6个猪品种CD53基因中结构变异SV184分布频率的研究

基于香猪全基因组重测序数据,在白细胞表面抗原CD53基因中筛选到一个结构变异（structure variant,SV）184,为了探索不同猪品种间此结构变异是否存在多态性变化,本试验选择香猪、大白猪、糯谷猪、柯乐猪、江口萝卜猪和荣昌猪作为试验动物,采用PCR方法对猪群中SV184的分布频率进行比较研究,应用RT-PCR技术,分析CD53基因的原初转录本序列结构,研究SV184对CD53基因的转录是否有直接影响。结果显示,6个猪品种中SV184的分布频率存在明显差异,香猪以VN基因型为主,其他5个猪品种是NN基因型占优势;计算两种等位基因的频率,香猪以缺失的V等位基因为主,其他5个猪品种以正常的N等位基因为主。选择3种基因型个体的血液样本,逆转录后分析CD53基因的原初转录本的序列,从中检测到缺失型和不缺失的两种转录本形式,提示SV184对CD53基因的转录无直接影响。SV184可作为分子标记用于区分香猪和其他猪品种。     

3个地方猪品种BOLL和STRA8基因结构变异位点的群体分布研究

为了探讨BOLL-I6-sv285（15号染色体BOLL基因第6内含子）和STRA8-I4-sv447（18号染色体STRA8基因第4内含子）这两个结构变异在地方猪与欧洲猪品种中的群体分布差异,试验采用PCR方法对从江香猪、江口萝卜猪、荣昌猪和大白猪4个猪品种进行基因分型,并通过在线软件miRBase、UCSC、RegRNA 2.0对结构变异序列所含的功能元件进行分析。结果显示,BOLL-I6-sv285为一段285 bp的插入,经群体验证,3个地方猪品种以DI和DD基因型为主,I等位基因的频率高于大白猪,插入基因型（Ⅱ）对应较高的产仔数,基因型与从江香猪产仔数之间呈显著相关（P<0.05）;STRA8-I4-sv447为一段447 bp的插入突变,群体中DD基因型占优势,从江香猪、江口萝卜猪和大白猪的I等位基因频率高于荣昌猪,插入基因型（Ⅱ）对应较低的产仔数,基因型与从江香猪产仔数之间呈显著相关（P<0.05）。本试验鉴定的BOLL-I6-sv285和STRA8-I4-sv447两个结构变异可作为从江香猪产仔数的候选分子标记。     

Analysis on Polymorphisms of Three CNV Regions in Five Pig Breeds

The paper was aimed to investigate the polymorphism of copy number variation (CNV) in different pig breeds.Three CNV regions of CNVR91,CNVR92 and CNVR143 were chosen from the porcine SNP60 chip genotyping results.The polymorphisms of three CNVs were determined by Real-time quantitative PCR method,taking five pig breeds as samples,including Yorkshire pig,Xiang pig,Kele pig,Nuogu pig and Rongchang pig breeds.The results showed that the dominant status of CNVR91 was loss in Xiang pig,while it was normal in other four pig breeds.The major type of CNVR92 was deletion in Xiang pig,Yorkshire pig,Kele pig and Rongchang pig breeds,with a high normal percent in Nuogu pig.For CNVR143,the dominant event was gain in Xiang pig and Nuogu pig breeds,but it was not diverse in other three pig breeds.These results indicated that three CNV regions emerged with polymorphism in five pig breeds,which might have effects on gene expression in CNV regions and physiological function by dosage effect especially in Xiang pig,Nuogu pig and Kele pig breeds.     

5个猪品种3个拷贝数变异区的多态性分析

为了探究拷贝数变异(copy number variation,CNV)在不同猪品种间的多态性,本试验根据猪SNP60芯片检测结果,以大白猪、香猪、柯乐猪、糯谷猪、荣昌猪为主要研究对象,采用实时荧光定量PCR方法,对5个猪品种基因组中3个CNV区域(CNVR91、CNVR92和CNVR143)的拷贝数进行测定。结果显示,香猪的CNVR91以拷贝数缺失为主,其他4个猪品种的拷贝数以正常为主;香猪、大白猪、柯乐猪、荣昌猪的CNVR92以拷贝数缺失为主,糯谷猪以拷贝数正常为主;香猪和糯谷猪的CNVR143以拷贝数增加为主,其他3个猪品种以拷贝数正常为主。表明3个拷贝数变异区在5个猪品种之间具有多态性,可能通过剂量效应影响香猪、糯谷猪和柯乐猪等相关基因的表达和生理功能。     

