7 : GOLA-DQB breeding of goats. Keywords: goats; MHC-DQB1; PCR-RFLP; immune trait; correlation analysis (Major histocompability complex, MHC) M

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1 HEREDITAS (Beijing) , 30(7): ISSN 研究报告 DOI: /SP.J GOLA-DQB1 2 邢凤, 李培培, 李珏, 李成渤, 王建民, : 利用 PCR-RFLP 技术, 对莱芜黑山羊鲁波山羊和波尔山羊 3 个山羊种群共 175 只个体的 GOLA-DQB1 基因外显子 2 进行遗传多态性研究, 并对山羊种群的血液免疫指标的效应进行了分析结果表明 : 3 个山羊种群共检测到 (AA BB CC AB AC BC DD)7 种基因型, GOLA-DQB1 基因外显子 2 的第位的碱基表现出多态性多数指标品种效应是主要效应莱芜黑山羊中, BC 基因型的淋巴细胞百分比 (W-SCR) 显著高于 AC CC 基因型 (P<0.05), 中性球比例 (W-LCR) 显著低于 CC 基因型 (P<0.05), 大型白细胞数 (W-LCC) 低于 AC CC 基因型, 但差异不显著 (P>0.05) 波尔山羊中, BC 基因型的 W-LCC 低于 AA AB BB 基因型, 但差异不显著 (P>0.05) 鲁波山羊中, BC AC 基因型的 W-LCC 显著低于 AA 基因型 (P<0.05) 揭示 GOLA-DQB1 基因与血液免疫性状有一定的相关性 : 山羊 ;MHC-DQB1;PCR-RFLP; 免疫性状 ; 相关分析 Association of polymorphisms of exon 2 of GOLA-DQB1 gene with immune traits in goats XING Feng, LI Pei-Pei, LI Jue, LI Cheng-Bo, WANG Jian-Min College of Animal Science and Technology, Shandong Agricultural University, Tai an , China Abstract: PCR-RFLP was used to analyze the polymorphisms of exon 2 of GOLA-DQB1 gene in Laiwu black goats, Lubo goats, and Boer goats. The effects of genotypes of exon 2 of GOLA-DQB1 gene on immune traits were estimated. Seven genotypes (AA, BB, CC, AB, AC, BC, and DD) were detected by analyzing restriction maps. Polymorphic sites were detected at base position 24, 151 of exon 2 of GOLA-DQB1 gene. The results showed that the effects of breed were major effect. W-SCR in Laiwu black goats with genotype BC was significantly higher than that with genotype AC and CC (P<0.05), W-LCR in Laiwu black goats with genotype BC was significantly lower than that with genotype CC (P<0.05); W-LCC in Laiwu black goats with genotype BC was lower than that with genotype AC, CC (P>0.05). W-LCC in Boer goats with genotype BC was lower than that with genotype AA, AB, and BB (P>0.05); W-LCC in Lubo goats with genotype BC and AC was significantly lower than that with genotype AA (P<0.05). It was concluded from the results that GOLA-DQB1 was the gene affecting the immune traits. These results may be applied to marker assisted selection in disease resistance 收稿日期 : ; 修回日期 : 基金项目 : (: SDLZ06RY0203)(: nyhyzx07-037)[supported by the Project of Shandong Province Good Agricultural Breeds (No. SDLZ06RY0203), and National Scientific and Technological special program of Commonweal industry (No. nyhyzx07-037) 作者简介 : (1981 ),,,, : xingfeng2001@126.com 通讯作者 : (1963 ),,,, : Tel: ; wangjm@sdau.edu.cn

