Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

用焦磷酸测序技术研究猪线粒体细胞色素B基因单倍型

选择43头大白猪,79头长白猪,66头皮特兰猪和60头清平猪作试验材料,采用焦磷酸测序技术分析猪线粒体细胞色素b(CytB)基因单倍型。研究结果显示CytB基因可分为4种单倍型E1,E2,A1和A2。清平猪仅存在于A1单倍型(100%),大白猪和长白猪存在于E1(49.19%,79.25%)和A1(55.81%,20.25%)单倍型,皮特兰则存在于E1(57.58%)和A2(42.42%)单倍型。 Abstract：To detect porcine mitochondrial cytochrome b (CytB) gene haplotypes, Pyrosequencing, which is a novel DNA sequencing method, has been used to analyze SNPs selected Large White, Landrace, Pietrain and Qingping pigs. The pyrosequencing analysis of CytB gene displayed four distinct haplotypes E1, E2, A1 and A2 respectively. Qingping pigs are only present in haplotype A1, Large White and Landrace pigs are present in haplotype E1 and A1, and Pietrain pigs are present in haplotupe E1 and A2.     

New SNP of the porcine Perilipin 2 (<Emphasis Type="Italic">PLIN2</Emphasis>) gene,association with carcass traits and expression analysis in skeletal muscle

PLIN2 (perilipin 2) is a cytosolic protein that promotes the formation and stabilization of the intracellular lipid droplets, organelles involved in the storage of lipid depots. Porcine PLIN2 gene represents a biological and positional candidate for fat deposition, a polygenic trait that affects carcass and meat quality. The aim of the present study was to screen PLIN2 gene for polymorphisms, to evaluate the association with carcass quality traits, and to investigate the gene expression in skeletal muscle. Six new single nucleotide polymorphisms (SNP) were detected by sequencing 32 samples from five pig breeds (Italian Large White, Italian Duroc, Italian Landrace, Belgian Landrace, Pietrain). Two SNP localized in introns, two in the 3′-untranslated region (UTR), and two missense SNP were found in exons. A 3′-UTR mutation (GU461317:g.98G>A), genotyped in 290 Italian Duroc pigs by High Resolution Melting, resulted significantly associated (P < 0.01) with average daily gain, feed conversion ratio, lean cuts and hams weight estimated breeding values. PLIN2 gene expression analysis in skeletal muscle of Italian Large White and Italian Duroc pigs divergent for backfat thickness and visible intermuscular fat showed a trend of higher expression level in pigs with higher intermuscular fat. These results suggest that PLIN2 can be a marker for carcass quality in pigs. Further investigation at both gene and protein level could elucidate its role on fat deposition.     

Polymorphisms in an obesity-related gene (PCSK1) are associated with fat deposition and production traits in Italian heavy pigs

The proprotein convertase subtilisin/kexin type 1 (PCSK1) gene encodes the prohormone convertase 1/3 enzyme that processes prohormones into functional hormones that, in turn, regulate central and peripheral energy metabolism. Mutations in the human PCSK1 gene cause severe monogenic obesity or confer risk of obesity. We herein investigated the porcine PCSK1 gene with the aim of identifying polymorphisms associated with fat deposition and production traits in Italian heavy pigs. By re-sequencing about 5.1 kb of this gene in 21 pigs of different breeds, we discovered 14 polymorphisms that were organized in nine haplotypes, clearly distributed in two clades of putative European and Asian origin. Then we re-mapped this gene on porcine chromosome 2 and analysed its expression in several tissues including gastric oxyntic mucosa of weanling pigs in which PCSK1 processes the pre-pro-ghrelin into ghrelin, which in turn is involved in the control of feed intake and energy metabolism. Association analyses between PCSK1 single-nucleotide polymorphisms (SNPs) and production, carcass and several other traits were conducted on five groups of pigs from three different experimental designs, for a total of 1221 animals. Results indicated that the analysed SNPs were associated (P < 0.01 or P < 0.05) with several traits including backfat thickness and visible intermuscular fat in Italian Duroc (ID) and growth performances in Italian Large White (ILW) and in ILW × Italian Landrace pigs. However, the effects estimated in the ILW were opposite to the effects reported in the ID pigs. Suggestive association (P < 0.10) was observed with muscle cathepsin B activity, opening, if confirmed, potential applications to reduce the excessive softness defect of the green hams that is of particular concern for the processing industry. The results obtained supported the need to further investigate the PCSK1 gene to fully exploit the value of its variability and apply this information in pig breeding programmes.     

