山羊GOLA-DQA2基因多态性与血液免疫性状的相关分析

文章采用PCR-RFLP技术对莱芜黑山羊、波尔山羊、鲁波山羊3个山羊种群的GOLA-DQA2基因外显子2进行遗传多态性研究, 并对其血液免疫指标的效应进行分析。结果表明, 3个山羊种群共检测到4种基因型, 由3个等位基因控制; GOLA-DQA2基因外显子2的第77、79、80和169位的碱基表现出多态性; 多数血液免疫指标品种效应是主要效应; 鲁波山羊中, AB基因型的红细胞计数、白细胞计数分别显著高于BB基因型、BC基因型(P<0.05); AB基因型的红细胞压积显著高于BB、BC 基因型(P<0.05); BC基因型的噬中性粒细胞比率显著高于BB基因型(P<0.05); 波尔山羊和莱芜黑山羊中, 基因型与血液免疫指标之间也有一定的相关性, 但没有达到显著水平。由上述结果初步推测, 在鲁波山羊中, AB、BC基因型是影响红细胞计数、白细胞总数、噬中性粒细胞比率等血液免疫指标的重要因素。研究结果揭示GOLA-DQA2基因与血液免疫指标之间有一定的相关性, 可为山羊的抗病育种提供一定的参考。     

FSHβ 基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究

采用PCR-SSCP技术检测促卵泡素b亚基(follicle-stimulating hormone β, FSHβ)基因5′调控区、外显子1和外显子2在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊、安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 山羊与绵羊的FSHβ 基因该段核苷酸序列同源性为98%。9对引物中, 只有P9的扩增片段存在多态性。P9的扩增片段在济宁青山羊和辽宁绒山羊中检测到AA、AB和AC 3种基因型; 在波尔山羊中检测到AA、CC和AC 3种基因型; 在安哥拉山羊中检测到AA、BB、CC、AB、AC和BC共6种基因型。测序分析发现BB型与AA型相比在外显子2的第94 bp处有G→A突变, 并引起氨基酸改变(丙氨酸→苏氨酸); CC型与AA型相比在外显子2的第174 bp有一处C→T沉默突变。济宁青山羊AA、AB和AC基因型频率分别为0.686、0.137和0.177。AA基因型济宁青山羊产羔数最小二乘均值比AB基因型的多0.78只(P<0.05), 比AC基因型的多0.64只(P<0.05)。     

鸡apoA5基因单核苷酸多态性及其与屠体性状的关联研究

以丝羽乌骨鸡和隐性白洛克正反交产生的F2代为实验群体, 采用PCR-SSCP和DNA测序的方法检测鸡载脂蛋白A5(apoA5)基因的单核苷酸多态性(SNPs), 并将所发现的SNPs与体重、胸肌重、腿肌重、心脏重、肝脏重和腹脂重等屠体性状进行关联分析。结果发现, 鸡apoA5基因5′-调控区C-169T, 外显子2 C600T、T635C, 外显子3 C841G、C914T、C1142G、C1394T共7个突变位点。其中外显子2突变位点C600T、T635C对12周龄腹脂重、腹脂率、肝脏重和心脏重有显著影响(P<0.05), 根据PCR-SSCP的结果将其分为6种基因型(AA, AB, AC, BB, BC, CC): 其中CC型个体的腹脂重和腹脂率显著高于AA型、AB型、AC型、BB型、BC型 (P<0.05); AC型个体的肝脏重显著低于AA型、AB型、BB型、BC型和CC型的肝脏重(P<0.05); BC型个体的心脏重显著低于BB型的心脏重(P<0.05)。     

山羊MyoD基因家族多态性及与体尺性状的相关性

用PCR-SSCP技术研究了波尔山羊和徐淮山羊2个群体共147个个体MyoD基因家族中3个基因座位的单核苷酸多态性(single nucleotide polymorphism, SNP)。结果表明, 在徐淮白山羊群体中, myf-5基因座发现有3种基因型AA、AB和BB, 波尔山羊均为AA型。在myf-6基因座和myoD 5′侧翼区基因座, 两个山羊群体均检测到了AA和AB型个体。对山羊myf-5、myf-6基因座, myoD 5′侧翼区基因座不同基因型与两品种山羊体尺性状相关分析表明, myf-5基因座对管围和管围指数的效应差异显著(P<0.05)。myf-6基因座对徐淮白山羊体尺性状的效应均不显著(P>0.05), 而对波尔山羊的体高和管围指数效应差异显著(P<0.05)。两个山羊品种myoD 5′侧翼区不同基因型个体的体尺性状差异均不显著。     

