猪a1-岩藻糖转移酶基因(FUT1)在26个中外猪种中的遗传变异研究

肠毒素大肠杆菌F18(ECF18)是引起仔猪断奶后水肿和腹泻病的主要病原菌,a1—岩藻糖转移酶基因(FUT1)是ECF18侵染猪小肠的受体蛋白候选基因。通过采用PCR—RFLP方法检测了5个西方商业猪种以及21个中国地方猪种(群)1458个个体在FUT1基因开放阅读框架的307核苷酸位点的G-A点突变(M307^G-A)遗传变异。结果表明：5个外来猪种以及中国地方猪种中的临高猪在该FUT1基因位点存在多态性,其他中国地方猪种均表现为极端的单态分布,只有易感的GG基因型,没有多态性。由此提示：1)如果猪FUT1 M307^G-A点突变是决定猪小肠上ECF18受体表达与否的关键因素,则绝大部分中国地方猪种均不具备抵抗ECF18的遗传基础,这除了表明ECF18抗性基因有可能起源于西方猪种外,同时也表明对中国地方猪种中在这个位点惟一存在多态性的海南临高猪的品种资源保存具有非常重要的意义。2)一般而言,在中国的养猪生产实践中,中国地方猪种的仔猪抗水肿与腹泻病能力普遍强于外来猪种,研究的结果提示有必要对中国地方猪种所具备的上述遗传抗性做更深入的研究,寻找、定位其相应的QTL或／和抗性基因。     

猪a1-岩藻糖转移酶基因(FUT1)M857位点遗传变异分析

肠毒素大肠杆菌 (ETEC) F18是引起仔猪断奶后水肿和腹泻病的主要病原菌, a-1岩藻糖转移酶(FUT1)基因是ECEC F18侵染猪小肠的受体蛋白基因。利用PCR-RFLP方法检测了1个野猪以及20个中外家猪猪种(群)共696个个体在FUT1基因开放阅读框架的857核苷酸位点的遗传变异, 结果表明: 在所有猪种中, 均未检测到抗性的AA型纯合子, 在外来猪种杜洛克和约克夏、国内猪种临高猪和杂交猪种中检测到AG型杂合子, 外来猪种中的皮特兰、长白猪以及除临高猪外的所有国内猪种和野猪均表现为极端的单态分布, 只有易感的GG基因型。研究结果提示, 中国地方猪种不具备抵抗ETEC F18大肠杆菌的遗传基础, 与外来猪种确实存在差异, 这种差异可能与各自不同的起源有关, ETEC F18抗性基因可能起源于欧洲野猪; 并推测猪种的生长速度与ETEC F18大肠杆菌病的发生具有密切的关系。     

猪MyoG基因的PCR-RFLP多态性分析

以杜洛克、长白、大约克、南昌白、二花脸、梅山猪、玉山黑猪、乐平花猪、金华两头乌及上高两头乌等中外10个猪种共计561头猪为研究材料,采用3对引物（PCR1、PCR2、PCR3）分别扩增猪肌细胞生成素（MyoG）基因的不同区域,扩增产物经限制性核酸内切酶MspⅠ酶切后发现：（1）在PCR1 MspⅠ-RFLP位点上,外来品种杜洛克、长白、大约克及培育品种南昌白中极大多数个体表现为AA型,个别为BB型;而6个中国地方猪种除乐平花猪外均以BB型居多。（2）在PCR2 MspⅠ-RFLP位点上,6个中国地方猪种除一头玉山黑猪表现为MN型外,其余均为MM型;而外来品种以NN型占大多数,培育品种南昌白更趋向于外来品种。（3）在PCR3 MspⅠ-RFLP位点上,所有猪种均可得到扩增产物,但无MspⅠ酶切位点。（4）在梅山猪及与其亲缘关系较近的二花脸猪中,没有发现Soumillion等(1997)报道的梅山猪特异性MspⅠ多态性酶切位点。     

Porcine insulin-like growth factor 1 (IGF1) gene polymorphisms are associated with body size variation

