Porcine growth differentiation factor 9 gene polymorphisms and their associations with litter size

Growth differentiation factor 9 (GDF9) is expressed in oocytes and is thought to be required for ovarian folliculogenesis.Given this function,GDF9 may be considered as a candidate gene controlling pig ovulate rate.In this study,the complete coding sequence was cloned (encoding a 444 amino acid),intron sequence and partial 5'-UTR of pig GDF9.RT-PCR results showed that GDF9 mRNA is expressed in a wide range of tissues of the ruttish Erhualian pig.The expression levels of GDF9 mRNA in pituitary,ovary,uterus and oviduct are higher in the Erhualian pigs than those in Duroc pigs,especially in pituitary with a significant difference (P＜0.05).Comparative sequencing revealed 12 polymorphisms,including 8 single nucleotide polymorphisms (SNPS) and one 314 bp indel in noncoding regions,and the other 3 SNPS in coding regions.Four polymorphisms,G359C,C1801T,T1806C and 314 bp indel,were developed as markers for further use in population variation and association studies.The G359C polymorphism segregates only in Chinese native pigs,Erhualian and Dahuabai,on the contrary,314 bp indel segregates only in Duroc and Landrace.C1801T and T1806C sites seem to be completely linked and segregate in Erhualian,Dahuabai and Landrace.In a word,GDF9 may be not associated with pig litter size in extensive populations as per the studies of allele distributions of the four polymorphisms and pilot association in four breeds.     

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。     

Polymorphisms on SSC15q21-q26 Containing QTL for reproduction in Swine and its association with litter size

Several quantitative trait loci (QTL) for important reproductive traits (ovulation rate) have been identified on the porcine chromosome 15 (SSC15). To assist in the selection of positional candidate swine genes for these QTL on SSC15, twenty-one genes had already been assigned to SSC15 in a previous study in our lab, by using the radiation hybrid panel IMpRH. Further polymorphism studies were carried out on these positional candidate genes with four breeds of pigs (Duroc, Erhualian, Dahuabai and Landrace) harboring significant differences in reproduction traits. A total of nineteen polymorphisms were found in 21 genes. Among these, seven in six genes were used for association studies, whereby NRP2 polymorphism was found to be significantly (p < 0.05) associated with litter-size traits. NRP2 might be a candidate gene for pig-litter size based on its chromosome location (Du et al., 2006), significant association with litter-size traits and relationships with Sema and the VEGF super families.     

Bi-PASA genotyping of a new polymorphism in the APOB gene shows no evidence for an association with fatness in pigs

Sequence analysis of PCR products of a 343-bp fragment from exon 29 of the porcine APOB gene of four Erhualian and four Landrace pigs revealed a missense G/C substitution at position of 6117 in this gene. Two allele-specific primers were designed to genotype this polymorphism using the Bi-PASA technique. Genotyping of 146 animals from Erhualian, Hampshire, Large White, Landrace and Duroc breeds revealed large breed differences in allele frequency. No association with fatness was observed within each of the four European breeds, where animals had been selected to be those with the highest and those with the lowest backfat depth at 100 kg liveweight from a large carcass dissection project.     

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.     

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.     

Characterization of porcine leptin receptor polymorphisms and their association with reproduction and production traits

Fatness in pigs is of prime economic importance due to market incentives for production of lean pork and elevated fatness increasing the feed costs. Leptin (LEP) action, mediated through its specific receptors (LEPR), was reported to be involved in the regulation of fatness via feed intake, energy expenditure, and whole-body energy balance in pigs. In this study, we have designed 17 primer sets based on the human and mouse LEPR sequences and successfully amplified coding regions of 15 porcine LEPR exon fragments by polymerase chain reactions (PCR). Four single nucleotide polymorphisms (SNP) of Intron 2, Exons 2, 6, and 18 were found in Landrace, Yorkshire, and Duroc by mutagenetically separated-PCR (MS-PCR) and PCR-restriction fragment length polymorphisms (PCR-RFLP). Chi-square statistics was used to calculate homogeneity of genotypic frequencies of 4 gene polymorphisms for three breeds of animals. Effects of Intron 2, Exon 2, and Exon 18 polymorphisms on the reproduction trait such as litter sizes of sows were evident (p < 0.05) in Duroc and Yorkshire. There was no (p > 0.05) significant influence on the production trait of average daily gain due to four candidate gene polymorphisms in three porcine breeds. However, effects of Exon 6 and 18 polymorphisms on the production trait of backfat thickness were significant (p < 0.05) in Landrace and Yorkshire, respectively. Effects of Exon 18 polymorphisms on feed efficiency were also evident (p < 0.05) in Duroc.     

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   

两种家猪心脏组织基因表达谱的分析

选取丹麦成年长白母猪的心脏、肌肉、主动脉、左心室和中国成年二花脸母猪心脏共5种组织,建立相应的cDNA文库,在此基础上测定了35180条表达序列标签。通过比较分析长白猪4种组织的基因表达,找出了长白猪心脏组织中3个显著高表达的功能群和47个显著高表达的基因。长白猪心脏组织在分子伴侣活动、马达活动和生理过程中表达的基因较多,47个显著高表达的基因都是和运动、运动调节、能量及保护有关的基因。两种家猪心脏组织表达差异显著的基因有74个,并且绝大部分是在长白猪心脏中表达更高,显示出长白猪心脏组织生理活动的高效与旺盛。     

Genetic polymorphisms and preliminary association analysis with production traits of the porcine <Emphasis Type="Italic">SLC27A4</Emphasis> gene

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).     