2 7 : GOLA-DQB breeding of goats. Keywords: goats; MHC-DQB1; PCR-RFLP; immune trait; correlation analysis (Major histocompability complex, MHC) MHC, [1~3] MHC MHC MHC,,, MHC MHC,, [4~8], MHC-DQB [9~14],, MHC-DQB MHC GOLA, GOLA GOLA-DRB3 [15~17] GOLA-DQB1, Amills [18] PCR-RFLP GOLA-DQB1 2,, , (Laiwu black goat, LBG) 71, (Boer goat, BG) 63, (, )( Lubo goat, LG) 41 1~2, 10 ml /,, 1 ml,, 20 : (RBC), (WBC), (HGB), (HCT), (MCV), (MCH), (MCHC), (W-SCR), (W-LCR), (W-SCC), (W-LCC) 基因组 DNA 的提取与检测 2 ml,, PBS, r/min 10 min,, 1 ml, 37 1~2 h, K 100 μg/μl, 55 3, 2 DNA,, 70%,, TE 0.8% DNA,, 引物合成及 PCR 扩增 Amills [ 18], : : 5 -CCCCGCAGAGGATTTCGTG-3 : 5 -ACCTCGCCGCTGCCAGGT-3 PCR, : ddh 2 O 15.8 μl, 10 PCR buffer 2.5 μl, dntps (2.5 mmol/l) 2.0 μl, MgCl 2 (2.5 mmol/l)1.5 μl, (20 μmol/l) 1 μl, (20 μmol/l) 1 μl, Taq 0.2 μl, 1 μl: 94 5 min 33 (94, 30 s 65, 30 s 72, 45 s) 72, 5 min 4 PCR 1.5 % 限制性内切酶酶切与检测 HaePCR, 10 μl: PCR 4.0 μl, Hae 0.5 μl, 10 buffer 1.0 μl, ddh 2 O 4.5 μl, 37 3 h, 3.5% 1.3 PopGene 1.32(FrancisC.Yeh, Ph.D) (Ho) (He) (Ne) Hardy-Weinberg (Polymorphism information content, PIC) Bostein [19], 3,, :

872 HEREDITAS (Beijing) 2008 30 Yijl=μ +Ai+Gj+Eijl, Yijl : ; μ: ; Ai: i ; Gj: j ; Eijl:, SAS (8.0) GLM GOLA-DQB1 2 24 151 2 2.1 PCR PCR 1.

2 PCR-RFLP 3 GOLA-DQB1 2 3 Hae, 2, PCR Hae, 7 ( 1) 179/77/24 bp, 127/77/52/24 bp, 203/77 bp, 151/77/52 bp, 179/127/77/52/24 bp, 203/ 179/77/24 bp,

BB; 11: BC ; 14: PCR ; M: 20 bp DNA ladder Fig.

3 872 HEREDITAS (Beijing) Yijl=μ +Ai+Gj+Eijl, Yijl : ; μ: ; Ai: i ; Gj: j ; Eijl:, SAS (8.0) GLM GOLA-DQB PCR PCR 1.5%, bp, 8 bp 1, 2, 2 bp PCR-RFLP 3 GOLA-DQB1 2 3 Hae, 2, PCR Hae, 7 ( 1) 179/77/24 bp, 127/77/52/24 bp, 203/77 bp, 151/77/52 bp, 179/127/77/52/24 bp, 203/ 179/77/24 bp, 203/127/77/52/24 bp 24 bp 52 bp, 7 : AA BB CC DD AB AC BC 4 (A B C D) : 图 1 GOLA-DQB1 基因外显子 2 的 Hae 酶切电泳图 1: CC; 2,5,10: AA; 3,9,12,13: AB; 4,7: DD; 6: AC; 8: BB; 11: BC ; 14: PCR ; M: 20 bp DNA ladder Fig. 1 Exon2 of GOLA-DQB1 gene digested with Hae in goats 1: CC; 2,5,10: AA; 3,9,12,13: AB; 4,7: DD; 6: AC; 8: BB;11: BC; 14: PCR product; M: 20 bp DNA ladder. 2.3 PCR, BB PCR,, DNA 3 Hae, 24 bp 151 bp 203 bp ( 2) 图 2 BB 基因型 151 和 203 处酶切位点 Fig. 2 Digestion sites at the base position of 151 bp and 203 bp 2.4 GOLA-DQB1 2,, 3, (Ho) (He) (PIC)(Ne) ( 1 2) 1, 3 7, 3 AB, A 1 CC, 2 DD, 1 BB, DD, 3 Hae Hardy-Weinberg (P<0.05),, [20] (Ne),