Previous studies have confirmed that insulin growth factor-1 (IGF1) plays important roles in growth and body size in humans and animals. However, whether single nucleotide polymorphisms (SNPs) within the IGF1 gene affects body size and growth in pigs has been unclear. We identified IGF1 SNPs among 5 pig breeds (Berkshire, Duroc, Landrace, Yorkshire and Korea Native Pig) and found that the G allele of SNP (c.G189A) was associated with higher body weight and was more predominant in western pig breeds, while the Korean Native Pig is the breed with the highest frequency of the A allele. Four haplotypes (–GA–, –GG–, –AG–, and –AA–) were constructed using the 2 identified SNPs. The GA haplotype was most frequently observed, except in the Berkshire breed. In addition, these SNPs and haplotypes were significantly associated with body size (final weight), average daily gain, and backfat thickness (P < 0.05) in 2 intercrossed F₂ pig populations (KNP × YS F₂ and KNP × LR F₂). Furthermore, the major GA haplotype had a significant additive effect on body size and average daily gain. In conclusion, specific SNPs within the porcine IGF1 gene may contribute to the smaller body size and lower growth rate of Korea Native Pigs.     

猪MyoG基因3'端PCR-SSCP遗传多态性及其遗传效应

采用PCR—SSCP方法对长白猪、大白猪、杜洛克猪、山西黑猪和马身猪共636头猪的肌细胞生成素（Myogenin,简称MyoG）基因3’端的遗传多态性进行检测,分析MyoG基因对猪的初生体质量、断奶体质量、6月龄体质量和背膘厚的影响。根据已发表的猪MyoG基因3’端侧翼序列设计3对引物,发现F1／R1引物对扩增的片段有多态性。统计结果发现：长白、大白、杜洛克猪种B基因为优势基因,其基因频率分别为0．8807、0．7256和0．8581：山西黑猪种4基因为优势基因,其基因频率为0．9359;马身猪种只检测到4基因。χ^2独立性检验表明,基因型分布在外来猪种（长白猪、大白猪、杜洛克猪）与地方猪种（山西黑猪、马身猪）间存在极显著差异（P〈0．01）。固定效应模型分析结果表明,初生体质量基因型闻差异显著（P〈0．05）,而断奶体质量、6月龄体质量和背膘厚基因型闻差异不显著（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质量,同似和AB型比较差异极显著（P〈0．01）。因此,推测MyoG基因对个体的初生体质量存在一足的影响,选择带有B等位基因的个体有望提高个体的初生体质量。     

The footprint of recent and strong demographic decline in the genomes of Mangalitza pigs

The Mangalitza pig breed has suffered strong population reductions due to competition with more productive cosmopolitan breeds. In the current work, we aimed to investigate the effects of this sustained demographic recession on the genomic diversity of Mangalitza pigs. By using the Porcine Single Nucleotid Polymorphism BeadChip, we have characterized the genome-wide diversity of 350 individuals including 45 Red Mangalitza (number of samples; n=20 from Hungary and n=25 from Romania), 37 Blond Mangalitza, 26 Swallow-belly Mangalitza, 48 Blond Mangalitza × Duroc crossbreds, 5 Bazna swine, 143 pigs from the Hampshire, Duroc, Landrace, Large White and Pietrain breeds and 46 wild boars from Romania (n=18) and Hungary (n=28). Performance of a multidimensional scaling plot showed that Landrace, Large White and Pietrain pigs clustered independently from Mangalitza pigs and Romanian and Hungarian wild boars. The number and total length of ROH (runs of homozygosity), as well as F_ROH coefficients (proportion of the autosomal genome covered ROH) did not show major differences between Mangalitza pigs and other wild and domestic pig populations. However, Romanian and Hungarian Red Mangalitza pigs displayed an increased frequency of very long ROH (>30 Mb) when compared with other porcine breeds. These results indicate that Red Mangalitza pigs underwent recent and strong inbreeding probably as a consequence of severe reductions in census size.     