Polymorphism of SNP Site rs80894395 and its Association with the Body Size of Xiang Pig

To explore the functional genes affecting the growth and development of Xiang pig, a single nucleotide polymorphism (SNP) site rs80894395, located at intron 10 of polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2) gene was screened out by Illumina Porcine SNP60K chip, which showed a weak association with the abdomen circumference of pig.Further, the genotypes of site rs80894395 in 520 female Xiang pigs were detected using allele-specific PCR(AS-PCR) method, taking 43 Yorkshire pigs and 36 Kele pigs as control groups.All of CC, CT and TT genotypes were detected from the three pig breeds.The dominant genotype of Xiang pig was CT genotype and it was positively weak-associated with its body measurement indexes including body weight, body length, and abdomen circumference.The CC genotype was dominant genotype in Yorkshire pig, and the frequency of T allele was extremely significant decreased compared with that of Xiang pig (P<0.01).There was no difference in the frequencies of CC and CT genotypes in Kele pig, and its allele frequencies was not significantly different compared with that of Xiang or Yorkshire pig breeds (P>0.05).A splice-site nearby site rs80894395 was found out using bioinformatics prediction software, which might affect the splicing process of GALNT2 gene.It suggested that site rs80894395 might have a connection with the growth and development of Xiang pig.     

香猪SNP位点rs80894395的多态性及其与体尺指标之间的关联研究

为了探究影响香猪生长发育的功能基因,本试验利用Illumina Porcine SNP60K芯片筛选出N-乙酰氨基半乳糖转移酶2(polypeptide N-acetylgalactosaminyltransferase 2,GALNT2)基因内含子10中一个与腹围呈弱相关的单核苷酸多态性(single nucleotide polymorphism,SNP)位点rs80894395。在此基础上应用等位基因特异性PCR(allele-specific PCR,AS-PCR)技术检测520头雌性香猪群体中该位点的基因型,并与43头大白猪、36头柯乐猪相比较。结果表明,从3个猪群体中检测到rs80894395位点的CC、CT和TT3种基因型;其中香猪群体以CT为优势基因型,且基因型与香猪的体重、体长和腹围呈弱的正相关;大白猪群体以CC基因型为主,T等位基因频率较香猪极显著减少(P<0.01);柯乐猪群体中CC与CT基因型频率相等,与香猪和大白猪的基因频率间的差异不显著(P>0.05)。生物信息学分析表明,rs80894395位点可能影响GALNT2基因的剪接过程。rs80894395与香猪的生长发育可能有一定的联系。     

贵州地方猪品种对氧磷脂酶1基因的多态性研究

本试验以欧洲猪种为参照,研究贵州地方猪对氧磷脂酶1(paraoxonase,PON1)基因的多态性,探讨基因多态性与地方猪的脂肪沉积之间是否存在相关性。研究采用锚定聚合酶链反应技术对香猪、糯谷猪、萝卜猪和可乐猪共138个贵州地方猪品种PON1基因的单核苷酸多态性(single nucleotide polymorphisms,SNP)进行研究。结果表明,猪群PON1基因的163/165位点出现A/T、G/A变异,515位点检测到C/T突变,均以3种基因型存在。经卡方(χ²)检验,贵州地方猪群PON1基因515位点的基因型频率和基因频率与欧洲猪品种间的差异不显著(P>0.05);163/165位点中,贵州地方猪种A等位基因的频率为83.7%,明显高于欧洲猪品种(25.2%)。贵州地方猪品种PON1基因的163/165位点A等位基因占优势,编码的第55位氨基酸为甲硫氨酸(Met55),与脂代谢异常人群的基因型相同;欧洲猪种以B等位基因为主(P<0.01),编码的第55位是亮氨酸(Leu55),与正常人群的基因型一致。以人PON1蛋白的晶体结构为模板,推导出香猪PON1蛋白的三维结构。地方猪PON1基因变异导致的Met55突变可能影响PON1水解酶活性中心的疏水环境,可能使蛋白的水解酶活性下降,使得地方猪品种的脂肪沉积量和肉质不同于欧洲猪种。由此推测,贵州地方猪品种PON1基因163/165位点存在多态性,可能与地方猪种的脂肪沉积和肉质有一定的相关性。     

