Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Molecular characterization, expression analysis and association study with meat quality traits of porcine TTID gene.

Titin immunoglobulin domain protein (TTID) is localized to the Z-line and binds to alpha-actinin, gamma-filamin. It plays an indispensable role in stabilization and anchorage of thin filaments. In this study, the full-length cDNA sequence was isolated by the reverse transcription-polymerase chain reaction (RT-PCR) and the rapid amplification of cDNA ends (RACE). The TTID sequence was deposited into the Genbank under the accession no. DQ157551. The deduced protein of 499 amino acids showed 93 % identity to the corresponding human and rat sequence. Semi-quantitative RT-PCR revealed porcine TTID gene was expressed highest level in skeletal muscle, at second-highest level in the heart, but only low expression in the fat was detected. Bioinformatics analysis shows the molecular weight of the TTID protein is 55.747 kD with a PI of 9.26. It contains the protein function site of two potential Ig-like domain profiles, six N-myristoylation sites, six potential Casein kinase II phosphorylation sites, eight protein kinase C phosphorylation sites, three N-glycosylation sites, a tyrosine kinase phosphorylation site and a cell attachment sequence site. No putative base substitution was detected in the coding region by comparing sequences of Large White, Landrace and Meishan pig breeds. A T978C single nucleotide polymorphism in the intron 6 of porcine TTID gene was detected by a HinfI PCR-restriction fragment length polymorphism. Study showed allele frequency differences among four purebreds. Association of the genotypes with meat quality traits showed that different genotypes of porcine TTID gene were significantly associated with meat pH (m.Biceps Femoris) (P < 0.05), meat color value (m.longissimus Dorsi) (P < 0.05) and Water Moisture (m.longissimus Dorsi) (P < 0.05).

Imprinting analysis of porcine MAGEL2 gene in two fetal stages and association analysis with carcass traits.

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.

Distribution and linkage disequilibrium analysis of polymorphisms of MC4R, LEP, H-FABP genes in the different populations of pigs, associated with economic traits in DIV2 line.

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.

Imprinting analysis of porcine DIO3 gene in two fetal stages and association analysis with carcass and meat quality traits.

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).

Temporal and spatial distribution of Bacillus and Clostridium histolyticum in swine manure composting by fluorescent in situ hybridization (FISH).

The temporal and spatial distribution of the genus Bacillus and Clostridium histolyticum group in swine manure composting was determined by fluorescent in situ hybridization using fluorescently labeled 16S rRNA-targeted oligonucleotide probes LGC353b and Chis150, respectively. The temporal distribution of total bacteria, Bacillus and C. histolyticum, detected in each layer of the composting pile was noticeable in that the number of them detected at the high-temperature stage was higher than that of the cooling stage. The number detected at the cooling stage was higher than that of the temperature-rising stage. The number of the total bacteria distributed in three locations achieved balance at the stage of cooling. The spatial distribution of the genus Bacillus cells was that the number and the relative abundance of Bacillus cells detected in the middle layer of composting pile were the lowest at each stage of composting. However, the minimum value of the relative abundance exceeded 8%. Compared with Bacillus spp., the C. histolyticum group displayed higher relative abundance in the same layer at different stages of composting except in the top layer at the stage of high temperature. However, the characteristic of the spatial distribution was not noticeable. The detected limits of the genus Bacillus and C. histolyticum group were both found to be the high cell density of 10(6) cells g(-1) (wet weight). These results indicated that the genus Bacillus and C. histolyticum group were the predominant bacteria in the swine manure composting process and may play important role in this complex environment.

Molecular phylogenetic diversity of Bacillus community and its temporal-spatial distribution during the swine manure of composting.

In order to obtain the diversity and temporal-spatial distribution of Bacillus community during the swine manure composting, we utilized traditional culture methods and the modern molecular biology techniques of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and -denaturing gradient gel electrophoresis (PCR-DGGE). Bacillus species were firstly isolated from the composting. Based on temperature changes, the temporal-spatial characteristics of total culturable Bacillus were remarkable that the number of the culturable Bacillus detected at the high-temperature stage was the highest in each layer of the pile and that detected in the middle layer was the lowest at each stage of composting respectively. The diversity of cultivated Bacillus species isolated from different composting stages was low. A total of 540 isolates were classified by the RFLP method and partial 16S rDNA sequences. They affiliated to eight species including Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus megaterium, Bacillus licheniformis, Bacillus pumilus, and Bacillus circulans. The predominant species was B. subtilis, and the diversity of culturable Bacillus isolated in the middle-level samples at temperature rising and cooling stages was the highest. The DGGE profile and clone library analysis revealed that the temporal-spatial distribution of Bacillus community was not obvious, species belonging to the Bacillus were dominant (67%) with unculturable bacteria and B. cereus was the second major culturable Bacillus species. This study indicated that a combination of culture and culture-independent approaches could be very useful for monitoring the diversity and temporal-spatial distribution of Bacillus community during the composting process.