猪肌细胞生成素基因对部分生长性状的影响

采用PCR-SSCP方法对长白猪、大白猪、杜洛克猪、山西黑猪和马身猪的肌细胞生成素（Myogenin,简称MyoG）基因进行单核苷酸多态性检测,并分析MyoG基因对猪的初生重、断奶重、6月龄重和背膘厚的影响。根据猪MyoG基因的DNA序列设计10对引物,发现In2-3引物扩增的片段有多态性。统计结果发现,3种基因型（AA、AB、BB）在各品种中的分布不一致。χ^2独立性检验表明,基因型频率在外来猪种（长白猪、大白猪、杜洛克猪）与地方猪种（山西黑猪、马身猪）间存在显著差异（P〈0．05）。固定效应模型分析结果表明,初生重和背膘厚基因型间差异显著（P〈0．05）,而断奶重和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基因型与其他两种基因型比较有较小的初生重,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;AA基因型与其他两种基因型比较有较小的背膘厚,同AB和BB型比较差异极显著（P〈0．01）,3种基因型在背膘厚的大小排列顺序为AA〈AB〈BB。因此,推测基因型对个体的初生重和胴体瘦肉率存在一定的影响,选择带有A等位基因的个体有望提高个体的初生重和胴体的瘦肉率。     

Construction of multiple shRNAs expression vector that inhibits FUT1 gene expression and production of the transgenic SCNT embryos in vitro

Enterotoxigenic Escherichia coli F18 is a major pathogen that causes postweaning diarrhoea and edema disease in piglets. The alpha(1,2)-fucosyltransferase (FUT1) gene has been identified as an ideal candidate gene for controlling the expression of the receptor for ECF18 bacteria. Therefore, the use of RNA interference (RNAi) to study the function of the FUT1 gene and to produce FUT1 knockdown transgenic pig would be highly beneficial. We developed an effective strategy for the expression of multiple small hairpin RNA simultaneously using multiple RNA polymerase III (hU6, hH1, mU6 and h7SK) promoters in a single vector to knockdown the FUT1 gene. Stable FUT1 knockdown transgenic fibroblast lines were generated by transfecting porcine fetal fibroblasts with the constructed vectors. Real-time RT-PCR indicated that the mRNA level of FUT1 in the transgenic fibroblast lines was significantly lower than that in the control, as much as 29 %. Finally, we successfully obtained transgenic SCNT porcine embryos. Overall, the results demonstrated that this vector-based RNAi expression system is an efficient approach to knockdown FUT1 gene expression in porcine fetal fibroblast cells, which could thereby provide donor cells for somatic cell nuclear cloning and the potential production of a marker-free transgenic pig resistant to F18 related diseases. Furthermore, it also provides strong evidence that this approach could be useful both in the production of transgenic livestock resistant to disease, and in the development of effective strategies for the suppression of gene expression in clinical gene therapy.     

The effect of mutation at M307 in <Emphasis Type="Italic">FUT1</Emphasis> gene on susceptibility of <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 and gene expression in Sutai piglets

Escherichia coli F18 (ECF18) is a common porcine enteric pathogen. The pathogenicity of ECF18 bacteria depends on the existence of ECF18 receptor in the brush border membranes of piglet’s small intestinal mucosa. Alpha (1) fucosyltransferase gene (FUT1) has been identified as the candidate gene controlling the adhesion to ECF18 receptor. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting resistant pigs. The piglets were divided into three groups, AA, AG and GG, according to the genotypes present at M307 of FUT1. Small intestinal epithelium cells of piglets with AA, AG and GG genotypes were selected to test the adhesion capability of the wild type E.coli expressing F18ab fimbriae, the recombinant E. coli expressing F18ac fimbriae or the recombinant E. coli secreting and surface-displaying the FedF subunit of F18ab fimbriae, respectively. Here, we examined the distribution and expression of porcine FUT1 mRNA in different tissues in Sutai pigs using real-time PCR. The results showed that piglets with AA genotype show resistance, whereas piglets with GG or AG genotypes are sensitive to the pathogenic E. coli F18 in Sutai piglets. FUT1 was expressed in all the tissues that were examined, and the gene’s expression was highest in the lungs. There was no significant difference in expression level among the three genotypes in the liver, lung, stomach and duodenum, where the gene expression was relatively high. The present analysis suggested that mutation at M307 in FUT1 gene determines susceptibility of small intestinal epithelium to E. coli F18 adhesion in Sutai piglet and the expression of FUT1 gene may be regulated by other factors or the mutation was likely to be in linkage disequilibrium with some cis-regulatory variants.     