贵州地方猪品种毛色与KIT基因拷贝数之间的关联分析

为了研究KIT基因的拷贝数与贵州地方猪毛色之间的关系,本试验以黔北黑猪、柯乐猪、糯谷猪、香猪4个地方品种为试验材料,采用实时荧光定量PCR方法检测基因组中KIT基因的拷贝数变异,并与荣昌猪和大白猪2个白毛色猪品种相比较.结果显示,4个贵州地方猪种中,全黑/全棕毛色个体的KIT基因拷贝数以缺失为主,黑色带白斑个体的KIT基因拷贝数全部缺失,而棕色带白个体的KIT基因拷贝数增加、正常各半.大白猪的KIT基因拷贝数显著增加,荣昌猪KIT基因拷贝数以正常类型为主.表明大白猪的白毛色由KIT基因拷贝数增加决定,但荣昌猪的白毛色可能与KIT基因的拷贝数变异之间没有直接的联系;4个贵州地方品猪的全黑/全棕色毛色与KIT基因拷贝数的缺失有关,但柯乐猪和香猪中带白斑个体的KIT基因拷贝数并未增加,提示贵州地方猪品种的白斑毛色的形成机制有一定的特殊性.     

The Genetic Diversity and Phylogenetic Relationships of Five Guangxi Pig Breeds and Three Exotic Pig Breeds by Analysis of mtDNA D-loop Sequences

In order to compare the genetic diversity and phylogenetic relationships between Guangxi pig breeds and three exotic pig breeds,one pair of primers was used to amplify 85 mtDNA D-loop sequences of five Guangxi pig breeds (Bama Xiang pig,Dongshan pig,Guizhong spotted pig,Luchuan pig,Huanjiang Xiang pig),and we downloaded 45 mtDNA D-loop sequences of three exotic pig breeds (Duroc,Yorkshire,Landrace) from GenBank.Then the haplotype,genetic diversity and phylogenetics analysis were carried out.The results showed that 38 haplotypes were identified in 130 sequences,and the mean distances among Guangxi pig breeds were smaller than that among Duroc,Landrace and Guangxi pig breeds.The NJ phylogenetic tree classified all samples into two clusters.One contained all of Guangxi pig breeds and partial Yorkshire,the other was composed wholly of exotic pig breeds,which mean there were two distinct maternal origins.In the cluster of Guangxi pig breeds,Luchuan pig were clustered,and the others were rather dispersive.It indicated that Luchuan pig had simple maternal bloods,and the other breeds had complex maternal bloods,and they had gene flow with each other.The maternal pig resources in Guangxi were not affected by the introduction of exotic pig breeds,and they were still suitable for use as a female parent in their development and utilization.     

不同猪种肉质相关基因Hal、RN和FTO的多态性研究

为了明确肉质相关基因氟烷基因(Hal)、酸肉基因(RN)和脂肪肥胖相关基因(FTO)在国内外不同猪种群体间的遗传变异特性,本研究以大约克、长白、杜洛克和金华猪等猪种群体为研究对象,应用PCR-RFLP技术分别检测了Hal、RN和FTO基因的多态性,并分析了FTO基因上2个多态位点g.276G＞T和c.594C＞G的遗传变异情况.结果表明:(1)在杜洛克猪中发现了Hal的基因型HalN Haln,其频率为0.166,但未发现基因型Haln Haln,其它猪种群体中仅检测到了基因型HalN HalN;(2)在已检测的所有猪种群体中只检测到RN基因的rn/rn基因型,未发现rn/RN和RN/RN基因型;(3)在FTO的g.276G＞T位点上,金华猪呈现单态,其它猪种群体均呈现多态.在FTO的c.594C＞G位点上,4个猪种群体均呈现多态,3个外来猪种群体均以CC基因型频率较高,而金华猪则以GG基因型频率较高.研究结果提示,由于Hal基因在养猪生产中的利弊双重性,因此在某些猪种群体中仍然存在较高频率的HalN Haln基因型.此外,结合FTO基因的生理功能和已有的研究结果,可在特定的猪群中将其作为影响猪肉质性状的候选基因.本研究发现FTO的基因型分布在我国优良地方猪种金华猪与国外3个种猪群间存在较大差异,为深入研究不同品种猪的肉质形成机理提供了基础数据.     