Association of corticotropin-releasing hormone gene variation with performance and meat quality traits in commercial pig lines

The porcine corticotropin-releasing hormone(CRH) gene is a functional-positional candidate for quantitative tract loci on porcine chromosome 4 with major effects on growth and carcass composition. In addition, the central role of CRH in the neuroendocrine response to stress implicates the CRH gene as a functional candidate for meat quality. Association of a single nucleotide polymorphism (SNP) in the promoter region of the porcine CRH gene (g.233C > T) with several growth, carcass and meat quality traits was examined using more than 2000 individuals from four commercial lines: German Landrace (LR), Pietrain (Pi), German Large White x German Landrace (F1) and the German commercial fattening pig cross of Pietrain x F1 (PiF1). Significant association of the CRH SNP was found with feed conversion ratio in the PiF1 line, with carcass length in the LR line and with lean content in the F1, LR and Pi lines. Moreover, significant association with meat colour was found in the Pi and LR lines; however, the effects were in opposite directions. The presented results indicate that sequence variation in the porcine CRH gene has no major effect on growth and carcass composition in commercial pig lines, although it may significantly contribute to variation in meat quality. The g.233C>T SNP may be in incomplete linkage disequilibrium with causal mutations and/or exhibit effects in the context of DNA variation at other interacting loci.     

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。     

Characterization of the porcine acyl-CoA synthetase long-chain 4 gene and its association with growth and meat quality traits

Summary Long-chain acyl-CoA synthetase (ACSL) catalyses the formation of long-chain acyl-CoA from fatty acid, ATP and CoA, activating fatty acids for subsequent reactions. Long-chain acyl-CoA synthetase thus plays an essential role in both lipid biosynthesis and fatty acid degradation. The ACSL4 gene was evaluated as a positional candidate gene for the quantitative trait loci (QTL) located between SW2456 and SW1943 on chromosome X. We have sequenced 4906 bp of the pig ACSL4 mRNA. Sequence analysis allowed us to identify 10 polymorphisms located in the 3'-UTR region and to elucidate two ACSL4 haplotypes. Furthermore, a QTL and an association study between polymorphisms of the ACSL4 gene and traits of interest were carried out in an Iberian x Landrace cross. We report QTL that have not been previously identified, and we describe an association of the ACSL4 polymorphisms with growth and percentage of oleic fatty acid. Finally, we have determined allelic frequencies in 140 pigs belonging to the Iberian, Landrace, Large White, Meishan, Pietrain, Duroc, Vietnamese, Peccary and Babirusa populations.     

Linkage mapping of gene-associated SNPs to pig chromosome 11

Single nucleotide polymorphisms (SNPs) were discovered in porcine expressed sequence tags (ESTs) orthologous to genes from human chromosome 13 (HSA13) and predicted to be located on pig chromosome 11 (SSC11). The SNPs were identified as sequence variants in clusters of EST sequences from pig cDNA libraries constructed in the Sino-Danish pig genome project. In total, 312 human gene sequences from HSA13 were used for similarity searches in our pig EST database. Pig ESTs showing significant similarity with HSA13 genes were clustered and candidate SNPs were identified. Allele frequencies for 26 SNPs were estimated in a group of 80 unrelated pigs from Danish commercial pig breeds: Duroc, Hampshire, Landrace and Large White. Eighteen of the 26 SNPs genotyped in the PiGMaP Reference Families were mapped by linkage analysis to SSC11. The EST-based SNPs published here are new genetic markers useful for linkage and association studies in commercial and experimental pig populations. This study represents the first gene-associated SNP linkage map of pig chromosome 11 and adds new comparative mapping information between SSC11 and HSA13. Furthermore, our data facilitate future studies aimed at the identification of interesting regions on pig chromosome 11, positional cloning and fine mapping of quantitative trait loci in pig.     

