性状 Traits	AG型 AG genotype (n=488)	GG型 GG genotype (n=158)	P值 P-value
腹脂 AF(g)	8.618±0.498	6.310±0.924	0.0292
腹脂率 AFR(%)	0.936±0.051	0.692±0.096	0.0264
皮下脂肪厚度 SFT(cm)	0.452±0.007	0.486±0.014	0.0339
葡萄糖 GLU(mmol·L^-1)	8.617±0.164	9.471±0.319	0.0191
乳酸脱氢酶 LDH(U·L^-1)	2811.195±21.164	2707.856±41.082	0.0277
盲肠长度 CL(cm)	16.125±0.117	15.619±0.218	0.0427