皱皮香猪WIF1基因结构变异WIF1-I8-sv889的研究

为探究WIF1（Wnt inhibitory factor 1）基因中WIF1-I8-sv889结构变异位点变异与皱皮香猪躯干被毛形成的关系,本试验采用冰冻组织切片观察皱皮香猪皮肤毛囊的组织学形态,采用PCR方法分析WIF1-I8-sv889位点在猪群中的分布频率,并通过在线软件UCSC、RegRNA 2.0对结构变异序列所含的功能元件进行分析。组织学研究显示,与正常香猪和大白猪皮肤相比,皱皮香猪毛囊毛根鞘伸入真皮层,形成棘突,且皱褶凹陷处毛囊聚集,相反皱褶凸起处毛囊较少;皱皮香猪皮肤毛囊中的毛球宽度显著增大（P<0.05）,单个毛囊中的毛干数极显著增多（P<0.01）。在WIF1-I8-sv889结构变异断点两端设计特异性引物,扩增片段长1383 bp,与参考基因组比,1383 bp中889 bp为结构变异区间,缺失581 bp,倒置308 bp。群体分布结果显示,猪群中WIF1-I8-sv889位点呈现出丰富的多态性,检测到3种基因型：正常的Ⅱ型、杂合的DI型和缺失的DD型,皱皮香猪中未检测到Ⅱ型;与正常香猪和大白猪相比,皱皮香猪中D等位基因占优势（94.44%）;经卡方检验,皱皮香猪D等位基因显著或极显著高于正常香猪和大白猪（P<0.05;P<0.01）。结果提示,WIF1基因中的WIF1-I8-sv889结构变异可能与皱皮香猪毛囊的形态发生有关。     

6个猪种胰岛素样生长因子-1(IGF-1)基因座位遗传多态性检测

本文采用ＰＣＲ－ＲＦＬＰ分析方法对我国地方猪种民猪、香猪、桃源猪、内江猪、姜曲海猪以及用作对照的国外猪种杜洛克猪的ＩＧＦ－１基因座位进行研究。结果发现：ＩＧＦ－１基因ＰＣＲ扩增片段存在２个限制性内切酶ＨｈａＩ酶切位点,其中１个酶切位点产生遗传多态。此片段上共产生２个等位基因。在ＩＧＦ－１基因位点上,香猪除与桃源猪基因频率差异不显著之外,与其它猪种之间基因频率差异极显著（Ｐ＜０．０１）,而其它各猪种之间差异均不显著     

香猪SRY基因的克隆及序列分析

试验参照猪SRY基因保守序列设计了1对引物,对香猪基因组DNA进行PCR扩增。将扩增产物通过T—A互补法克隆到质粒pGEM—T载体中,筛选阳性克隆进行DNA测序。测序结果显示,香猪SRY基因开放阅读框全长627bp,编码227个氨基酸。其中核心区域HMG—box长237bp,并且编码79个氨基酸。与白鲸、人、牛、绵羊4种哺乳动物的SRY基因序列相比较,香猪和白鲸的SRY序列具有较高的相似性,相似度达82．1％。序列相似性和邻接法聚类树的结果均显示,在SRY基因中香猪和白鲸有着较近的亲缘关系,表明以白鲸为代表的鲸目和以香猪为代表的偶蹄目有较近的亲缘关系。     

MAPK信号通路2个结构变异多态性及其与香猪生长性状的关联分析

试验旨在探究丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号通路中MAPK10、MEF2C基因结构变异的遗传多样性及其与香猪生长性状的关联性,以期为贵州香猪的育种改良提供理论依据。采用PCR技术对233头香猪2个结构变异（structure variation,SV）位点（MAPK10-I₈-sv273和MEF2C-I₁-sv71）进行群体基因变异研究,利用Origin软件绘制香猪的生长曲线,利用在线软件计算出多态信息含量（PIC）、杂合度（He）、纯合度（Ho）及有效等位基因数（Ne）,并利用SPSS 26.0软件分析基因分型结果与香猪生长性状的关联性。结果显示,2个结构变异位点在香猪群体中存在多态性,均有3种基因型：DD、ID和II,2种等位基因：D和I。其中,MAPK10-I₈-sv273的D等位基因频率明显高于I等位基因,DD基因型频率最高（0.65）,且DD基因型个体的平均日增重、体高和生长速度均大于II基因型个体,表明该位点D等位基因具有提高香猪生长性能的遗传效应;MEF2C-I₁-sv71的I等位基因频率高于D等位基因,II基因型频率最高（0.51）,II基因型个体的平均日增重、胸围、总体生长速度均大于DD基因型个体,表明该位点的I等位基因可提高香猪的生长性能。综上,结构变异MAPK10-I₈-sv273和MEF2C-I₁-sv71与香猪的生长发育相关,可作为生长性状选育的候选分子标记。     

