Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (16): 2880-2890.doi: 10.3864/j.issn.0578-1752.2019.16.013

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

The Variation/Heteroplasmy of Chicken Mitochondrial ND1 Gene and Its Association with Traits

XU YuanYuan1,HOU LingLing2,JI JieFei1,WANG HuanJie1,LI SiLu1,CHEN Wen1,KANG XiangTao1,HUANG YanQun1()   

  1. 1 College of Livestock Husbandry and Veterinary Engineering, Henan Agricultural University, Zhengzhou 450002
    2 Xuchang Animal Husbandry Bureau (Municipal Animal Health Inspection Institute) , Xuchang 461099, Henan
  • Received:2018-09-18 Accepted:2019-07-03 Online:2019-08-16 Published:2019-08-21
  • Contact: YanQun HUANG E-mail:hyanqun@aliyun.com

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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

Table 1

The primers used in the research"

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

Fig. 1

Gel electrophoresis results of PCR amplification products of primer ND1-1"

Fig. 2

The sequencing results of mt.ND1 gene (DNA mixed pool as a template)"

Fig. 3

Electrophoresis analysis of ND1-2 PCR products"

Table 2

Distribution of mitochondrial mt.A4589G variation/heteroplasmy in eight breeds of chicken"

基因型
Genotype		 频率Frequency 品种（样本数）Breed 1)（sample number）
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）

Fig. 4

PCR-RFLP detection of mt.A4589G variation"

Fig. 5

Validation of 5 heteroplasmic individuals detected by PCR-RFLP using Sanger sequencing 1) Left is the breeds and heteroplasmic level of the five heterogeneous individuals detected by the PCR-RFLP method, and right is the Sanger sequencing peak map corresponding samples (GS1, GS2 - Gushi chicken, BC1 - Broiler chicken, HC1, HC2 - Henan Cockfighting chicken). 2) The red arrow is the mt.A4589G site"

Table 3

The distribution of mt.A4589G variation in Gushi chicken resource population"

基因型
Genotype		 F0 F1 F2 总分布 Total
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

Table 4

Correlation analysis between mt.A4589G variation/heteroplasmy and traits of Gushi chicken F2 generation resource group (families containing AA and AG genotypes)"

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

Table 5

Correlation analysis between mt.A4589G variation/heteroplasmy and traits of Gushi chicken F2 generation resource group (families containing GG and AG genotypes)"

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

Table 6

Correlation analysis between different grades of the proportion of mt.4589A in mt.A4589G and traits of Gushi chicken F2 generation resource population"

性状
Traits		 1 级=0%
(n=165)		 0%<2级≤15%
(n=226)		 15%<3级≤45%
(n=247)		 45%<4级<100%
(n=67)		 5级=100%
(n=66)		 P值P-value
腹脂重Abdominal fat (g) 5.968±0.929A 8.857±0.768 7.313±0.720a 12.004±1.392Bb 9.448±1.897 0.0048
腹脂率Abdominal fat rate(%) 0.668±0.096A 0.958±0.080 0.811±0.075a 1.284±0.145Bb 1.055±0.197 0.0067
腿肌率Leg muscle rate(%) 32.569±0.143 32.587±0.118a 32.117±0.110b 32.506±0.213 32.087±0.296 0.0157
脾重Spleen weight (g) 2.823±0.088a 2.874±0.073 2.948±0.068 3.294±0.132b 3.056±0.180 0.0661
脾率Spleen rate(%) 0.212±0.006 0.214±0.005 0.220±0.005 0.244±0.010 0.234±0.013 0.0717
肌胃率Masticatory stomach rate (%) 2.056±0.023 2.063±0.019 2.075±0.018 2.030±0.035 2.181±0.047 0.0984
盲肠长度Cecal length(cm) 15.508±0.212 16.196±0.176 15.884±0.164 15.980±0.322 16.737±0.438 0.0410
屠宰率Slaughter rate(%) 89.586±0.170 89.863±0.142 89.526±0.131 90.184±0.255 90.182±0.343 0.0910
8周胫长8-weeks shank length(cm) 7.830±0.054 7.945±0.045 7.940±0.042 8.076±0.082 7.766±0.110 0.0406
12周胸深12-weeks chest depth(cm) 7.985±0.063 7.913±0.052 7.814±0.049 8.019±0.095 7.743±0.129 0.0680
12周胸角12-week chest angle(°) 78.515±0.340 78.418±0.283 79.275±0.263 79.763±0.512 79.225±0.694 0.0704
葡萄糖GLC(mmol·L-1) 9.472±0.319 8.576±0.251 8.911±0.239 7.878±0.458 7.887±0.677 0.0370
淀粉酶Amylase(U·L-1) 410.247±18.096a 405.599±14.427a 423.412±13.735 504.657±25.784b 519.791±40.013 0.0106
乳酸脱氢酶 LDH(U·L-1) 2714.742±40.803 2761.307±32.411 2845.511±30.532 2827.621±58.904 2820.090±85.306 0.0996
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