猪TLR4基因外显子1新等位基因的分离及遗传变异分析

文章采用PCR-SSCP方法对亚洲野猪、3个引进的商业化品种和10个中国地方猪品种共893个个体TLR4基因外显子1的遗传变异进行了检测,旨在系统分析国内外猪种TLR4基因的多态性,为探讨该基因在免疫和防御系统中发挥的作用提供依据。结果,在猪TLR4基因外显子1中分离到新的等位基因,共检测到3个等位基因,6种基因型。其中杜洛克检测到AA、BB、CC、AB、AC、BC基因型,有杜洛克血统的苏太猪中检测到BB、CC、BC基因型,长白猪、约克夏中检测到CC、BC基因型,野猪及所有10个中国地方猪品种TLR4基因外显子1高度保守,只检测到CC基因型,中国地方猪品种和引进品种TLR4基因外显子1多态性存在极显著的差异。3种基因型中CC型与GenBank中的序列一致,BB和AA基因型分别存在G93C同义突变位点和G194A无义突变位点,这2个变异位点与抗逆性和一般抗病力的关系值得进一步深入研究。     

猪IGF-Ⅰ基因的一个遗传多态性及其遗传效应分析

采用PCR-SSCP方法对长白猪(87头)、大白猪(79头)和马身猪(102头)的胰岛素样生长因子-Ⅰ(IGF-Ⅰ)基因外显子3和外显子4分别进行单核苷酸多态性分析.发现外显子3上有多态性,且存在3种基因型(AA、AB、BB).统计结果表明,3种基因型在各品种中的分布不一致,多重比较差异极显著(P＜0.01).固定效应模型分析结果表明,背膘厚基因型间差异显著(P＜0.05),而初生重、断奶重和6月龄重基因型间差异不显著(P＞0.05).最小二乘分析结果表明,BB基因型与其它2种基因型比较有较大的初生重,同AA和AB型比较差异极显著(P＜0.01),3种基因型在初生重的大小排列顺序为AA＜AB＜BB;BB基因型与AA基因型比较有较小的背膘厚,且差异极显著(P＜0.01).因此,推测IGF-Ⅰ基因对个体的初生重和胴体瘦肉率存在一定的影响.选择带有B等位基因的个体,有望提高个体的初生重和胴体的瘦肉率.     

猪IGF2基因外显子4b的遗传多态性及其遗传效应

采用PCR-SSCP方法检测猪胰岛素样生长因子2(insulin-like growth factor 2,IGF2)基因外显子4b的多态性,并分析其对初生重、断奶重、6月龄重和背膘厚的遗传效应。根据猪IGF2基因的DNA序列(AY044828)设计引物,在exon4b上发现了一个多态性位点,并对纯合子进行测序,发现13位C→A转换,且存在3种基因型(AA、AB、BB)。统计结果表明,3种基因型在各品种中的分布不一致,长白猪与大白猪比较,莱芜猪与大薄莲猪比较,沂蒙黑猪和里岔黑猪比较差异不显著(P>0.05);其他猪种间基因型分布的差异均显著(P<0.05)。固定效应模型分析结果表明,初生重,断奶重和背膘厚基因型间差异显著(P<0.05),而6月龄重基因型间差异不显著(P>0.05)。最小二乘分析结果表明,BB基因型个体同AA和AB、基因型个体比较初生重和断奶重的差异显著(P<0.05),3种基因型在初生重和断奶重的大小排列顺序为AA>AB>BB;AA基因型个体同AB和BB基因型个体比较背膘厚的差异显著(P<0.05),3种基因型在背膘厚的大小排列顺序为BB>AB>AA。因此,推测IGF2基因对个体的生长速度和胴体瘦肉率存在一定的影响,将IGF2基因应用于猪育种过程中的标记辅助选择可以加快猪的育种进程。     

生长激素基因多态性与山羊体重性状的关系

以鲁北白山羊、波尔山羊以及波尔山羊与鲁北白山羊的杂交一代、回交一代共计224只山羊为材料,根据山羊生长激素基因5′调控区的序列(GenBank登录号:D00476)设计两对引物,用PCRSSCP法进行多态性分析。多态性片段的纯合基因型经克隆测序,发现共有5处突变。对突变位点产生的不同基因型与体重、体尺性状进行分析表明:第一对引物扩增片段波尔山羊AA型个体的初生重、周岁重显著高于BB型和AB型(P<0.05);杂交一代中断奶重、周岁重也以AA型偏高,但不同基因型间的差异均不显著(P>0.05);而鲁北白山羊BB基因型的体重相对于另外两种基因型的偏低,且断奶重的差异达到显著水平(P<0.05)。第二对引物扩增片段不同基因型对体重、体尺没有显著影响。由此初步推断生长激素基因可能是影响山羊体重性状的主基因或与主基因相连锁,可以用该位点对山羊体重性状进行标记辅助选择。     