猪垂体特异性转录因子基因多态性的研究

对香猪、上海白猪、大约克夏猪的垂体特异性转录因子(PIT-1)基因进行了单核苷酸多态性(SNP)分析。结果显示:在3个猪种PIT-1基因的第5和第6外显子中均未发现SNP;而在第4外显子中出现SNP,即在公布序列的第272位上有1/2的大约克夏猪为碱基C,其余1/2的大约克夏猪和全部的香猪、上海白猪均发生了C→T突变,这是一个沉默突变。根据研究结果推测,PIT-1基因第4外显子中的碱基突变及这3个外显子的序列状况可能与香猪的矮小性无关。     

Genetic structure of pig breeds from Korea and China using microsatellite loci analysis

To understand molecular genetic characteristics of Korean pigs, the genetic relationships of nine pig breeds including two Korean pigs (Korean native pig and Korean wild pig), three Chinese pigs (Min pig, Xiang pig, and Wuzhishan pig), and four European breeds (Berkshire, Duroc, Landrace, and Yorkshire) were characterized from a 16-microsatellite loci analysis. The mean heterozygosity within breeds ranged from 0.494 to 0.703. Across multiple loci, significant deviation from Hardy-Weinberg equilibrium was observed in most pig breeds, except for two Chinese pigs (Min pig and Wuzhishan pig). This deviation was in the direction of heterozygote deficit. Across population loci, 36 of 144 significantly deviated (P < 0.05) from Hardy-Weinberg equilibrium. The mean FST, a measure of genetic divergence among subpopulations, of all loci indicated that 26.1% of total variation could be attributed to the breed difference. Relationship trees based on the Nei's DA genetic distance and scatter diagram from principal component analysis consistently displayed pronounced genetic differentiation among the Korean wild pig, Xiang pig, and Wuzhishan pig. Individual assignment test using a Bayesian method showed 100% success in assigning Korean and Chinese individual pigs into their correct breeds of origin and 100% exclusion success from all alternative reference populations at P < 0.001. These findings indicate that the Korean native pig has been experiencing progressive interbreeding with Western pig breeds after originating from a North China pig breed with a black coat color. Considering the close genetic relationship of Korean pigs to the Western breeds such as Berkshire and Landrace, our findings can be used as valuable genetic information for the preservation and further genetic improvement of the Korean native pig.     

皱皮香猪3个基因组拷贝数变异的多态性分布

为探究皱皮香猪个体产生皮肤变异的根本原因,本文基于重测序数据,采用生物信息学分析皱皮香猪基因组中的拷贝数变异(CNV),通过荧光定量 PCR 方法检测皱皮香猪中 3 个 CNVs 的拷贝数变异规律,并与香猪和大白猪进行比较。结果表明:3 个 CNVs 在猪群中的拷贝数变异呈现出多态性变化,皱皮香猪中CNV1以拷贝数缺失型为主,其中的 MPC1和 RSK3基因可能与脂肪沉积有关;皱皮香猪 CNV2和 CNV3以拷贝数增加型占优势,其中的 CCL17和 CX3CL1基因参与皮肤等组织的炎症反应,可能通过剂量效应引起香猪皮肤产生褶皱。     

不同品种猪α1-岩藻糖转移酶基因遗传变异初析

本实验采用PCR RFLP方法对 5个瘦肉型猪种杜洛克、长白猪、大白猪、汉普夏猪和皮特兰猪共计 2 5 0头猪的α1 岩藻糖转移酶基因 (FUT1)进行多态性分析。结果表明 :本研究中的 5个猪种在该FUT1基因座位存在多态性 ,均分布着三种基因型 (AA ,AG和GG) ,抗性基因型AA分布频率为 0 .0 2 8,易感基因型AG与GG分布频率分别为0 .2 4 4和 0 .72 8。卡方检验结果表明 ,长白猪与大白猪基因型频率差异极显著 (P <0 .0 1) ,与皮特兰猪之间差异显著(P <0 .0 5 ) ,其它猪种之间差异均不显著。