4 7 : GOLA-DQB 表 1 Table 1 山羊 GOLA-DQB1 基因外显子 2 内切酶 Hae 的基因型频率和基因频率 Genotypes and gene frequencies of exon2 of GOLA-DQB1 gene digested with Hae in goats Breeds Genotypes No. Genotype frequencies Alleles LBG AA A BB B CC C AB AC BC Allele frequencies LG AA A BB B DD C AB D AC BC BG AA A BB B CC C AB AC BC LGB: (Laiwu black goat); LG: (Lubo goat); BG: (Boer goat). 表 2 山羊 GOLA- DQB1 基因遗传多态性分析 Table 2 The genetic polymorphism parameters of GOLA- DQB1 gene in goats Breeds Ho He Ne PIC LBG BG LG ,,,, 2, (He), ,,, 2,,,, ,,, (PIC) PIC,,, ; PIC,, PIC> < PIC <0.5 ; PIC< PIC ~0.5545,, 3,, 2.5 GOLA-DQB1,,, ;,,,,, SAS 8.0,,, 3 4 3, WBC HGB, (P>0.05); RBC, (P<0.05), HCT (P<0.05), W-LCC (P<0.05) W-SCC, (P>0.05), W-LCR (P<0.05) MCV (P<0.05), MCH MCHC (P<0.05)

5 874 HEREDITAS (Beijing) 表 3 不同种群血液免疫指标的最小二乘均值及方差分析 Table 3 Least square means and standard error and analysis of normal blood indes of different goat breeds and different genotypes Breeds Traits BG LBG LG WBC 13.29± ± ±0.49 RBC 2.36±0.15 b 3.68±0.13 a 1.98±0.20 b HGB 86.96± ± ±1.71 HCT 0.07±0.01 b 0.12±0.04 a 0.09±0.02 ab MCV 37.47±3.02 b 31.17±0.05 b 46.36±4.70 a MCH 36.56±1.49 ab 25.55±0.89 b 66.02±30.44 a MCHC 1165±47.45 ab 819±28.56 b 1566± a W-SCR 1.07± ± ±1.12 W-LCR 0.53±0.01 a 0.41±0.02 b 0.42±0.03 b W-SCC 8.69± ± ±1.49 W-LCC 7.41±0.38 a 5.17±0.39 b 6.44±0.76 ab : (P<0.05); (P>0.05) Notes: Values in each line with different case superscripts are at P<0.05; Values in each line with the same case superscripts are at P> ,, BC W-SCR AC CC (P<0.05), W- LCR CC (P<0.05), W-LCC AC CC, (P>0.05);, BC W-LCC AA AB BB, (P>0.05), BC AC W-LCC AA (P<0.05),, 3 BC W-LCC, 1 CC, 1 BB, 2 DD,,,,, 表 4 山羊 GOLA-DQB1 基因不同基因型对免疫指标的效应分析 Table 4 Effect of different genotypes of GOLA-DQB1 gene exon2 on immune traits in three goat breeds Genotypes Breeds Traits AA AB BB BC AC CC LBG WBC 19.84± ± ± ± ± ±14.85 RBC 3.36± ± ± ± ± ±0.29 HGB 88.3± ± ± ± ± ±5.94 HCT 0.10± ± ± ± ± ±0.01 MCV 31.15± ± ± ± ± ±0.16 MCH 27.17± ± ± ± ± ±3.52 MCHC 871± ± ± ± ± ±111.4 W-SCR 0.66±0.05 ab 0.54±0.05 ab 0.66±0.05 ab 0.73±0.10 a 0.54±0.05 b 0.45±0.05 b W-LCR 0.34±0.04 ab 0.45±0.05 ab 0.34±0.05 ab 0.27±0.10 b 0.46±0.05 ab 0.54±0.05 a W-SCC 15.25± ± ± ± ± ±0.64 W-LCC 3.84± ± ± ± ± ±1.50 BG WBC 13.56± ± ± ±1.31 RBC 2.41± ± ± ±0.42 HGB 83.00± ± ± ±3.24 HCT 0.075± ± ± ±0.01 MCV 38.74± ± ± ±0.70 MCH 32.61± ± ± ±3.35 MCHC 1049± ± ± ±107.9 W-SCR 0.39± ± ± ±0.02 W-LCR 0.60± ± ± ±0.37 W-SCC 5.52± ± ± ±0.50 W-LCC 8.53± ± ± ±0.50 LG WBC 13.95± ± ± ±1.73 RBC 2.73± ± ± ±0.35 HGB 90.28± ± ± ±6.01 HCT 0.07± ± ± ±0.01 MCV 42.70± ± ± ±0.08 MCH 32.18± ± ± ±6.69 MCHC 1029± ± ± ±219.7 W-SCR 0.55± ± ± ±0.05 W-LCR 0.33± ± ± ±0.17 W-SCC 8.4± ± ± ±0.03 W-LCC 7.67±0.64 a 6.47±0.43 ab 3.70±0.73 b 3.30±0.01 b : (P<0.05); (P>0.05) Notes: Values in each line with different case superscripts are at P<0.05, Values in each line with the same case superscripts are at P>0.05.