猪CAST基因的单核苷酸多态性及其对肉质性状的效应

CAST基因作为肉质性状的主要候选基因.以80头外来猪和190头地方猪为材料,在CAST基因内含子24上检测到两个多态性位点(A916G 和C1633G ).在916位点上,长白猪和大白猪以A基因为优势基因,其频率分别为0.88和1.00;莱芜猪,大薄莲猪,沂蒙黑猪和里岔黑猪以B基因为优势基因,其频率分别为0.93, 0.97, 0.78和0.68.在1633位点上,长白猪和大白猪以C基因为优势基因,其频率分别为0.82和0.79:莱芜猪,大薄莲猪,沂蒙黑猪和里岔黑猪以D基因为优势基因,其频率分别为1.00, 1.00, 0.88, 0.78.在试验猪种中,共检测到6种单倍型(AACC,AACD,AADD,ABCC,BBCC,BBDD).单倍型分布的多重比较结果表明,外来猪种(长白猪和大白猪)与地方猪种(莱芜猪,大薄莲猪,沂蒙黑猪和里岔黑猪)比较差异极显著(P < 0.01).固定效应模型分析结果表明,嫩度,屠宰45 min后pH值和滴水损失单倍型间差异显著(P < 0.05).最小二乘分析结果表明,外来猪种与地方猪种在嫩度,屠宰45 min后pH值和滴水损失间差异显著(P < 0.05).BBDD单倍型个体与其它单倍型个体比较,嫩度及滴水损失差异显著(P < 0.05);AADD,BBCC,BBDD单倍型个体与其它单倍型个体比较,屠宰45 min后pH值差异显著(P < 0.05).因此,在育种过程中将CAST基因应用于标记辅助选择,将有利于改善猪肉品质,加快育种进程.     

猪ATF4基因多态性与生产性状的关联及基因表达分析

为进一步了解和认识ATF4基因的功能,揭示ATF4对猪脂肪代谢的影响,寻找与肉质性状相关联的分子标记,文章采用PCR方法扩增了ATF4基因部分序列,通过序列比对发现在翻译起始密码子ATG下游159 bp处存在A159G转换,通过PCR-AluⅠ-RFLP对大白猪、长白猪、梅山猪和通城猪进行酶切分型,发现在大白猪和长白猪中均为AA基因型,在梅山猪和通城猪中均为GG基因型。进一步对大白猪×梅山F2群体资源家系进行了酶切分型,并分析该位点的多态性与生产性状的关系。结果表明,ATF4的多态性与臀部平均膘厚存在极显著相关(P<0.01),与胸腰椎间膘厚、平均膘厚、眼肌高、眼肌面积存在显著相关(P<0.05)。采用Real-time PCR分析了ATF4基因在大白猪与梅山猪背最长肌不同发育阶段的表达模式。结果表明,ATF4基因在大白猪和梅山猪胚胎期65 d和出生后3 d中的表达水平相对都比较低,且在两品种间无明显差异;而在出生后60 d和120 d,ATF4基因在大白猪中与梅山猪均出现了上调表达,并且在梅山猪中的相对表达水平要显著高于大白猪。研究结果为进一步深入研究猪ATF4基因在脂肪代谢中的分子机理奠定了基础。     

Characterization and physical mapping of the porcine CDS1 and CDS2 genes

CDP-diacylglycerol synthase (CDS) catalyzes the conversion of phosphatidic acid to CDP-diacylglycerol, an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We amplified and sequenced 2,053 bp of the pig CDS1 mRNA. The structure of the pig CDS1 gene was determined, being very similar to that of the human, rat, and mouse genes with respect size and organization of the 13 exons. In addition, we identified three polymorphic positions in exons 10 and 11. One of them, the A/C1006, was genotyped in samples belonging to Iberian, Landrace, Large White, Pietrain, and Meishan pig breeds. Expression of this gene was also analyzed by real-time polymerase chain reaction (PCR) in different tissues showing a high CDS1 expression in testis. Moreover, a 1240-bp fragment of the pig CDS2 mRNA was amplified and sequenced. Finally, the CDS1 and CDS2 genes were physically mapped to porcine chromosomes 8 and 17, respectively, by using the INRA, University of Minnesota porcine Radiation Hybrid panel (IMpRH).     

Simultaneous identification of ryanodine receptor 1 (RYR1) and estrogen receptor (ESR) genotypes with the multiplex PCR-RFLP method in Polish Large White and Polish Landrace pigs

A method allowing simultaneous genotyping of two loci: ryanodine receptor 1 (RYR1) and estrogen receptor (ESR) is presented. In multiplex PCR amplification, two amplicons were simultaneously produced: a 272 bp fragment of RYR1 gene and a 185 bp fragment of ESR gene and were then subjected to "one-tube" restriction enzyme digestion with Hin6 I and Ava I, respectively. A total of 122 Polish Large White and Polish Landrace pigs were genotyped by this method, demonstrating its reliability, convenience and lower costs. This method may be useful in the wide-scale genotyping of both loci in pig breeding programmes.     

