利用DNA条形码对海南特种野猪的鉴定研究

试验旨在探讨以猪的细胞色素氧化酶Ⅰ(cytochrome C oxidase subunitⅠ,COXⅠ)基因的特定区段作为DNA条形码识别海南特种野猪与其他品种的可行性和有效性。以海南特种野猪、海南野猪、屯昌猪、五指山猪及长白猪为研究对象,测定其COXⅠ基因,同时在NCBI中下载已发表的具有地方代表性的13个猪品种的COXⅠ基因序列,分析海南特种野猪与其他品种的遗传多样性、遗传距离,并构建系统进化树。结果发现,猪COXⅠ基因序列长1 419bp,5个猪品种45个个体COXⅠ基因共检测到67个变异位点,占分析位点的4.7%,其中简约信息位点22个;5个猪品种的种内遗传距离为0.001~0.019,其中海南特种野猪的种内遗传最大,为0.019;5个猪品种的种间遗传距离为0.009~0.123,其中海南特种野猪与长白猪的遗传距离最大,为0.123;海南特种野猪杂交情况比较明显,与海南本地品种五指山猪、屯昌猪和海南野猪都有聚类,与武夷黑猪、三都黑猪、陆川猪位于一个分支,另一部分与大花白猪位于一个分支,与长白猪没有聚类。本试验结果为海南特种野猪的进一步选育提供了参考依据。     

三个国外猪种线粒体COXⅠ基因SNPs筛选及序列分析

本研究对三个国外猪种216个样本(大白猪、长白猪和杜洛克)mt DNA COXⅠ基因构建混合池,利用直接测序技术获得3个猪种COXⅠ基因的序列,进行SNPs筛选及序列分析,并采用MEGA 6.0分析软件基于Kimurk双参数模型应用邻接法构建系统发生树。结果表明,3个猪种mt DNA COXⅠ基因序列共存在16个突变位点,且有3个突变位点为3个猪种共有。长白猪与杜洛克猪的多态性较丰富,含有13个相同位点的突变,核苷酸的转换数(si)和颠换数(sv)的比值(R)分别为12和15,序列替换远未达到饱和。系统进化树和遗传距离分析显示长白猪及杜洛克亲缘关系较近,先聚为一支,再与大白猪、及野猪聚为一支。大白猪与中国东北地区野猪聚为一支,这可能与大白猪在培育过程中亲本遗传背景较复杂有关。本研究在一定程度上为了解大白猪的育成史及合理利用国外猪种提供了理论依据。     

三个地方猪种线粒体COXⅠ基因SNPs筛选及序列分析

本研究对西北地区三个地方猪种471个样本(藏猪、甘肃黑猪和八眉猪)mt DNA COXⅠ基因构建混合池,利用直接测序技术获得3个猪种COXI基因的序列,进行SNPs筛选及序列分析,并采用MEGA 6.0分析软件基于Kimurk双参数模型应用邻接法构建系统发生树。结果表明,3个猪种mt DNA COXI基因序列仅存在5个突变位点,且无相同突变位点,多态性较贫乏。系统进化树和遗传距离分析显示,八眉猪、藏猪、黑猪及野猪聚为一支,再与杜洛克、欧洲伯克希尔猪及大白猪聚为一支,而后与同属为偶蹄目的牛、绵羊聚为一支。西北猪种应有共同的母系祖先,品种内母系遗传多样性较贫乏,外来猪种引入我国后主要是用作父本以提高生产速度和瘦肉率等,对本地猪种未有母系贡献。本研究在一定程度上为我国地方猪品种的有效保护和合理利用提供理论依据。     