6 7 : GOLA-DQB (P<0.05) MHC-DQB 2 MHC, MHC, Amills [18] GOLA-DQB1 2, 5 GOLA-DQB1 2, 7, 4 (A B C D), 3 0.5,, 3, Amills [18] GOLA-DQB1 2 3 : (1),,,,, ; (2),, ; (3),,, 3,,, 3 Hae Hardy- Weinberg 3,, 3.2 GOLA-DQB1,,,,, [21] DQB, SLA-DQB-Hae SAS 8.0,, (GLM) 3 11,,, BC W-SCRAC CC (P<0.05), W-LCR CC (P<0.05),, BC AC W-LCCAA (P<0.05), 3 BCW-LCC GOLA-DQB1,, DNA, (References): [1] Apanius V, Penn D, Slev PR, Ruff LR, Potts WK. The nature of selection on the major histocompatibility complex. Crit Rev Immunol, 1997, 17: [2] Edward S, Hedrick PW. Evolution and ecology of MHC molecules: From genomics to sexual selection. Trends Ecol Evol, 1998, 13(8): [DOI] [3] Hedrick PW, Kim TJ. Genetics of Complex Polymorphisms: Parasites and Maintenance of the Major Histocompatibility Complex Variation. In: Singh RS, Krimbas CK, ed. Evolutionary Genetics From Molecules to Morphology. New York: Cambridge University Press, [4] ZHANG Fu-Qian, ZHENG Xiao-Min, TANG Da-Wei, ZHAO Peng, SHI Yuan-Gang. Research on polymorphism of BoLA-DRB3 gene in Holstein and the relationship between different genotypes and resistance to dairy mastits. Acta Veterinaria et Zootechnica Sinica, 2007, 38(2): ,,,,. BoLA-DRB3., 2007, 38(2): [5] Aarestrup FM, Jensen NE, Ostergard H. Analysis of association between major histocompatibility complex (BOLA)