Effects of FUT1 gene mutation on resistance to infectious disease

Alpha-(1,2)-fucosyltransferase (FUT1) gene has been identified as a candidate gene for regulating the expression of Escherichia coli F18 receptor gene (ECF18R) which promotes adherence of Enterotoxigenic (ETEC) and Verotoxigenic (VTEC) Escherichia coli (E. coli) via F18 fimbriae. In order to illustrate the polymorphisms of FUT1 and their effects on resistance to natural infection by Porcine Respiratory and Reproductive Symdrome Virus (PRRSV) and Haemophilus parasuis, the distributions of different genotypes and the relative risks of disease incidence in pigs were investigated. A total of 1,041 pigs representing three European breeds (Duroc, Landrace and LargeWhite), five Chinese local breeds (Wild pig, Small MeiShan, QinPing, JinHua, and JianLi) and three commercial populations (LargeWhite?×?JianLi, Duroc?×?Landrace?×?LargeWhite and Duroc?×?wild pig) were selected to analyze the genotype of the FUT1 gene by PCR-RFLP. Only the GG genotype associated with susceptibility to ECF18 bacteria was detected in Chinese local pig breeds and a population of LargeWhite?×?JianLi, while the AA genotype which confers resistance to ECF18 was detected in two European breeds (Duroc and LargeWhite), two populations of Duroc?×?wild pig and Duroc?×?Landrace?×?LargeWhite. Regarding relative risk of incidence, Duroc?×?Landrace?×?LargeWhite with genotypes GG or AG showed greater relative risk (OR?=?2.040, P?=?0.025; OR?=?1.750, P?=?0.081, respectively) than those with genotype AA during natural infection by both PRRSV and Haemophilus parasuis. It can be concluded that the mutation of FUT1 gene might play a role in pig infection by multi-pathogens, and that AA may be a favourable genotype for increasing the resistance to disease.     

Genome-Wide Association Analysis for Blood Lipid Traits Measured in Three Pig Populations Reveals a Substantial Level of Genetic Heterogeneity

Serum lipids are associated with myocardial infarction and cardiovascular disease in humans. Here we dissected the genetic architecture of blood lipid traits by applying genome-wide association studies (GWAS) in 1,256 pigs from Laiwu, Erhualian and Duroc × (Landrace × Yorkshire) populations, and a meta-analysis of GWAS in more than 2,400 pigs from five diverse populations. A total of 22 genomic loci surpassing the suggestive significance level were detected on 11 pig chromosomes (SSC) for six blood lipid traits. Meta-analysis of GWAS identified 5 novel loci associated with blood lipid traits. Comparison of GWAS loci across the tested populations revealed a substantial level of genetic heterogeneity for porcine blood lipid levels. We further evaluated the causality of nine polymorphisms nearby or within the APOB gene on SSC3 for serum LDL-C and TC levels. Of the 9 polymorphisms, an indel showed the most significant association with LDL-C and TC in Laiwu pigs. But the significant association was not identified in the White Duroc × Erhualian F₂ resource population, in which the QTL for LDL-C and TC was also detected on SSC3. This indicates that population-specific signals may exist for the SSC3 QTL. Further investigations are warranted to validate this assumption.     

Growth hormone gene polymorphisms and growth performance traits in Duroc, Landrace and Tao-Yuan pigs

This study investigated the restriction fragment length polymorphism (RFLP) of the porcine growth hormone (pGH) gene in Duroc, Landrace, and Tao-Yuan pigs and its effects on growth performance and levels of plasma growth hormone in peripheral circulation. Genomic DNA extracted from 81 Tao-Yuan, 60 Landrace and 48 Duroc pigs were subjected to Southern blot hybridization with a pGH cDNA probe. Polymorphism was detected with the restriction enzymes TaqI and DraI. A comparison of these three breeds showed significant differences in allelic frequencies. Blood samples for radioimmunoassay (RIA) of GH were collected biweekly during the experimental period from pigs 12 to 40 weeks of age. Tao-Yuan pigs showed a mean plasma GH level (2.51 +/- 1.23 ng/mL) that was much lower than that of the Landrace (3.80 +/- 1.52 ng/mL) and Duroc (4.20 +/- 1.03 ng/mL) pigs (P < 0.05). Moreover, the Tao-Yuan pigs also showed poorer growth performance than the Landrace and the Duroc pigs both in the daily weight gain (0.37 +/- 0.06 vs. 0.67 +/- 0.05 and 0.70 +/- 0.05 kg/day, P < 0.01) and feed efficiency (3.12 +/- 0.28 vs. 2.60 +/- 0.14 and 2.52 +/- 0.12, P < 0.05). These results suggest that the growth performance trait in these pigs is highly correlated with their growth hormone genotype.     

西部地区主要猪种H-FABP基因多态性,IMF含量及不同基因型脂肪细胞脂滴量的关系

H-FABP是FABP家族成员之一,在长链脂肪酸的吸收和代谢平衡中发挥关键作用,但它对猪IMF含量的影响还知之甚少,对中国西部地区的猪种更是如此.文章利用PCR-RFLP（HinfⅠ、HaeⅢ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,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,并运用猪脂肪细胞培养,油红O染色和TG测定等技术检测了H-FABP基因不同基因型脂肪细胞内脂滴的形态和沉积的量.结果表明：（1）在HinfⅠ-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HaeⅢ-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＜Dd＜dd,AA＜Aa＜aa,遗传效应值分别为：3.89,3.42,3.17,2.27,2.49,2.91,2.28,2.70,2.95,H-FABP基因可显著地提高IMF含量（P＜0.05）;（3）aaddHH型的脂肪细胞脂滴的形态,密度和含量与其他基因型细胞差异显著（P＜0.05）.结果提示：可通过提高＂aaddHH＂基因型的频率来增加IMF含量,达到改善猪肉质的目的.