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.     

中国美利奴羊H-FABP基因多态性及其与部分肉质性状的相关性

利用聚合酶链式反应-单链构象多态（PCR-SSCP）技术检测中国美利奴羊（Ovis aries var. Merino）心型脂肪酸结合蛋白基因（H-FABP）外显子2的单核苷酸多态性（SNPs）和遗传多态性,分析其与肌内脂肪（IMF）含量、肌纤维直径和肌纤维密度的相互关系,为该品种绵羊的分子标记辅助选择提供理论依据。结果显示,H-FABP基因外显子2有AA、AB和BB 3种基因型,AA型和BB型在778位均发生了C缺失,939位均发生了A→G转换,BB型还在789位发生了T→C转换,该突变导致所编码氨基酸发生了缬氨酸→丙氨酸的替换;BB型为IMF的优势基因型,与AB型相比差异显著（P<0.05）,与AA型相比差异极显著（P<0.01）;BB型对肌纤维直径存在负相关。结果提示,中国美利奴羊H-FABP基因外显子2具有多态性,该基因可能是中国美利奴羊肉质性状的主效基因,或者与控制肉质性状的主效基因相连锁。     

猪钙蛋白酶抑制蛋白基因的基因型与肉质性状的关联性分析

钙蛋白酶抑制蛋白基因是影响猪肉质性状的候选基因之一。本研究以125头地方猪和117头外来猪为材料,研究CAST基因的多态性。结果在CAST基因上检测到一个多态性位点(A876G),并引起了氨基酸残基的改变Lys250 Arg。在地方猪种中仅检测到G (Arg)等位基因,而在外来猪种中A (Lys) 和 G (Arg)两个等位基因均检测到。基因型与肉质性状的关联性分析结果表明,CAST基因型与肌肉的嫩度,屠宰45 min后的pH值及滴水损失存在强相关,又由于地方猪种与外来猪种的肉质性状间存在显著差异。因此,在CAST基因上检测到的多态型位点Lys250Arg的基因型效应有待于进一步研究,并将其有效应用于商品猪生产中。     

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.     

Cloning,chromosomal localization,SNP detection and association analysis of the porcine IRS-1 gene

Insulin receptor substrate-1(IRS-1) gene is one member of the Insulin receptor substrate (IRS) gene family, which plays an important role in mediating the growth of skeletal muscle and the molecular metabolism of type 2 diabetes. Here, we cloned a 3,573 bp fragment of the partial CDS sequence of porcine IRS-1 gene by in silicon cloning strategy and RT-PCR method. The porcine IRS-1 gene was assigned to SSC15q25 by using IMpRH. Sequencing of PCR products from Duroc and Tibetan pig breeds identified one SNP in exon 1 of porcine IRS-1 gene (C3257A polymorphisms). Association analysis of genotypes with the growth traits, anatomy traits, meat quality traits and physiological biochemical indexes traits showed that different genotypes at locus 3,257 of IRS-1 have significant differences in carcass straight length in pigs (P = 0.0102 < 0.05).     

山猪群体钙调蛋白酶抑制蛋白基因遗传变异研究

目的：研究猪钙调蛋白酶抑制蛋白（CAST）基因在山猪群体的遗传变异情况,为山猪肉质研究奠定基础。方法：应用PCR-SSCP技术和测序方法检测山猪及其杂种猪CAST基因的遗传多态性,并与其他品种猪相应序列进行比较。结果：用pCT1引物在山猪及其杂种群体中检测到2个多态位点（A,B）,用pCT2引物检测到3个多态位点（C,D,E）;序列分析表明,C和E位点共同拥有6处变异。结论：通过与其他猪品种比较,发现山猪的CASTMsp基因型分布与梅山猪的基因型分布完全一样,而与国外品种猪的基因型分布差异明显。     

Genetics of fat tissue accumulation in pigs: a comparative approach

Fatness traits are important in pig production since they influence meat quality and fattening efficiency. On the other hand, excessive fat accumulation in humans has become a serious health problem due to worldwide spread of obesity. Since the pig is also considered as an animal model for numerous human diseases, including obesity and metabolic syndrome, comparative genomic studies may bring new insights into genetics of fatness/obesity. Input of genetic factors into phenotypic variability of these traits is rather high and the heritability coefficient (h ²) of these traits oscillates around 0.5. Genome scanning revealed the presence of more than 500 QTLs for fatness in the pig genome. In addition to QTL studies, many candidate gene polymorphisms have been analyzed in terms of their associations with pig fatness, including genes encoding leptin (LEP) and its receptor (LEPR), insulin-like growth factor 2 (IGF-2), fatty acid-binding proteins (FABP3 andFABP4), melanocortin receptor type 4 (MC4R), and theFTO (fat mass and obesity-associated) gene. Among them, a confirmed effect on pig fatness was found for a well-known polymorphism of theIGF-2 gene. In humans the strongest association with predisposition to obesity was shown for polymorphism of theFTO gene, while in pigs such an association seems to be doubtful. The development of functional genomics has revealed a large number of genes whose expression is associated with fat accumulation and lipid metabolism, so far not studied extensively in terms of the association of their polymorphism with pig fatness. Recently, epigenomic mechanisms, mainly RNA interference, have been considered as a potential source of information on genetic input into the fat accumulation process. The rather limited progress in studies focused on the identification of gene polymorphism related with fatness traits shows that their genetic background is highly complex.     