7 876 HEREDITAS (Beijing) class I haplotypes and subclinical mastitis of dairy cows. Dairy Science, 1995, 78(8): [6] JIA Bin, SHEN Hong, YU Zhi-Yong, CHEN Ming-Hui, LIU Hong-Kun, LI Hai, CHEN Yu-Lin, PENG Lin-Ze, LIN Ying-Chun. Polymorphism of the ovine major histocompatibility complex class DRB1gene and the genetic susceptibility to hydatidosis. Chinese Journal of Zoonoses, 2007, 23(10): , 1012.,,,,,,,,. MHC-DRB1., 2007, 23(10): , [7] Sayers G.G, Hanrahan B, Ryan JP, Angles M, Sweeney JM, Título T. Major histocompatibility complex DRB1 gene: its role in nematode resistance in Suffolk and Texel sheep breeds. Parasitology, 2005, 131(3): [DOI] [8] LI Guo-Qin, LU Li-Zhi, WANG De-Qian, SHEN Jun-Da, TAO Zheng-Rong, ZHAO Ai-Zhen, YUAN Ai-Ping. Advance in association of major histocompatibility complex (MHC) gene polymorphisms with resistance character to infectious disease in chicks. Hereditas (Beijing), 2006, 28 (7): ,,,,,,. MHC., 2006, 28(7): [9] SUN Dong-Xiao, ZHANG Yuan, Li Ning. Polymorphism analysis of BOLA-DRB3 and DQB gene in mongolian. Journal of Agricultural Biotechnology, 2001, 9(4): ,,. MHC-DRB3 DQB PCR-RFLP., 2001, 9(4): [10] FANG Mei-Ying, HU Xiao-Xiang, LI Ning, WU Chang-Xin. Polymorphism analysis of the exon 2 of SLA-DQB gene in Xiao Meishan, Zhong Meishan and Yorkshire pigs with PCR-RFLP. Acta Genetica Sinica, 2002, 29(8): ,,,. SLA-DQB 2 PCR-RFLP., 2002, 29(8): [11] LI Hua, ZHANG Ya-Ping, QIU Xiang-Pin. The genetic diversity of the second exon of SLA-QB in some Chinese native pigs. Hereditas (Beijing), 2005, 27(2): ,,. SLA-DQB 2., 2005, 27(2): [12] WANG Kun, SUN Dong-Xiao, XU Ru-Hai, WANG Zhi-Gang, ZHANG Yuan. Polymorphism in upstream rgulatory rgion of BoLA-DQB gnes. Acta Veterinaria et Zootechnica Sinica, 2005, 36(6): ,,,,. BoLA-DQB., 2005, 36(6): [13] BAO Wen-Bin, WU Sheng-Long, CHEN Guo-Hong, JU Hui-Ping, HUA Jin-Di, HUANG Xue-Gen. Polymorphism of the exon2 of SLA-DQB gene and its relationship with reproductive performance in Sutai pigs. Acta Veterinaria et Zootechnica Sinica, 2007, 8(7): ,,,,,. SLA-DQB 2., 2007, 38(7): [14] XU Ru-Hai, WANG Kun, SUN Dong-Xiao, DING Xiang- Dong, LIU Pei-Qiong, ZHANG Qin. Sequences and teir polymorphisms of swine MHC-DQB, DRB proximal promoter region. Acta Genetica Sinica, 2005, 32(3): ,,,,,. MHC-DQB DRB., 2005, 32(3): [15] SUN Dong-Xiao, ZHANG Yuan, LI Ning. Polymorphism analysis of the GOLA-DRB3 gene digested with Hae in Mongolian goat and Kazakh goat. Hereditas (Beijing), 2004, 26(1): ,,. GOLA-DRB3 Hae., 2004, 26(1): [16] LI Meng-Hua, LI Kui, Kantanen Juha, FENG Zheng, FAN Bin, ZHAO Shu-Hong. Allelic variations in exon 2 of caprine MHC class RB3 gene in Chinese indigenous goats. Small Rumin Res, 2006, 66(1): [DOI] [17] YANG Yi, XU Jin-Rui, WANG Jie, YANG Xue, ZHENG Yu-Cai. The polymorphism of exon 2 of MHC-DRB3 gene in four goats of Sichuan. Heilongjiang Animal Science and Veterinary Medicine, 2006, (1): ,,,,. 4 MHC-DRB3 2., 2006, (1): [18] Amills M, Sulas C, Sànchez A, Bertoni G, Zanoni R, Obexer-Ruff G.. Structure characterization of the caprine major histocompatibility complex class DQB1 gene. Mol Immunol, 2004, 418: [19] Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet, 1980, 32: [20] LI Ning, CHEN Hong, ZHAO Xing-Bo, XU Ning-Ying, HUANG Lu-Sheng, XIE Zhuang. Animal Genetics. Beijing: China Agricultural Press, 2003.,,,,,.. :, [21] LIU Bang. The SNPs of 4 gene of MHC class II in 15 pig breeds and their association with immune traits [Dissertation]. Huazhong Agricultural University, MHC 4 SNPs []., 2003.

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