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PCR-RFLP was used to analyze the polymorphisms of MC4R, LEP, H-FABP genes in a swine breed composite (DIV2) and 4 swine breeds (Yorkshire, Landrace, Meishan, Bamei). The association study of these polymorphisms with several economic traits was carried out on a DIV2 population. The results obtained showed that MC4R/TaqI genotype had an effect for average backfat thickness (P < 0.05) and lean meat percentage (P < 0.05). At locus LEP/HinfI animals of AA genotype had lower test daily gain than that of BB (P < 0.01) or AB genotype (P < 0.05). At the H-FABP/HaeIII locus lean meat percentage of the individuals with genotype DD were higher than that with genotype dd (P < 0.05). Linkage disequilibrium analysis among MC4R, LEP and H-FABP revealed that these genes were independent. This represented two or more genes that could be combined together within one genotype in order to facilitate breeding for objective traits. In addition, a method allowing simultaneous detection of fragments of MC4R and LEP gene was developed.

Imprinted genes play important roles in mammalian growth, development and behavior. In this study, we obtained 1568 bp mRNA sequence of porcine DIO3 (deiodinase, iodothyronine, type III), and also identified its imprinting status during porcine fetal development. The complete open reading frame (ORF) encoding 278 amino acids. The porcine DIO3 mRNA was expressed predominantly in backfat, mildly in liver, uterus, kidney, heart, small intestine, muscle and stomach, and almost absent in spleen and lung. A single nucleotide polymorphism in exon (A/C (687)) was used to investigate the allele frequencies in different pig breeds and the imprinting status in porcine embryonic tissues. The results indicate that DIO3 was imprinted in all the tested tissues. Statistical analysis showed the DIO3 gene polymorphism was significantly associated with almost all the fat deposition and carcass traits, including lean meat percentage (LMP), fat meat percentage (FMP), ratio of lean to fat (RLF), shoulder fat thickness (SFT), sixth-seventh rib fat thickness (RFT), buttock fat thickness (BFT), loin eye area (LEA), and intramuscular fat (IMF).

Imprinted genes have important effects on the regulation of fetal growth, development, and postnatal behavior. However, the study of imprinted genes has been limited in mammalian species other than human and mouse. Therefore, the study of porcine imprinted genes is useful for defining the extent of conservation of genomic imprinting among different species. In this study, the imprinting status of porcine NDN, MAGEL2 and MEST genes was determined by direct sequencing of the cDNAs and detection of single nucleotide polymorphisms (SNPs) identified in individuals from reciprocal crosses between Meishan and Large White pigs for allele discrimination. The analysis was carried out in 13 different tissues (skeletal muscle, fat, pituitary gland, heart, lung, liver, kidney, spleen, stomach, small intestine, uterus, ovary and testis) from 12 two-month-old piglets. Imprinting analysis showed that NDN and MAGEL2 were paternally expressed in all tissues where the genes were expressed as in human and mouse. Interestingly, MEST showed tissue-specific imprinting, being paternally expressed in skeletal muscle, fat, pituitary gland, heart, kidney, lung, stomach and uterus, and maternally expressed in spleen and liver.

Imprinted genes play an essential role in the regulation of fetal growth, development and function of the placenta, however only a limited number of imprinted genes have been studied in swine. In this study, we cloned and characterized porcine MAGEL2 (melanoma antigen-like gene 2), and also identified its imprinting status during porcine fetal development. The complete open reading frame (ORF) encoding 1,193 amino acids was isolated and two single nucleotide polymorphisms (SNPs) (g.2592A>C and g.3277T>C) in the coding region were identified. The reciprocal Yorkshire×Meishan F1 hybrid model and the RT-PCR/RFLP method were used to detect the imprinting status of porcine MAGEL2 gene at two developmental stages of day 30 and 65 of gestation. Imprinting analysis showed that porcine MAGEL2 was paternally expressed in day 65 fetal tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, brain and placenta. Interestingly, we observed an imprinting variance of MAGEL2 gene in 30 dpc fetuses produced by the cross of Yorkshire boar×Meishan sow, in which seven heterozygous fetuses were monoallelically expressed from the paternal allele but two were biallelically expressed from both the paternal and maternal alleles. Association analysis in a Yorkshire×Meishan F2 resource population showed that the mutation of g.2592A>C was significantly associated with dressed carcass percentage (P<0.05) and buttock fat thickness (P<0.05). Our results suggest that MAGEL2, as a novel imprinted gene in pig, might be a candidate gene affecting carcass traits and could provide important information for the functional study of imprinted genes during porcine development.