Genetic variation in the porcine myogenin gene locus

The myogenin (MYOG) gene fulfills a key function in muscle differentiation by controlling the onset of myoblast fusion and the establishment of myofibers. In meat-producing animals like pigs and cattle, myofiber numbers have been related to growth capacity. We have characterized the porcine MYOG gene to detect genetic variation at this locus and to relate it to growth characteristics. MYOG gene fragments were isolated by PCR on genomic DNA and by screening a genomic library with a mixture of the four human MyoD cDNA fragments. Both the exons and promoter region were very similar to the human and mouse genes. Southern blot analysis of 105 unrelated pigs revealed three polymorphic MspI sites, located in the promoter region, the second intron, and at the 3′ side of the gene. PCR-RFLP tests detecting four MYOG alleles were developed. PCR analysis of a panel of pig–rodent somatic cell hybrids confirmed the genetic localization of MYOG on pig Chromosome (Chr) 9. The PCR-RFLP tests and microsatellite markers on Chr 9 offer the possibility to genotype large numbers of pigs for studies of genetic linkage to meat deposition and growth characteristics. Received 13 October 1996 / Accepted: 16 March 1992     

丙酸通路基因多态性与猪肉质及胴体性状的关联分析

为了挖掘新的猪肉品质及胴体性状的候选基因,揭示猪肉质及胴体性状的遗传机制,文章将丙酸代谢通路作为候选通路,将通路内基因与猪肉质及胴体性状进行关联分析。实验采用37头三元杂交商品猪作为研究对象,首次针对丙酸通路中7个基因的36个SNP位点利用SNaPshot方法进行基因分型,分别用最小二乘模型及MB-MDR模型与肉质及胴体性状进行关联分析。结果发现,基因PCCB、MUT、MCEE及ACSS2上的4个SNP位点分别与肌内脂肪含量、背膘厚等性状显著相关(P<0.05),ACSS2与猪脂肪含量显著相关;MCEE及MUT与猪的背膘厚显著相关;PCCB基因与脂重显著相关。通过MB-MDR方法检测到多个SNP位点具有互作效应,并与背膘厚、水分含量、脂肪含量显著相关(P<0.05)。另外,丙酸代谢通路中的基因间的互作效应对猪肉品质有显著影响。     

Polymorphisms of the 5′ regulatory region of the porcine <Emphasis Type="Italic">PPARGC1A</Emphasis> gene and the effects on muscle fiber characteristics and meat quality

The purpose of this study was to determine the structure of the porcine PPARGC1A 5′ upstream region, and to find suitable molecular markers for improved meat quality and good lean meat production. Ten DNA polymorphisms, including 7 SNPs, 2 microsatellites, and 1 insertion or deletion were newly found in the 5′ upstream region of PPARGC1A. Three SNPs that had restriction enzyme site were evaluated for associations with muscle fiber characteristics and production traits. Two hundred fifty-two pigs (Yorkshire and Landrace) were used in this analysis. The c.-2894G>A genotypes was significantly associated with muscle fiber characteristics, including the number of fiber type I and IIb composition (P < 0.05), mean cross-sectional area of fibers (P < 0.01), and fiber number per unit area (P < 0.05). The animals with the GG genotype had a higher percentage of type I fibers and a lower percentage of type IIb fibers with better meat quality [higher pH value (P < 0.05) and lower drip loss (P < 0.05)] and lean meat production [larger loin eye area (P < 0.05)]. Moreover, the mRNA expression levels of PPARGC1A among genotypes were significantly different with the highest level of GG genotype. The c.-2885G>T and c.-1402A>T sites showed similar results that had significant effects on the mean cross-sectional area (CSA; P < 0.05), fiber number per unit area (P < 0.05) and loin eye area (P < 0.01). Therefore, we suggest that the c.-2894G>A polymorphism in the 5′ upstream region of the porcine PPARGC1A gene can be used as a meaningful molecular marker for simultaneous improvement of lean meat production and quality traits.     