Evaluation of M307 of <Emphasis Type="Italic">FUT1</Emphasis> gene as a genetic marker for disease resistance breeding of sutai pigs

Alpha (1,2) fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ETEC F18. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting ETEC F18 resistant pigs. The polymorphisms of M307 in FUT1 of breeding base group for ETEC F18 resistance of Sutai pigs (Duroc × Meishan) was detected and their correlations to some immune indexes, growth and development ability, carcass traits and meat quality were also analyzed, which aimed to investigate feasibility of further breeding for diseases resistance based on M307 of FUT1 for Sutai pigs. After digested by Hin6 I, M307 of FUT1 gene could be divided into three kinds of genotypes, AA, AG, and GG. The frequencies were 0.235, 0.609, and 0.156, respectively. The results indicated that Sutai pigs with the AA genotype in M307 of FUT1 gene not only have relatively strong general disease resistance ability in piglets, but also have higher growth and development ability and stable carcass traits and meat quality. It is entirely feasible to raise the new strains of Sutai pigs resistant to Escherichia coli F18 based on genetic marker of the M307 position in FUT1gene.     

Molecular characterization of differentially expressed TXNIP gene and its association with porcine carcass traits

Thioredoxin interacting protein (TXNIP), which plays a regulatory role in lipid metabolism and immune regulation, is down-regulated expressed in F₁ hybrids Landrace?×?Yorkshire skeletal muscle. Here we described the molecular characterization of porcine TXNIP gene. The full-length cDNA contains a coding sequence of 1,176?bp nucleotides with untranslated regions of 263?bp at 5′-end and 441?bp at 3′-end, respectively. The predicted molecular mass and isoelectric point of porcine TXNIP is 43.81?kDa and 7.385, respectively. The deduced 391 amino acids exhibit high identity with other mammalian TXNIP. The TXNIP gene contains eight coding exons and seven non coding introns, spans approximately 3,348?bp. The expression of porcine TXNIP mRNA is almost absent in Landrace?×?Yorkshire and lower level in 6-month-old pigs during skeletal muscle development. Other stages and breeds were high level expressed. Statistical analysis showed the TXNIP gene polymorphism (c.575-4T>C) was different between F₁ hybrids and their parents, was highly associated with dressing percentage (DP) and thorax–waist fat thickness (TFT) in the Yorkshire?×?Meishan F₂ population. The possible role of TXNIP was discussed.     

白色基因座(I)在中国地方猪种毛色遗传中的作用研究

通过利用PCR—RFLP和PCR—SSCP技术对中国地方猪种KIT基因内含子17、18的序列进行多态性分析。结果表明：内含子17上的替换突变(G→A)发生于毛色为白色的个体——白色五指山猪、大白猪、长白猪上,其基因型(AB型)频率分别为1、1和0．8;其他中国地方猪种的此基因型频率均为0。内含子18上的缺失突变(AGTT)也同样发生在上述3个猪种的白色个体中,其基因型(AA型)频率分别为1、1和0．93;而且同样在其他的地方品种中其基因型频率均为0。这充分证明KIT基因对于猪的白毛色有重要的调控作用,而且I基因座对于其他的经典遗传基因座有上位作用。另一方面,中国地方猪种荣昌猪虽然在表型上与引入猪种大白猪、长白猪相似(白毛色),但是在KIT基因上发生的突变完全不同,推测它们分别属于不同的毛色遗传体系。     

SIM1基因第8外显子的SNP对猪背膘厚的影响

采用PCR-SSCP方法检测了约克夏、杜洛克、皮特兰、长白猪、嘉兴黑猪和金华猪6个品种共169头猪的SIM1基因外显子8的SNP及其基因型频率。结果共发现CC、CT、TT 3种基因型, 其基因型频率在国内外猪品种之间具有较大差异。其中, 国内猪种嘉兴黑猪和金华猪只存在TT基因型, 而国外猪种约克夏、杜洛克、皮特兰、长白猪则都存在3种基因型。用最小二乘法分析SNP对长白猪、约克夏猪和杜洛克猪的背膘厚的效应的结果表明, 纯合基因型个体的背膘厚大于杂合基因型个体。SIM1基因型对国外猪种背膘厚有显著效应(P< 0.05), 并且不同部位效应不同。     