基于测序技术的海南地方猪TLR4基因多态性分析

为了了解海南地方猪TLR4基因的多态性,试验采用PCR技术对五指山猪、临高猪和屯昌猪的TLR4基因组序列进行了克隆、测序,应用DNAStar和BioEdit软件分析其序列,SMART、SOPMA、SWISS-MODEL在线软件分析TLR4蛋白的结构,并预测二级结构和三级结构。结果表明:获得的三个品种海南地方猪TLR4基因DNA序列全长均为10 435 bp,其中编码区长2 526 bp,编码841个氨基酸。三个品种猪TLR4基因序列种内比对时,五指山猪在种内有1个核苷酸位点存在多态性;屯昌猪在种内有6个核苷酸位点存在多态性,2处位于编码区;临高猪在种内有3个核苷酸位点存在多态性,1处位于编码区。三个品种猪TLR4基因序列种间比对时,有27个核苷酸位点存在多态性,其中2个为错义突变,导致氨基酸改变。三个品种猪的TLR4基因序列的同源性在99.8%～99.9%之间,高度保守。海南猪TLR4基因在7 209位点的G→T突变和7 781位点的G→A位点突变引起了TLR4蛋白二级结构和三级结构发生改变。说明海南地方猪TLR4基因存在多态性,并且其多态性引起的结构改变可能使TLR4基因功能发生变化。     

基于测序技术的海南地方猪TLR2基因多态性分析

为了解海南地方猪TLR2基因的多态性,本研究采用PCR法从五指山猪、临高猪和屯昌猪3种海南地方猪的血液中克隆Toll样受体2(TLR2)基因,并进行测序及序列分析。结果显示,3种海南地方猪TLR2基因的扩增长度均为2649bp,编码区长2358bp。3种猪TLR2基因序列的种内比对结果显示,五指山猪种内有7个核苷酸位点存在多态性,2处位于编码区;屯昌猪种内有5个核苷酸位点存在多态性,均在编码区外;临高猪种内有4个核苷酸位点存在多态性,1处位于编码区。种间比对结果显示3种猪有12个核苷酸位点存在多态性,其中3处为错义突变,导致氨基酸改变。同源性分析结果显示,3种猪的TLR2基因序列高度保守,同源性在99.6%~99.9%之间。蛋白质结构预测分析显示,海南猪TLR2基因在876、1454位点呈现的AG突变均引起了其蛋白质二级结构和三级结构的改变,提示可能存在TLR2功能的变化。本研究填补了海南地方猪TLR2基因多态性空白,为深入研究TLR2基因多态性与猪疫病易感性的关系提供基础研究资料。     

广东省现有5个地方猪种基于SNP芯片的遗传多样性分析

本研究以广东省现有的5个地方猪品种为对象,利用SNP芯片进行SNP分型,对比欧洲野猪、亚洲野猪以及4个西方商业猪种,对广东省现有地方猪品种的遗传多样性、遗传距离以及群体结构进行分析。结果表明:两广小耳花猪、大花白猪和蓝塘猪的遗传多样性低于西方商业猪种以及粤东黑猪和梅花猪;广东省地方猪与亚洲野猪遗传距离较近,与西方猪种遗传距离较远;作为大花白猪一个类群的梅花猪与大花白猪遗传距离较远,主成分分析以及Structure分析表明梅花猪与大花白猪遗传差异较大,梅花猪具有独特的遗传结构并存在西方猪血液的渗入;粤东黑猪与西方猪种遗传距离较近,粤东黑猪也存在西方猪种血液的渗入。综上所述,两广小耳花猪、大花白猪和蓝塘猪的遗传多样性较低,梅花猪和粤东黑猪存在西方猪种血液的渗入。     

猪TLR4基因2个突变位点的PCR-SSCP分析

为了研究Toll样受体4(TLR4)的多态性与动物对相关病原的免疫力的相关性,试验选择北京黑猪、长白猪、野猪、杜洛克猪、大白猪、民猪6个中外猪种,利用PCR-SSCP和克隆测序方法对TLR4基因第3外显子1 824位点的G/A和3’UTR 208位点的T/C多态进行群体遗传学分析。结果表明:1 824位点北京黑猪与野猪、民猪、杜洛克猪、长白猪和大白猪的基因型分布存在显著差异(P<0.05);3’UTR 208位点基因型分布在猪种间存在极显著差异(P<0.01)。     