猪MyoG基因内含子1的一个遗传多态性及其遗传效应分析

以长白猪(73),大白猪(68),沂蒙黑猪(57)和莱芜猪(83)为研究对象,采用PCR-SSCP方法 对猪MyoG基因遗传多态性进行分析,并研究基因型与初生重、日增重、肌肉嫩度和背膘厚的相关性.根据猪MyoG 基因的DNA序列（M14331）设计引物,结果在内含子1扩增的片段上发现了一个多态性,检测到2个等位基因(A、B),3种基因型（AA、AB、BB）,并对纯合子进行测序,发现2　943位G→C突变.基因型在不同猪种分布的多重比较,结果表明,大白猪与长白猪、沂蒙黑猪和莱芜猪比较差异显著（P<0.05）,莱芜猪与长白猪和沂蒙黑猪比较差异显著（P<0.05）,长白猪与沂蒙黑猪比较差异不显著（P>0.05）.固定效应模型分析结果表明,初生重及嫩度基因型间差异显著（P<0.01）,而日增重及背膘厚基因型间差异不显著（P>0.05）.最小二乘分析结果表明,BB基因型个体与AA基因型个体比较肌肉嫩度的差异显著（P<0.05）,AA基因型个体与AB和BB基因型个体比较背膘厚的差异显著（P<0.05）.因此,推测MyoG基因对猪肉品质、生长速度及背膘厚存在一定的影响,将 MyoG基因应用于猪育种过程中的标记辅助选择将可以改善猪肉品质,提高生长速度,加快猪的育种进程.     

CXCR2基因多态性与奶牛乳房炎和乳品质的关联

采用PCR-SSCP技术研究了荷斯坦牛、西门塔尔牛和通江黄牛3个品种共160头个体CXCR2基因多态性与乳房炎抗性性状和对牛奶品质性状的遗传效应。结果表明: CXCR2基因有3个SNP多态位点, 分别位于序列的第685 bp、777 bp和861 bp位点, 确定了5个等位基因A、B、C、E和F。685 bp位点表现为BC和CC基因型, 777 bp位点表现为AA和AB基因型, 861 bp位点为CC和BC基因型。与奶牛乳房炎敏感性有关的基因型主要是BC、CC和FF, 可能对乳房炎有抗性的是AA、AB和EE基因型。据不同基因型对乳品质的遗传效应分析来看, AA、AB和EE基因型的乳品质性状极显著或显著优于其他基因型。     

Polymorphism of bone morphogenetic protein 4 gene and its relationship with litter size of Jining Grey goats

Two pairs of primers (P1 and P2) were designed to detect single nucleotide polymorphisms of exon 2 and intron 2 of bone morphogenetic protein 4 (BMP4) gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Boer, Angora and Inner Mongolia Cashmere goats) by single strand conformation polymorphism. Results showed that no polymorphism was detected for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2 (primer P2), three genotypes (AA, AB and BB) were detected in Jining Grey and Inner Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats, and only one genotype (AA) in Boer goats. Sequencing revealed one mutation (2203G>A) of BMP4 gene in the genotype BB in comparison to the genotype AA. The differences of litter size between AA, AB and BB genotypes were not significant (P > 0.05) in Jining Grey goats. A pair of primer (P3) was designed to detect polymorphism in the 3' flanking region of BMP4 gene that contained dinucleotide repeated sequence (CA) in the four goat breeds by microsatellite analysis. For primer P3, three genotypes (CC, CD and DD) were detected in four goat breeds. Sequencing revealed one more CA dinucleotide in genotype DD than in genotype CC. The Jining Grey does with genotype CC had 0.55 (P < 0.05) or 0.72 (P < 0.05) kids more than those with genotype CD or DD. These results preliminarily indicated that allele C of BMP4 gene is a potential DNA marker for improving litter size in goats.     