Associations between PRLR/AluI gene polymorphism with reproductive,growth, and meat traits in pigs

The aim of this study was to determine the associations between genotypes of the prolactin receptor gene (PRLR) and reproductive, growth, and meat traits in pigs. Genetic groups of sows were used: Large White (LW), Danish Landrace (LD), and Landrace × Yorkshire × Duroc hybrids (L × Y × D). Reproductive traits studied were the total number of born (TNB) and the number born alive (NBA). The growth traits were the number of days to 100 kg and the average daily gains (ADG). The meat traits were the average backfat thickness (BFT), half carcass weight (HCW), area of M. longissimus dorsi (MLT), and lean meat content (LM). The polymorphism was detected using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method. It was found that the genotype BB is associated with the best NBA. In sows LD, the AB genotype is associated with best number of days to 100 kg; in hybrids L × Y × D, the AA genotype is associated with better meat traits as compared to the genotype BB.     

Relationship Among H-FABP Gene Polymorphism, Intramuscular Fat Content, and Adipocyte Lipid Droplet Content in Main Pig Breeds with Different Genotypes in Western China

H-FABP (Heart fatty acid-binding protein), a member of FABP family, plays an essential role in long-chain fatty acid uptake and metabolic homeostasis. Its role in pig intramuscular fat content remains poorly understood, especially in local pig breeds in western China. In this study, the genetic variations of 5′-upstream region and the second intron in porcine H-FABP gene were investigated by PCR-RFLP in 256 pigs including Duroc, Large White, Landrace, Neijiang, Rongchang, Bamei pig, Hanjiang Black, Hanzhong White, and the wild ones. The effect of H-FABP gene on the IMF content was analyzed by the least square method. Lipid droplet morphology and content in adipocytes cultured from pigs with different H-FABP genotypes, were studied by oil red O staining and a triglyceride assay kit. Results showed a Hinf I -RFLP in these eight pig breeds and wild pigs, among which Large white, Bamei pig, Hanjiang Black, Hanzhong White, and wild pigs presented with low polymorphism while the other breeds had intermediate polymorphism. There was no Hae III or Msp I -RFLPs in the four Chinese local pig breeds tested, but Duroc, Landrace, Large White, Hanzhong White and wild pig had polymorphism. Landrace, Large White and wild pigs had low levels of Hae III-and Msp I -RFLP, whereas others had intermediate polymorphism. H-FABP genotypes significantly affected the IMF content (P<0.05). The IMF content ordered by H-FABP genotypes were HH>Hh>hh, DD<Dd<dd, and AA<Aa<aa. The genetic effect values were 3.89, 3.42, 3.17, 2.27, 2.49, 2.91, 2.28, 2.70, and 2.95, respectively. Fat deposition in adipocytes was stronger in the HH, dd and aa genotypes than in others. The results suggest that porcine meat quality may be improved by increasing the frequency of genotype aa-dd-HH in pig breeds.     

Genetic Variation and Association of Insulin-Like Growth Factor Binding Protein-3 with Performance in Swine

Genetic variation of insulin-like growth factor binding protein-3 was analyzed in 17 pig breeds (14 native Chinese and 3 European). Using PCR-single strand conformation polymorphism, we found a polymorphism in intron 2, and this SNP was the combined mutation of G897T-G903A-C911T. The Chinese breeds carried a higher TAT/TAT genotype frequency (over 50%), except for Bamei (22%), Yujiang Black (0.0%), and Erhualian (10.0%); the European breeds had a higher GGC/GGC genotype frequency (Large White 1.67%, Landrace 13.89%, Duroc 0.0%). The allelic frequency of TAT in Chinese breeds was over 50%, except for Yujiang Black (12.5%); the allelic frequency of GGC was over 50% in all European breeds. The effect of genotype on 43 performance traits was investigated in one population (Lantang × Landrace). Pigs with the TAT/TAT genotype had higher B-point and C-point back-fat thickness than pigs with the GGC/GGC genotype. The TAT/TAT pigs also scored higher in meat color than the GGC/GGC pigs. These results implied that IGFBP-3 may affect meat quality and carcass traits.     