Ladybird-like genes were recently identified in mammals. The first member characterized, Lbx1, is expressed in developing skeletal muscle and the nervous system. However, little is known about the porcine Lbx1 gene. In the present study, we cloned and characterized Lbx1 from porcine muscle. RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. Lbx1 gene expressed at higher levels in biceps femoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.-1559C>G. Association analysis in our experimental pig populations showed that the mutation of g.752A>G was significantly associated with loin muscle area (P<0.05) and internal fat rate (P<0.05). Our results suggest that the Lbx1 gene might be a candidate gene of carcass traits and provide useful information for further studies on its roles in porcine skeletal muscle.

The lecithin cholesterol acyltransferase gene (LCAT) plays an important role in lipoprotein metabolism, especially in the process termed 'reverse cholesterol transport'. In this study, we obtained the 1,434 bp mRNA sequence of porcine LCAT including the full coding region and encoding a protein of 472 amino acids. The sequence was deposited into the GenBank under the accession no. EU717835. The genomic sequence of this gene which contains six exons and five introns, is 3,712 bp in length (GQ379050). Bioinformatic analysis of the 5' regulatory region has revealed that some transcription factor Sp1, AP-1, AP-2 and NF-kappaB were represented in this region. Tissue expression analysis showed that the porcine LCAT gene is ubiquitously expressed in all examined tissues. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, we found a single nucleotide polymorphism (SNP, C/G266) in intron 1 of the LCAT gene and association analysis showed that it was significantly associated with ratio of lean to fat (P < 0.05), caul fat weight (P < 0.01), leaf fat weight (P < 0.05), carcass length (P < 0.05) and bone percentage (P < 0.05). Our study will lay the groundwork for the further investigations on the detailed physiological function of LCAT in pig models.

ATP-citrate lyase (ACL), one of the lipogenic enzymes, catalyses the formation of acetyl-coenzyme A (CoA) involved in the synthesis of fatty acid and cholesterol. In pig, very little is known about the ACL gene. In this work, the mRNA differential display technique was used to analyse the differences in gene expression between Meishan and Large White pigs and the F1 hybrids of both direct and reciprocal crosses. Our results show that among the differentially expressed genes ACL is up-regulated in the backfat of the F1 hybrids. After cloning and analysing the full-length cDNA and the 870 bp 50-flanking sequence of the porcine ACL gene, a C/T mutation at position -97 bp upstream of the transcription site was detected. Luciferase activity detection showed that this mutation changed the transcriptional activity. In F1 hybrids, the heterozygous genotype CT was more frequent than the homozygous genotypes CC and TT. Real-time PCR analysis showed that in Meishan pigs, ACL mRNA expression was more abundant in individuals with genotype CT than in those with genotype CC or TT or in Large White pigs. These results indicate that the C/T mutation affects ACL mRNA expression, probably via the activator protein 2.

Although expression and epigenetic differences of imprinted genes have been extensively characterised in man and the mouse, little is known on livestock species. In this study, the polymorphism-based approach was used to detect the imprinting status of NNAT and DIRAS3 genes in five heterozygous pigs (based on SNP) of Large White and Meishan F(1) hybrids. The results show that both genes were paternally expressed in all the tested tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary). In addition, the NNAT gene had two transcripts in all tested tissues, which is consistent with its counterpart in man and cattle.

In order to investigate porcine heterosis on the molecular basis, Large White (L), a European purebred, and Meishan (M), a Chinese indigenous purebred, were hybridized directly and reciprocally to produce F1 hybrids, Large WhitexMeishan (LM) and MeishanxLarge White (ML) pigs. Using mRNA differential display, we found an expression sequence tag (EST) differentially expressed in F1 hybrids and their parents, designated as EST55, which was homologous to human and murine skeletal muscle protein (SMPX), and the full-length cDNA of porcine SMPX was cloned by the rapid amplification of cDNA end (RACE) method. Translation of the mRNA transcript revealed an open reading frame (ORF) of 86 amino acid residues encoding a nuclear location signal peptide, two overlapping casein kinase II phosphorylation sites and one N-glycosylation site with theoretical molecular weight of 9.3 kDa. Alignment analysis revealed that the deduced protein sequence shared 94%, 83% and 78% homology with that of its human, mouse and rat counterparts, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that it was expressed predominantly in skeletal and heart muscles, whereas at a moderate level in backfat, spleen, stomach and uterus tissues. Two single nucleotide polymorphism (SNPs), located in 5'- and 3'-untranslated region (UTR), respectively,were identified by PCR and sequencing. Phylogenetic tree and the secondary structure prediction were also performed. The possible relationship between porcine SMPX and heterosis was discussed.