猪PLSCR4基因外显子区PCR-RFLP遗传多态性分析

本研究利用PCR-RFLP技术对我国6个地方猪种、中国野猪以及杜洛克、约克夏和长白3个国外猪品种,共307头猪的磷脂爬行酶基因(PLSCRs)外显子区的遗传变异进行了研究。结果表明:PLSCR4第7外显子处存在T68C的同义突变,第8外显子处存在G4A的错义突变;在T68C突变位点处,杜洛克、约克夏、长白猪、二花脸、五指山和民猪6个猪群优势等位基因为T,荣昌和野猪群体中优势等位基因为C;在G4A突变位点处,杜洛克、约克夏、长白猪、二花脸、五指山和民猪群体中优势等位基因为G,藏猪、荣昌、金华及野猪群体中优势等位基因A。     

海南3个地方猪种SLA-DQB基因外显子2的PCR-RFLP多态性分析

采用PCR-RFLP法对海南五指山猪、临高猪及屯昌猪的SLA-DQB基因外显子2的PCR产物进行多态性分析。结果表明:五指山猪和临高猪SLA-DQB基因外显子2经限制酶RsaⅠ酶切后出现7种基因型,均由4个复等位基因A、B、C、D控制。屯昌猪中仅出现4种基因型,由3个复等位基因A、C、D控制。经HaeⅢ酶切结果出现8种基因型,这8种基因型在五指山猪群体中均存在,由E、F、G、H4个复等位基因控制;临高猪、屯昌猪经HaeⅢ酶切分别出现6种和5种基因型,均由3个复等位基因E、F、G控制。χ2适合性检验结果表明,五指山猪的RsaⅠ和HaeⅢ酶切位点突变未达到Hardy-Weinberg平衡。临高猪SLA-DQB基因外显子2RsaⅠ酶切位点不符合Hardy-Weinberg平衡状态,而HaeⅢ酶切位点处于Hardy-Weinberg平衡状态,屯昌猪SLA-DQB基因外显子2的2个酶切位点均处于Hardy-Weinberg平衡状态。     

海南特种野猪与屯昌黑猪肌肉挥发性风味化合物比较研究

为了研究海南特种野猪和屯昌黑猪的肌肉挥发性风味特性的差异,试验采用顶空固相微萃取法吸附提取样品的挥发性风味组分,经过顶空固相微萃取-气相色谱-质谱串联技术分析检测背最长肌的挥发性风味成分,并根据总离子流图,由计算机Nist和Willey库检索定性检出的化合物,采用色谱峰面积归一化法对检出的化合物进行定量并用相对含量表示;采用SAS 9.0软件整理数据并对成分相同且相对含量大于1%的挥发性风味物质进行主成分分析,观测两种杂交猪肉品质的挥发性风味化合物差异,结果用百分比和特征向量表示。结果表明:在这两种杂交猪的肌肉中,主要检测出醛类、醇类、酮类、烃类、酯类、酸类、杂环化合物等。脂质的氧化产物醛、醇以及酮类化合物是海南特种野猪肌肉最主要的挥发性风味成分,共占总挥发性化合物的85.95%,其中醛类物质含量最多,占总体化合物的60%以上,而碳氢化合物、酸类、酯类、酚类、杂环化合物含量较低。肉品中重要的风味呈味物质是含硫、含氮及杂环化合物,在两种肉品中检出对猪肉香气有重要贡献的2-烷基呋喃,并且含量相对较高。与屯昌黑猪相比,海南特种野猪肉中碳氢化合物种数相对含量总数小于屯昌黑猪,主要以庚烷、β-蒎烯、1,3-二甲基-环戊烷为主。十一醛、十四醛、顺-2-壬烯-1-醇、2,4-己二烯-1-醇、1-苯基-1,2-丙二酮、β-蒎烯等多种化合物只存在于海南特种野猪中,因此推测脂质的氧化产物醛、醇以及酮类化合物是造成海南特种野猪和屯昌黑猪肉风味差异的主要挥发性风味成分。     

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.     