Polymorphisms in BMP-2 gene and their associations with growth traits in goats

BMPs plays an important role in skeletal development. In this study, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of caprine BMP-2 gene in 299 goats from three breeds (Boer goat, Chinese Xuhuai white goat and Chinese Haimen goat). Three fragments of BMP-2 gene were investigated, only 3′ flanking region of BMP-2 gene showed polymorphism. The alignment between nucleotide sequences of AY714781.1 in GenBank and the sequencing results of three PCR products with different patterns revealed that there was one mutation (AY714781.1: g.A172G) in 3′ flanking region of BMP-2 gene, which constructed three genotypes (AA, AB, BB). The frequencies of allele A and genotype AA were dominant in all three breeds. Frequency of allele B in Haimen goat breed was significantly lower than that of the other two breeds. The genotype distributions were in good agreement with Hardy-Weinberg equilibrium (P>0.05) in each breed. The PIC (Polymorphism Information Content) values in three populations were 0.3261, 0.2558, 0.1663 for Boer goat, Xuhuai White goat and Haimen goat respectively. It was indicated that individuals of Boer goats with genotype BB were significantly higher than those of individuals with genotype AA in body trunk index (P<0.01). No polymorphism was detected in the exon3.     

牛生长激素受体基因多态性与体尺、体重指标的相关性分析

利用PCR-SSCP技术对南阳牛、利木赞、盖洛威共100头牛的生长激素受体基因的外显子10(GHR10)部分序列进行单核苷酸多态性研究, 并分析了该基因的不同基因型与3个品种牛生产性状的关系。结果表明: 南阳牛、利木赞、盖洛威3个品种中共存在6种基因型(AA、BB、CC、AB、AC、BC), c2检验表明该实验群体在这一位点上处于Hardy-Weinberg平衡状态(P > 0.05), 利木赞和盖洛威的PIC表现为中度多态,而南阳牛表现高度多态。测序结果显示: 所扩增GHR10部分片段共有5处碱基突变, 分别是495 bp (A/T), 622 bp(C/T), 650 bp(A/C), 702 bp(T/C), 730 bp(A/G); 并导致3处氨基酸替代: 其中622 bp Pro/Ser(脯氨酸/丝氨酸), 650 bp Asn/Thr(天冬酰胺/苏氨酸), 730 bp Ser/Gly(丝氨酸/甘氨酸)。最小二乘分析表明: 基因型AB、BC所对应的12月龄体重最小二乘均值显著高于基因型CC所对应的最小二乘均值(P < 0.05, P < 0.01); 18月龄体重BC基因型显著高于CC基因型(P < 0.01), 基因型AB所对应的18月龄胸围最小二乘均值显著高于基因型CC所对应的最小二乘均值(P < 0.05)。     

Polymorphisms of BMPR-IB gene and their relationship with litter size in goats

The bone morphogenetic protein receptor IB (BMPR-IB) gene was studied as a candidate gene for the prolificacy of goats. According to mRNA sequence of ovine BMPR-IB gene, ten pairs of primers were designed to detect single nucleotide polymorphisms (SNPs) of exon 1, exon 2, exon 6 to exon 10 and 3′ untranslated region (UTR) of the BMPR-IB gene in both high prolificacy breed (Jining Grey goat) and low prolificacy breeds (Wendeng Dairy and Inner Mongolia Cashmere goats) by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method. Only the products amplified by primers P8 and P10 of the 3′UTR displayed polymorphisms. For primer P8, three genotypes (AA, AB and BB) were detected in Jining Grey and Wendeng Dairy goats, two genotypes (AA and AB) were in Inner Mongolia Cashmere goats. Sequencing revealed one mutation (71C→T) of the BMPR-IB gene in genotype BB compared with AA. The differences of least squares mean (LSM) for litter size between genotypes AA, AB and BB were non-significant (P > 0.05) in Jining Grey goats. For primer P10, three genotypes (CC, CD and DD) were detected in Jining Grey and Wendeng Dairy goats and one genotype (CC) in Inner Mongolia Cashmere goats. Sequencing revealed one mutation (130T→C) of the BMPR-IB gene in genotype DD compared with CC. The differences of LSM for litter size between genotypes CC, CD and DD were non-significant (P > 0.05) in Jining Grey goats. These results preliminarily showed that the detected loci of the BMPR-IB gene had no significant effect on prolificacy of Jining Grey goats.     