西部地区主要猪种和野猪H-FABP基因分子标记

利用PCR-RFLP(Hinf I、Hae Ⅲ和Msp I 3种限制性内切酶)分子标记技术,检测了杜洛克猪、长白猪、大白猪、内江猪、荣昌猪、汉江黑猪、汉中白猪、八眉猪和野猪共计265头猪H-FABP基因5′-上游区和第二内含子区的遗传变异,并利用最小二乘模型分析了H-FABP基因对猪肌内脂肪含量的遗传效应。结果表明：（1）在Hinf I-RFLP位点上,上述品种和野猪均存在多态性,其中大白猪、八眉猪、汉江黑猪、汉中白猪和野猪表现为低度多态,杜洛克、长白猪、内江猪和荣昌猪为中度多态;除汉江黑猪（P<0.05）和野猪（P<0.01）外,其他猪种基因频率和基因型频率都处于Hardy-Weinderg平衡状态（P>0.05）;而在Hae Ⅲ-RFLP和Msp I-RFLP位点上,仅内江猪、荣昌猪、汉江黑猪和八眉猪为单态;（2）9种基因型对肌内脂肪（IMF）含量的影响,HH>Hh>hh,DD相似文献   

Effects of the MyoG Gene on the Partial Growth Traits in Pigs

Single-nucleotide polymorphisms of the MyoG gene were tested using PCR-SSCP in different pig breeds including Landrace, Large White, Duroc, Shanxi Black, and Mashen pigs, and the effects of the MyoG gene on the birth weight, the weaning weight, the 6-month body weight, and the backfat thickness were also analyzed. On the basis of the published sequence of the porcine MyoG gene, ten pairs of primers were designed, and one polymorphism was found in the PCR product amplified with In2-3 primers. The results showed that: (1) the Landrace, the Large White, and the Duroc breeds differ significantly (P < 0.05) in genotype distribution from the Shanxi Black and the Mashen breeds; (2) On the basis of the fixed effect model, significant differences were found in the birth weight and the backfat thickness among the different MyoG genotypes, whereas no significant differences existed in the weaning weight and the 6-month body weight; (3) Using least square analysis, it was seen that individuals of the BB genotype had significantly less (P < 0.01) birth weight than those of the AA and AB genotypes, with the order being AA>AB>BB; the pigs of the AA genotype had significantly lower (P < 0.01) backfat thickness than those of the AB and BB genotypes, with the order being AA<AB<BB. These results suggest that the genotype has significant effects on the individual birth weight and the backfat thickness, and that the selection of the A allele is favored with regard to the birth weight and the backfat thickness.     

H2AFZ,RNF4 and NR4A1 loci are associated with boar semen quality by population association studies

Abstract

Spermatogenesis is a complex process regulated by many genes. In this study, H2AFZ, RNF4 and NR4A1 genes were selected as candidate genes for boar semen quality traits based on their functions during spermatogenesis, and the associations of three loci (H2AFZ c.192?+?210–192?+?213delCGAT, RNF4 c.374?+?358 T?>?C and NR4A1 c.956?+?796 A?>?G) with sperm quality traits were analyzed in Duroc (n?=?185), Large White (n?=?87) and Landrace (n?=?49) pig populations. The results showed H2AFZ c.192?+?210–192?+?213delCGAT AA boars produced 1.52% lower abnormal sperm rate (ASR) than AB boars in Landrace pigs (p?<?0.05); RNF4 c.374?+?358?TC boars produced 0.31?×?10⁸/ml higher sperm concentration (SCON) than CC boars (p?<?0.05) in Large White pigs; NR4A1 c.956?+?796 A?>?G was associated with ASR in Duroc and Large White pigs and was associated with sperm motility (MOT) in Large White and Landrace pigs. This study indicated the H2AFZ, RNF4 and NR4A1 loci were the potential molecular markers for improving the semen quality traits in boars.