Characterization of MC1R and KIT Genotypes in Luchuan Pig Population

In order to assess the purity of Luchuan pig populations, four South Chinese local pig breeds including Putian Black pig, Yuedong Black pig, Dahuabai pig, Bama miniature pig and three foreign pig breeds including Duroc pig, Yorkshire pig and Landrace pig were studied as controls by sequencing of PCR products, MC1R and KIT genotypes in 56 Luchuan pigs were analyzed in this study. Sequencing results indicated that a splicing mutation (G>A) was presented in the first base in intron 17 of KIT gene in both Yorkshire pig and Landrace pig, in contrast, the wildtype GG of KIT gene was presented in Luchuan pig, four south Chinese local pig breeds and Duroc pig.Compared with Hainan wild boar, South Chinese local pig breeds had two missense mutations 95Val > Met and 102Leu > Pro in the coding region of MC1R gene;Compared with Yorkshire pig, Landrace pig and Duroc pig, South Chinese local pig breeds had 5 to 6 SNPs in MC1R gene 5'UTR, and in addtion, an A base deletion in MC1R gene 3'UTR. Furthermore, we found one litter of Luchuan pig with abnormal coat color.The results showed that the presentation of two distinct MC1R genotypes E^D1 and E^p in both litters and the sow,but only E^D1 in the boar. Considering E^p was derived from Pietrain pig, we preliminarily considered that the genome of the sow might be infiltrated with foreign pig breeds. In summary, we detected the genotypes of the coat color genes KIT and MC1R in eight pig breeds, confirmed the molecular differences of coat color between Chinese local pig breeds and foreign pig breeds, which could be useful for the further investigation of the molecular mechanism of pig coat color.     

陆川猪MC1R和KIT基因型鉴定

为了检测陆川猪种群的纯度,本研究以4个华南地方猪种（莆田黑猪、粤东黑猪、大花白猪、巴马香猪）和3个外来猪种（杜洛克猪、大白猪、长白猪）作为对照,利用PCR测序法对陆川猪群体56个样本的MC1R和KIT基因进行基因型鉴定。测序结果表明,大白猪和长白猪KIT基因内含子17的第1个碱基发生G>A剪接突变,而陆川猪和4个华南地方猪种,以及杜洛克猪中KIT基因型一样,均为野生型的GG基因型;MC1R基因编码区分析表明,以海南野猪MC1R基因型作为参考序列,陆川猪和4个华南地方猪种在MC1R基因编码区存在95Val > Met和102Leu > Pro两个错义突变;MC1R基因非编码区分析表明,与大白猪、长白猪和杜洛克猪相比,陆川猪和4个华南地方猪种MC1R基因的5'UTR分别存在5~6个SNPs,3'UTR存在1个A碱基的缺失。另外,试验对一窝毛色异常的陆川猪仔猪及其亲本的KIT和MC1R基因进行测序分析,结果表明,仔猪及其亲本的KIT基因型均为GG野生型,仔猪和母本的MC1R基因均存在E^D1和E^p两种不同的基因型,父本的MC1R基因型为E^D1,因E^p对应于皮特兰猪的斑块表型,所以初步判定母本渗入了外来猪种的血统。本研究通过检测陆川猪等8个猪种的毛色相关基因KIT和MC1R基因的基因型,证实了中外猪种间在毛色遗传上的分子差异,为今后深入研究猪毛色遗传的分子调控机理提供参考。     

藏猪Mx1基因外显子14的遗传变异分析

为了解藏猪抗病性能分子遗传基础,试验采用PCR-RFLP技术测定112头藏猪的Mx1-Hin6 I多态性。结果表明:藏猪群体中Mx1基因外显子14有长度为11 bp片段的缺失,Hin6Ⅰ酶切位点有AA、AB和BB 3种基因型,基因频率分别为73.21%、23.22%和3.57%,A和B等位基因频率分别为84.82%、15.18%,与报道的野猪和五指山猪基因频率分布相近,但与国外猪种和其他国内地方猪种基因频率分布存在一定差异,这可能与藏猪的抗病和抗逆性能有关。     

海南五指山猪Myf5与MyoD基因多态性分析

为研究海南五指山猪Myf5和MyoD基因的多态性分布情况,本试验通过PCR-RFLP方法对80头海南五指山猪进行了Myf5和MyoD基因型检测,并运用生物信息学方法对这两个基因的序列进行分析.结果发现,在Myf5基因第1外显子的HhaⅠ-RFLP位点上,五指山猪以CC和CG基因型为主;在Myf5基因第1外显子的Hsp92Ⅱ-RFLP位点上,五指山猪以AA基因型为主;在MyoD基因第1内含子的DdeⅠ-RFLP位点上,等位基因A在五指山猪中是优势等位基因.此外,Myf5和MyoD基因启动子区域和第1外显子处存在CpG岛.本研究得到海南五指山猪Myf5和MyoD基因的序列信息,为下一步五指山猪Myf5和MyoD基因的功能研究奠定基础.     