催乳素受体基因外显子10多态性及其与济宁青山羊高繁殖力关系的研究

设计5对引物, 采用PCR-SSCP技术检测催乳素受体(prolactin receptor, PRLR)基因外显子10及部分3′非翻译区在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊和安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 首次拼接出的山羊PRLR基因外显子10及部分3′非翻译区的核苷酸序列长度为1,118 bp, 与已公布的绵羊、牛、人PRLR基因mRNA相应序列的同源性分别为98.33%、93.92%、74.52%, 与已公布的羊驼PRLR基因部分序列的同源性为78.29%。引物P1、P2与P4扩增片段具有多态性, 其余2对引物的扩增片段不存在多态性。对于P1扩增片段, 在济宁青山羊和辽宁绒山羊中检测到AA型和AB型, 在安哥拉山羊中检测到AA型和AC型, 在波尔山羊中只检测到AA型; 克隆测序表明AB型与AA型相比有两处突变(186G→A和220T→C), 分别导致氨基酸由天冬氨酸变为天冬酰胺、亮氨酸变为脯氨酸; AC型与AA型相比有1处突变(140A→G), 该突变没有导致氨基酸变化; 济宁青山羊AA和AB基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P2扩增片段, 在济宁青山羊、辽宁绒山羊和波尔山羊中都检测到DD型和DE型, 而在安哥拉山羊中只检测到DD型; 克隆测序表明DE型和DD型相比有两处突变(52G→A和122G→A), 其中122 bp处的突变导致氨基酸由精氨酸变为甘氨酸; 济宁青山羊DD和DE基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P4扩增片段, 在济宁青山羊中检测到FF型和FG型, 在辽宁绒山羊中检测到FF型和GG型, 在波尔山羊中只检测到FF型, 在安哥拉山羊中检测到FF型、FG型和GG型; 克隆测序表明GG型和FF型相比在扩增片段的143 bp处发生1处碱基突变(A→G), 并导致氨基酸由蛋氨酸变为缬氨酸; FG基因型济宁青山羊产羔数最小二乘均值比FF基因型的多0.76只(P<0.05)。研究结果初步表明: PRLR基因可能是控制济宁青山羊多胎性能的一个主效基因或是与之存在紧密遗传连锁的一个标记。     

秦川牛脂联素基因SNPs检测及其与胴体、肉质性状的相关性

利用PCR-SSCP结合测序技术对405头24月龄秦川牛脂联素基因SNPs位点进行检测, 运用SPSS统计程序中的GLM模型将检测到的SNPs位点与部分胴体及肉质性状的相关性进行了分析。结果检测到AA、AB、BB、CC、CD 5种基因型, 其中AB、BB型个体在脂联素基因第2外显子 64 bp处发现G→C突变, CD型个体第3外显子50 bp处发现C→T的突变, G→C导致谷氨酸(GGA)转化为谷氨酰胺(GCA), C→T导致丝氨酸(TCA)转化为亮氨酸(TTA)。方差分析结果表明: AA型个体的宰前活重、胴体重、眼肌面积显著高于BB型(P<0.05), 而在胴体腿臀围方面, AA型个体极显著高于AB型、BB型个体(P<0.01)。CD型个体的宰前活重、胴体腿臀围、皮下脂肪厚、背膘厚、嫩度都显著优于CC型个体(P<0.05)。脂联素基因该位点可能是影响秦川牛胴体及肉质性状的主效QTL或与之紧密连锁, 可作为秦川牛高档牛肉生产的候选分子标记。     

Polymorphism in exon 3 of follicle stimulating hormone beta (FSHB) subunit gene and its association with litter traits and superovulation in the goat

The polymorphism in follicle stimulating hormone beta (FSHB) subunit gene was detected by PCR-SSCP and PCR-RFLP methods in 780 does from four goat breeds including Boer, Matou, Black and Boer-Matou crossbred (BM). The associations of FSHB genotypes with litter size (LS), litter weight at birth (LWB) and gestation length (GL) as well as superovulation performances were analyzed in relatively lower-prolific Boer and higher-prolific Matou breeds. A 1514 bp linear DNA sequence of goat FSHB gene covering the complete 3 exons was cloned by four pairs of primer. A new mutation (A2645G, GenBank Accession no: S64745) locating in exon 3 causing an amino acid change from glutamine (Gln) to arginine (Arg) at residue 115 was identified, which resulted in three genotypes named AA, AB and BB. The three genotypes were detected in the four studied goat breeds. The higher-prolific Matou breed had the highest frequency of genotype AA. Association analysis showed that Boer and Matou does with AA genotype have the largest LS both in average and in parities from first to fourth (P < 0.05). Furthermore, Boer does with AA genotype had the heaviest LWB compared to other two genotypes (P < 0.05). Matou does with AA genotype have more numbers of ova harvested, large follicles and corpus lutea on ovaries after superovulation than those with BB genotype (P < 0.05). Therefore, these results suggest that the FSHB gene is a candidate gene that affects reproduction traits in goats.