Genes with expression levels correlating to drip loss prove association of their polymorphism with water holding capacity of pork

Six genes that were known to exhibit expression levels that are correlated to drip loss BVES, SLC3A2, ZDHHC5, CS, COQ9, and EGFR have been for candidate gene analysis. Based on in silico analysis SNPs were detected, confirmed by sequencing, and used for genotyping. The SNPs were genotyped in about 1,800 animals from six pig populations including commercial herds of Pietrain (PI) and German Landrace (DL), different commercial herds of Pietrain × (German Large White × German Landrace) (PIF1(a/b/c)), and one experimental F₂-population Duroc × Pietrain (DUPI). Comparative and genetic mapping established the location of BVES on SSC1, of SLC3A2 and ZDHHC5 on SSC2, of CS on SSC5, of COQ9 on SSC6 and of EGFR on SSC9, respectively, coinciding with QTL regions for carcass and meat quality traits. BVES, SLC3A2, and CS revealed association at least with drip loss and with several other measures of water holding capacity (WHC). Moreover, COQ9 and EGFR were associated with several meat quality traits such as meat color and/or thawing loss. This study reveals statistic evidence in addition to the functional relationship of these genes to WHC previously evidenced by expression analysis. This study reveals positional and genetic statistical evidence for a link of genetic variation at these loci or close to them and promotes those six candidate genes as functional and/or positional candidate genes for meat quality traits.     

Functional dissection of the Drosophila Kallmann's syndrome protein DmKal-1

Background

The proportion of muscle fibre types and their size affect muscularity as well as functional properties of the musculature and meat quality. We aimed to identify QTL for microstructural muscle properties including muscle fibre size, their numbers and fibre type proportions as well as biophysical parameters of meat quality and traits related to body composition, i.e. pH, conductivity, area of M. longissimus dorsi and lean meat content. A QTL scan was conducted in a porcine experimental population that is based on Duroc and Berlin Miniature Pig.

Results

Least square regression interval mapping revealed five significant and 42 suggestive QTL for traits related to muscle fibre composition under the line-cross model as well as eight significant and 40 suggestive QTL under the half-sib model. For traits related to body composition and biophysical parameters of meat quality five and twelve significant plus nine and 22 suggestive QTL were found under the line-cross and half-sib model, respectively. Regions with either significant QTL for muscle fibre traits or significant QTL for meat quality and muscularity or both were detected on SSC1, 2, 3, 4, 5, 13, 14, 15, and 16. QTL for microstructural properties explained a larger proportion of variance than did QTL for meat quality and body composition.

Conclusion

Microstructural properties of pig muscle and meat quality are governed by genetic variation at many loci distributed throughout the genome. QTL analysis under both, the line-cross and half-sib model, allows detecting QTL in case of fixation or segregation of the QTL alleles among the founder populations and thus provide comprehensive insight into the genetic variation of the traits under investigation. Genomic regions affecting complex traits of muscularity and meat quality as well as microstructural properties might point to QTL that in first instance affect muscle fibre traits and by this in second instance meat quality. Disentangling complex traits in their constituent phenotypes might facilitate the identification of QTL and the elucidation of the pleiotropic nature of QTL effects.     

Porcine <Emphasis Type="Italic">CSRP3</Emphasis>: polymorphism and association analyses with meat quality traits and comparative analyses with <Emphasis Type="Italic">CSRP1</Emphasis> and <Emphasis Type="Italic">CSRP2</Emphasis>

CRP3 is the muscle-specific form of the cysteine and glycine-rich protein family and plays an important role in myofiber differentiation. Here we isolated and characterized its coding gene CSRP3 from porcine muscle. Phylogenic analyses demonstrated that CSRP3 diverged first and is distinguished from two other members, CSRP1 and CSRP2. CSRP3 mRNA was up-regulated during the development of porcine embryonic skeletal muscle, indicating its potential importance in muscle growth. Genetic variant analyses detected multiple variations in an approximately 400 bp region covering exon 4 and its downstream intron, and two haplotypes were identified by sequencing. One of synonymous substitutions C1924T was used for linkage and association analyses. It was revealed that the substitution of C1924T had significant associations with firmness (P < 0.01), Lab Loin pH, Off Flavor Score and Water Holding Capacity (P < 0.05), and a suggestive effect (P < 0.1) on Flavor Score and Average Glycolytic Potential in a Berkshire × Yorkshire F2 population. The association analyses results agreed with the gene’s localization to a QTL region for meat quality traits on porcine chromosome 2p14-17 demonstrated by both linkage mapping and RH mapping. These results provide fundamental evidence for CSRP3 as a functional candidate gene affecting pig meat quality.     

Non-Synonymous SNP in the ApoR Gene Associated with Pork Meat Quality

Two novel non-synonymous SNPs in the 2^nd and 3^rd exons of the porcine ApoR gene are reported. One was identified as a novel SNP significantly associated with multiple traits of pork meat quality. The data can provide a useful resource for developing a marker in the genetic improvement of pigs.