Genetic diversity analyses of 10 indigenous Chinese pig populations based on 20 microsatellites

To study the genetic diversity of Chinese indigenous pig breeds, a total of 403 pigs from 10 local populations and 1 exotic Duroc breed were genotyped for 20 microsatellite markers. Heterozygosity and Wright's F-statistics (F(IS), F(ST), and F(IT)) were calculated to determine the genetic variation in those populations. The observed heterozygosities were in the range of 0.31 (Duroc) to 0.66 (Shengxian). The F(IS) value was in a range of -0.07 to 0.48. The mean F(ST) showed that approximately 78% of the genetic variation was within-population and 22% was across the populations. The 10 Chinese local breeds were classified into two major groups according to the phylogenetic tree, which was based on standard genetic distance. Four pig populations, Jianli, Ganxi Two Ends Black, Shaziling, and Dongshan were grouped into one branch. Before the study, these four populations were all classified as Central China Two Ends Black according to coat color, shape of the head, and shape of the ear. The Jinhua pig, which also has the two-ends-black coat color, was also grouped to the same branch but was not traditionally classified into this type. The five populations were located in various provinces in central China. The other five populations, Nanyang Black, Hainan Spotted, Huainan Black, Jiaxing Black, and Shengxian Spotted (black body, white feet), were grouped into another branch. The two groups of pig breeds had the same F(ST) value (0.14) when calculated separately. This value was similar to that of Iberian pigs (0.13) but smaller than that of the European pigs (0.27) as reported by other researchers. Our study showed that large genetic differentiation exists in Chinese pig breeds. The grouping of the five two-ends-black populations into one branch of the phylogenetic tree may indicate that the number of conservation farms can be decreased for this type of pig.     

MC4R基因多态性在海南地方猪中的分布

通过PCR—RFLP方法对90头海南五指山猪和36头临高猪进行了MC4R基因型检测。结果表明,MC4R基因中的TaqI酶切位点在五指山猪中均为AA基因型,在临高猪中该位点A等位基因和G等位基因频率分别是0．92和0．08。该结果为海南地方猪的杂交选育提供理论依据。     

猪骨形态发生蛋白-6基因多态性及其与产仔性能的关联性分析

试验旨在研究猪骨形态发生蛋白-6（bone morphogenetic protein 6, BMP-6）基因多态性及其与产仔性能的关联。采用PCR-SSCP技术检测了野猪、民猪、长白猪、北京黑猪、法系大白猪5个猪种268个个体BMP-6基因的多态性,并分析了BMP-6基因多态性与法系大白猪产仔性能的关联性。结果表明,猪BMP-6基因CDS区1104位存在1个G→A的同义突变,产生AA、AB、BB 3种基因型,其中在野猪和民猪中,A等位基因频率高;在长白猪、北京黑猪和法系大白猪中,B等位基因频率高。不同基因型对法系大白猪产仔数和产活仔数的影响差异显著（P<0.05）,初步认为BMP-6基因多态性与产仔性能显著关联。     

猪骨形态发生蛋白-6基因多态性及其与产仔性能的关联性分析

试验旨在研究猪骨形态发生蛋白-6（bone morphogenetic protein 6, BMP-6）基因多态性及其与产仔性能的关联。采用PCR-SSCP技术检测了野猪、民猪、长白猪、北京黑猪、法系大白猪5个猪种268个个体BMP-6基因的多态性,并分析了BMP-6基因多态性与法系大白猪产仔性能的关联性。结果表明,猪BMP-6基因CDS区1104位存在1个G→A的同义突变,产生AA、AB、BB 3种基因型,其中在野猪和民猪中,A等位基因频率高;在长白猪、北京黑猪和法系大白猪中,B等位基因频率高。不同基因型对法系大白猪产仔数和产活仔数的影响差异显著（P<0.05）,初步认为BMP-6基因多态性与产仔性能显著关联。