Molecular characterization and expression patterns of Lbx1 in porcine skeletal muscle.

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.

Molecular characterization, expression profile and association analysis with carcass traits of porcine LCAT gene.

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.

[Cloning and expression analysis of porcine ACTA2 gene and its association with production traits].

To identify new DNA markers which have significant impact on pig production traits, the full coding sequence and partial genomic sequence of porcine ACTA2(Actin alpha 2)gene were isolated using in silico cloning and PCR. PCR-Hinf-RFLP was developed to detect C1554T substitution in intron 2. The frequency of allele C is higher than that of allele T in all the seven detected pig populations except for Large White and MeishanxLarge White. Association analysis of markers and production traits showed that the relation between ACTA2 gene and shoulder fat thickness, buttock fat thickness, fat meat percentage, lean meat percentage, meat pH (m.Biceps Femoris, BF), and intramuscular fat were significant or highly significant. Compared with CC genotype, TT had a higher lean meat percentage, a lower fat meat percentage and backfat thickness. Real-time RT-PCR analysis showed that the expression level of ACTA2 gene in the skeletal muscle of Large White and Meishan pigs decreased with the increasing of days. And during each period, the expression level was higher in Meishan pigs than in Large White pigs.

Association of MYF5 and MYOD1 gene polymorphisms and meat quality traits in Large White x Meishan F2 pig populations.

MYF5 and MYOD1 belong to the myogenic regulatory factor (MRF) gene family. They code for the basic helix-loop-helix transcription factors that play key regulatory roles in the initiation and development of skeletal muscle and the maintenance of its phenotype. In this work three single nucleotide polymorphisms (SNPs) in porcine MYF5 and one in porcine MYOD1 were detected in three pig breeds (Large White, Landrace, and Meishan) by means of a PCR-RFLP protocol. Analysis of the association of meat quality traits with the four polymorphisms in a series of three Large White x Meishan F2 populations, totaling 399 pigs, found: (1) MYF5 exon 1 Hsp92II polymorphism causing a Met --> Leu substitution was associated with intramuscular fat content (P = 0.04) and water moisture content (P = 0.0001) in the longissimus dorsi; (2) MYF5 exon 2 MspI polymorphism and an intron 1 HaeIII polymorphism, which were completely linked, were significantly associated with longissimus dorsi pH (P < 0.05); (3) MYOD1 intron 1 DdeI polymorphism was not significantly associated with any meat quality traits tested. Among these genetic variants (a novel SNP and three identified SNPs), our data suggested that the novel SNP of the MYF5 gene within exon 1 is valuable for pig breeding.

[Sequence and polymorphism analysis of porcine hormone-sensitive lipase gene 5'-UTR and exon I].

Hormone-sensitive lipase (HSL) is the key enzyme responsible for the mobilization of free acids from adipose tissue, and it is also the most important enzyme that affect fat deposition. In this paper, the porcine hormone-sensitive lipase gene 5'-UTR and exon I were sequenced. The sequence number in GenBank are AY332499, AY332497, AY332504, AY332505. A GC-CG in the DNA sequence -13 - -12 bp of porcine HSL gene 5'-UTR was detected between Duroc, Meishan, Qingping pig, Largewhite and Landrace. A G-->A missense mutation was detected in HSL gene exon I of different pig breeds. The characterization of the BsaH I PCR-RFLP polymorphism in exon I of the porcine HSL gene of different breeds and "Largewhite x Meishan" F2 group was analyzed. By association analysis between BsaH I PCR-RFLP polymorphism and GG, GG, AA genotypes of HSL gene exon I, a significant difference of pig eye area was found between AG and GG genotypes (P<0.05) in F2 group.

[Effects of FUT1 gene on meat quality and carcass traits in swine].

A total of 139 hybrid finishing pigs from Large White x Meishan were slaughtered at about 88kg body weight. Fourteen meat quality traits and 8 carcass traits were assayed for each pig. FUT1 gene was scored by PCR-RFLP. The values of meat pH and meat color for AA genotype pig were higher than those of AG genotype pig (P<0.05). Water holding capacity (WHC) of AA genotype pig was higher than that of AG genotype pig(91.02% VS 86.70%, P<0.05). Backfat at 6-7 vertebra (BV) and backfat at vertebra-lumbar (BVL) of AA genotype pig were lower than those of AG genotype pig 4.26 mm and 3.96 mm respectively (P<0.05). Meat factor of AA genotype pig was higher than that of AG genotype pig 3.31% (53.46% VS 50.15%, P<0.05). These results indicated that FUT1 gene had good genetic effects on pig meat quality and carcass traits.