猪整合素β1基因CRS-PCR多态性与产仔数的关联性分析

文章采用CRS-RFLP技术对长白猪、大白猪和杜洛克猪3个品种的整合素β1基因第5外显子T32207C位点及第7外显子A35230G位点进行单核苷酸多态性分析,并将基因多态性与猪的产仔数进行关联分析。结果表明:32207多态位点的基因型效应对3个品种的总产仔数(TNB)和产活仔数(NBA)影响均不显著;35230多态位点的基因型效应对大白猪和长白猪头胎、二胎及所有胎次的TNB和NBA的影响达到显著(P<0.05)或者极显著水平(P<0.01),基因型GG、AG与AA对产仔数的影响存在差异,其效应为GG,AG>AA。可见整合素β1基因35230位点的G等位基因对大白猪和长白猪的产仔数性状有显著影响。     

Molecular Cloning of Porcine SUN5 Gene and Association between a SNP with Litter Size Trait

SUN domain-containing protein 5 (SUN5) is an important reproduction related gene. In this study, we cloned the full-length coding sequence of porcine SUN5 gene through RT-PCR. Sequence analysis of this gene revealed that the pig SUN5 gene encodes a protein of 383?amino acids that has high homology with the SUN5 protein of eight species: wild Bactrian camel (95%), alpaca (95%), Yangtze River dolphin (94%), sperm whale (94%), sheep (93%), black flying fox (93%), goat (92%), and horse (91%). This gene is structured into 13 exons and 12 introns as revealed by computer-assisted analysis. The prediction of transmembrane helices showed that pig SUN5 protein might be a transmembrane protein. PCR-Taq I-RFLP was established to detect the GU475008:c.138 G>A substitution of porcine SUN5 gene coding sequence and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n?=?200) and Landrace (n?=?200) pig populations, and the results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P?相似文献   

Confirmation of the human cathepsin B gene (CTSB) assignment to chromosome 8

Summary Human cathepsin B gene (CTSB) has been mapped to two locations: 8p22 and 13q14. Here we confirm the chromosome 8 assignment by three independent methods: (1) analysis of human-hamster somatic cell hybrid DNA by polymerase chain reaction; (2) comparison of hybridization signals to cathepsin B in interphase nuclei of normal fibroblasts and fibroblasts with a chromosome 8 deletion; and (3) fluorescence in situ hybridization to metaphase spreads using cathepsin B cosmid clones. Our results indicate that human CTSB is located at 8p22-p23.1.     

Molecular Characterization,Polymorphism, and Association of Porcine GADD45G Gene

Growth arrest and DNA-damage-inducible gamma (GADD45G) is a reproduction related gene. In this study, the full-length cDNA sequence of porcine GADD45G gene was cloned through rapid amplification of cDNA ends (RACE) method. The porcine GADD45G gene encodes a protein of 159 amino acids that shares high homology with the GADD45G of nine species: chimpanzee (97%), sumatran orangutan (97%), white-tufted-ear marmoset (97%), northern white-cheeked gibbon (97%), cattle (97%), human (97%), rhesus monkey (97%), rat (96%), and mouse (95%). This novel porcine gene was assigned to GeneID: 100152997. Phylogenetic analysis revealed that the porcine GADD45G gene has a closer genetic relationship with the GADD45G gene of cattle. Computer-assisted analysis indicated that porcine GADD45G gene is structured in four exons and three introns. PCR-Rsa I-RFLP was established to detect an A/G mutation on the position of 294-bp of coding sequence and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and result demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P < 0.01). Therefore, porcine GADD45G gene could be a useful candidate gene in selection for increasing the litter size. These data serve as a foundation for further insight into this novel porcine gene.     

The porcine proteasome subunit A4 (PSMA4) gene: isolation of a partial cDNA, linkage and physical mapping

A partial cDNA clone encoding the porcine proteasome subunit A4 ( PSMA4 or proteasome subunit C9) has been isolated from a porcine muscle cDNA library and sequenced. A biallelic Taq I RFLP was identified in Large White, Landrace and Duroc breeds. Moreover, the 3'-untranslated region of the gene showed a triallelic SSCP. By linkage analysis the PSMA4 locus was assigned to pig chromosome 7 and by radioactive in situ hybridization this locus was mapped to the region 7q13–q14.