[Sequencing analysis on partial translated region of uncoupling protein-3 gene and single nucleotide polymorphism associations with the porcine carcass and meat quality traits].

Porcine uncoupling protein-3 gene was used to study its effect on carcass and meat quality traits. The UCP3 partial translated regions in skeletal muscle from four pig breeds were sequenced and the comparison of the fragment sequences among four pig breeds showed that there were three coding-region single nucleotide polymorphisms (cSNP), of which the mutation at 842 bp of the open reading frame can result in the amino acid chang between methionine and threonine, so we selected the mutation as polymorphic site. The detection of polymorphic fragment by the technique of single strand conformation polymorphism (SSCP) and chi2 analysis among three pig breeds indicated that the distribution of three genotypes(AA, AB and BB) was significantly different between Meishan and Large White or Meishan and Landrace( P < 0.01). In addition, association analysis of UCP3 polymorphism with carcass and meat quality traits was conducted in the F2 generation from the Large White x Meishan resource family, the results with GLM analysis showed that UCP3 polymorphism has significant effect on several carcass and meat quality traits and also additive effect on some carcass and meat quality traits predominated in the resource family. It implied that UCP3 gene could be a candidate gene locus or a linked marker to a major gene, which affects the porcine carcass traits and meat quality traits significantly.

[Effects of individual gene heterozygosity on growth traits in swine].

Genotypes of 24 microsatellite loci were assayed in total of 139 F2 finishing pigs from resource population(Large White x Meishan) in the present study. There were not significant correlation in sex, family and microsatellite loci effects on birth weight (BW), average daily gain (ADG), adjusted weight of 180 d (LWT180), relative growth rate (RGR) and kleiber ratio (KR). Individual gene heterozygosity (Hi) was estimated by using 24 microsatellites. The 139 individuals were classified into five clusters based on Hi by stepwise 0.05 and the traits mentioned above were compared between Hi levels. Birth weight decreased significantly from gene heterozygosity level 1(0.5101 Hi and 1.5185 kg) to level 2(0.5796 Hi and 1.3063 kg, P < 0.05), the difference was 0.2123 kg equal to 14.66% of population average birth weight (1.4479 kg). And then the birth weight increased significantly with the gene heterozygosity increasing (P < 0.05). There were nonlinear relationships between LWT180, ADG, RGR, KR and individual gene heterozygosity, and mostly like the pattern of sinusoidal curve. The peaks were located at about heterozygosity level 2(averagely Hi 0.5796). ADG, RGR and KR at heterozygosity level 2 were significantly higher than level 4(28.7 g/d, 0.0375 and 0.0003 respectively, P < 0.05). The differences were equal 7.38%, 3.99% and 2.20% to respective average population values.

Detection of quantitative trait loci for protein percentage in muscle in resource pig population.

An intercross between Meishan and Large White pig population was used to map quantitative trait loci(QTL) for protein percentage in muscle. In the present study, all 135 F2 animals, their parents and their grandparents were genotyped for 24 microsatellites, which were in 1, 2 and 6 chromosome. The marker genotypes were used to calculate the additive and dominant coefficients at fixed position in the genome of each F2 animal, and the protein percentage were regressed on to the coefficients in intervals of 1 cM. Three significant QTLs effects were found for protein percentage in muscle. They were located between markers Sw1332 and Sw2130 at chromosome 1, between Sw2516 and Sw1201 at chromosome 2 and between Sw322 and Sw607 at chromosome 6(P < 0.05), and the explained residual variance were 0.91%, 12.76% and 4.60%, respectively. The results show that QTLs, which affect protein percentage, may be located in different chromosomes.

[Mapping quantitative trait loci for meat quality trait in a Large White x Meishan cross].

To search for the chromosome regions for quantitative trait loci (QTL) affecting meat quality in pigs, a three-generation resource family was developed in China using three Large White grand sires and seven Meishan grand dams. A total of 147 F2 progenies derived from two populations in 1998 (n = 81) and 2000 (n = 66) were phenotyped for meat quality. All animals were typed for 48 microsatellite markers covering six chromosomes: 1, 2, 3, 4, 6 and 7. Linear model and least square analyses were used for interval mapping meat quality in jointed population and single population, permutation for empirical threshold. The strongest linkage at chromosome-wide level (P < 0.01) and genome-wide level (P < 0.05) on chromosome 4 affecting intramuscular fat (IMF) QTL from the population in 2000 was detected with explained phenotypic variance of 5.24%, and Meishan's QTL increased the intramuscular fat content. There was a suggestive QTL for IMF near the threshold of chromosome-wide in the same region in jointly population. The QTL for pH value in m. Semispinalis Capitis and m. Biceps Femoris were located on chromosomes 1 and 3, respectively. One and three QTL affecting WHC reached the thresholds of chromosome-wide level in the populations of 1998 and 2000, respectively. In the population of 1998, QTL for moisture content at chromosome-wide level was on SSC6 and in jointed populations were on SSC2, 6 and 7. There were imprinting effects in moisture content QTL, and Meishan and Large White pigs all had favorable effects influencing moisture on different chromosomes.

[Construction of linkage map of chromosomes 1 and 3 in large white x Meishan reference family].

A three-generation family of pigs has been constructed by using three Large White boars and seven sows of Meishan pigs as parents. In this family, five F1 males and twenty-three F1 females were intercrossed to generate 147 F2 offspring. According to the pig linkage map of USDA-MARC, eight and nine microsatellite markers selected on chromosomes 1 and 3 were chosen to span the entire chromosomes. The members of this family were genotyped. The characterization of these microsatellites was shown that they were polymorphic and could be used to construct linkage map and detect quantitative trait loci (QTLs). Linkage analyses were performed using the CRI-MAP software package. The lengths of the sex-averaged linkage map were 182.3 cM and 180.2 cM on chromosomes 1 and 3, respectively. There were some differences between the linkage maps in this study and of USDA-MARC. The linkage map of chromosome 1 in female was found to be shorter than in male, and the contrary was on chromosome 3.

[Detection of quantitative trait loci for growth in large white x Meishan intercross].

The development of molecular biology techniques and the application of these techniques to farm animals have progressed rapidly and have opened new vistas for investigators wishing to identify genes that control quantitative traits. Now that a comprehensive map has been developed for the porcine genome, genomic scans to detect quantitative trait loci (QTL) can begin. In order to locate the genetic regions in the swine genome that are responsible for economically important traits, a resource population was developed by intercross with three Large White and seven Meishan pigs. In subsequent generations, 66 F2 offsprings in 2000 were recorded for four growth traits including birth weight (BWT), body weight at 60 day (WT60), average daily gain from birth to 60 day (ADG1) and average daily gain from 60 day to the end of test (ADG2), and genotyped for 48 microsatellite markers spanning six chromosomes. Association analyses were performed using interval mapping by regression under an outbred line-cross model on chromosomes 1, 2, 3, 4, 6 and 7. The F threshold values were determined by permutation. A total of 12 QTL were detected at suggestive level for the four traits evaluated in this study. Of the 12 suggestive QTL, 3 and 1 QTL were significant at the chromosome-wise and genome-wise levels. There was a QTL for ADG2 at genome-wide level on chromosome 4 explained additive variance 2.19%. A chromosome-wide QTL affecting ADG1 and WT60 were detected on chromosomes 2 and 1 explained additive variance 0.01% and 26.01%, respectively.

[Mapping quantitative trait loci for fat deposition in carcass in pigs].

One of the major determining factors in the price of market hogs today is backfat depth. Therefore, identification of regions of the genome affecting this trait is necessary. Gene-mapping technologies have provided scientists the necessary reagents to conduct genomewide searches for genes affecting any phenotype determined in part by the genetic makeup of the animal. Over the past few years, several experimental crosses have been used to detect quantitative triat loci (QTL) for fatness and meat quality traits in pigs. In an intercross between Large White and Meishan pigs, 81 F2 progenies in 1998 were slaughtered and phenotyped for fat deposite traits. F2 animals, their parents, and their grandparents were typed for molecular markers covering chromosomes (SSC) 1,2,3,4,6 and 7, since previous studies had revealed QTL affecting fatness traits on these chromosomes. Linear model and least square analyses were used for mapping these traits. Furthermore, a QTL accounting for imprinting effects was used. A total of 14 QTL at chromosome-wide level were detected on SSC1, 3-4, 6 and 7 for 12 fatness traits. There were evidences for imprinting effects on SSC7 for four traits at 60 cM and nearby. There were QTL for average backfat thickness (BFT) on SSC1, 4 and 7. Among them, BFT QTL on SSC1 and 7 may be the common locus for Sus scrofa. QTL for internal fat percentage was on SSC7 and QTL for average sidefat on SSC6.