Skeletal muscle transcriptional profiles in two Italian beef breeds, <Emphasis Type="Italic">Chianina</Emphasis> and <Emphasis Type="Italic">Maremmana</Emphasis>, reveal breed specific variation

Chianina and Maremmana breeds play an important role in the Italian cattle meat market. The Chianina breed is an ancient breed principally raised for draught. Now this breed is the worldwide recognized producer of top quality beef, tasteful and tender, specifically the famous “Florentine steak”. The Maremmana characterized by a massive skeletal structure, is a rustic cattle breed selected for adaptability to the marshy land of the Maremma region. We used a high throughput mRNA sequencing to analyze gene expression in muscle tissues of two Italian cattle breeds, Maremmana (MM) and Chianina (CN) with different selection history. We aim to examine the specific genetic contribution of each breed to meat production and quality, comparing the skeletal muscle tissue from Maremmana and Chianina. Most of the differentially expressed genes were grouped in the Glycolysis/Gluconeogenesis pathways. The rate and the extent of post-mortem energy metabolism have a critical effect on the conversion of muscle to meat. Furthermore, we aim at discovering the differences in nucleotide variation between the two breeds which might be attributable to the different history of selection/divergence. In this work we could emphasize the involvement of pathways of post-mortem energy metabolism. Moreover, we detected a collection of coding SNPs which could offer new genomic resources to improve phenotypic selection in livestock breeding program.     

The chromosomal localization,expression pattern and polymorphism analysis of porcine <Emphasis Type="Italic">FSCN1</Emphasis> gene differently expressed from LongSAGE library

Fascin homologue 1 (FSCN1) has established roles in cell adhesion, motility, and cell–cell interactions. Our LongSAGE analysis suggested that FSCN1 was potentially differentially expressed in prenatal pig skeletal muscle. We have cloned the genomic DNA and mRNA sequence of FSCN1 gene and mapped it to SSC3p16-p17. The FSCN1 gene was differently expressed during prenatal skeletal muscle development and exhibited different expression pattern between Tongcheng and Landrace pigs. In Tongcheng pigs, FSCN1 expression was similar at 33 and 65 days post conception (dpc), and then sharply increased to a peak at 90 dpc. In Landrace pigs, however, expression increased between 33 and 65 dpc, peaked at 65 dpc, and was down-regulated thereafter. Significantly different expression levels between Tongcheng and Landrace were observed at 65 and 90 dpc. In postnatal pigs, it was strongly expressed only in the brain, but weakly in skeletal muscle and other tissues. We initially identified 32 SNPs through genomic DNA of FSCN1 gene. Association analysis suggested that the 6840^C/T mutation was significantly associated with the age at market weight (AGE) (p = 0.0004), average day gain from birth to market (ADG1) (p = 0.0002), and average day gain at testing period (ADG2) (p < 0.0001). Our study suggested that FSCN1 gene plays an in prenatal skeletal muscle development and was a candidate gene for meat production trait.     

SNP variation in the promoter of the PRKAG3 gene and association with meat quality traits in pig

ABSTRACT: BACKGROUND: The PRKAG3 gene encodes the gamma3 subunit of adenosine monophosphate activated protein kinase (AMPK), a protein that plays a key role in energy metabolism in skeletal muscle. Nonsynonymous single nucleotide polymorphisms (SNPs) in this gene such as I199V are associated with important pork quality traits. The objective of this study was to investigate the relationship between gene expression of the PRKAG3 gene, SNP variation in the PRKAG3 promoter and meat quality phenotypes in pork. RESULTS: PRKAG3 gene expression was found to correlate with a number of traits relating to glycolytic potential (GP) and intramuscular fat (IMF) in three phenotypically diverse F1 crosses comprising of 31 Large White, 23 Duroc and 32 Pietrain sire breeds. The majority of associations were observed in the Large White cross. There was a significant association between genotype at the g.-311A>G locus and PRKAG3 gene expression in the Large White cross. In the same population, ten novel SNPs were identified within a 1.3 kb region spanning the promoter and from this three major haplotypes were inferred. Two tagging SNPs (g.- 995A>G and g.-311A>G) characterised the haplotypes within the promoter region being studied. These two SNPs were subsequently genotyped in larger populations consisting of Large White (n = 98), Duroc (n = 99) and Pietrain (n = 98) purebreds. Four major haplotypes including promoter SNP's g.-995A>G and g.-311A>G and I199V were inferred. In the Large White breed, HAP1 was associated with IMF% in the M. longissmus thoracis et lumborum (LTL) and driploss%. HAP2 was associated with IMFL% GP-influenced traits pH at 24 hr in LTL (pHULT), pH at 45 min in LTL (pH45LT) and pH at 45 min in the M. semimembranosus muscle (pH45SM). HAP3 was associated with driploss%, pHULT pH45LT and b* Minolta. In the Duroc breed, associations were observed between HAP1 and Driploss% and pHUSM. No associations were observed with the remaining haplotypes (HAP2, HAP3 and HAP4) in the Duroc breed. The Pietrain breed was monomorphic in the promoter region. The I199V locus was associated with several GP-influenced traits across all three breeds and IMF% in the Large White and Pietrain breed. No significant difference in promoter function was observed for the three main promoter haplotypes when tested in vitro. CONCLUSION: Gene expression levels of the porcine PRKAG3 are associated with meat quality phenotypes relating to glycolytic potential and IMF% in the Large White breed, while SNP variation in the promoter region of the gene is associated with PRKAG3 gene expression and meat quality phenotypes.     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.     

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (≥1.2‐fold, p<0.05), but only one of them, GDP‐dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender‐related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.     

Altered microRNA expression in bovine skeletal muscle with age

Age‐dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next‐generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty‐six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR‐1, miR‐206 and let‐7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up‐regulated and 22 miRNAs were down‐regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta‐mir‐10020. A dual luciferase reporter assay was used to demonstrate that bta‐mir‐10020 directly targeted the 3′‐UTR of the bovine ANGPT1 gene. The overexpression of bta‐mir‐10020 significantly decreased the DsRed fluorescence in the wild‐type expression cassette compared to the mutant type. Using three computational approaches – miranda , pita and rnahybrid – these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Effect of castration on carcass quality and differential gene expression of <Emphasis Type="Italic">longissimus</Emphasis> muscle between steer and bull

The effect of castration on carcass quality was investigated by ten Chinese Simmental calves. Five calves were castrated randomly at 2 months old and the others were retained as normal intact bulls. All animals were slaughtered at 22 months old. The results showed that bulls carcass had higher weight (P < 0.05), dressing percentages and bigger longissimus muscle areas (P < 0.05) than steers. But steer meat had lower shear force values and was fatter (P < 0.05) than bull. Furthermore, in order to discover genes that were involved in determining steer meat quality, we compared related candidate gene expression in longissimus muscle between steer (tester) and bull (driver) using suppressive subtractive hybridization. Ten genes were identified as preferentially expressed in longissimus muscle of steer. The expression of four selected differentially expressed genes was confirmed by quantitative real-time PCR. Overall, a 1.96, 2.41, 2.89, 2.41-fold increase in expression level was observed in steer compared with bull for actin, gamma 2, smooth muscle, tropomyosin-2, insulin like growth factor 1 and hormone-sensitive lipase, respectively. These results implied that these differentially expressed genes could play an important role in the regulation of steer meat quality.     

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 andTNNI2) with meat quality traits

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin AT Pase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of theTNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of theTNNI2 gene with 11 meat quality traits were studied on 334 Large White × Meishan F₂ pigs. In theTNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in theTNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.     

Cloning and analysis of differentially expressed ESTs in swine muscle tissue

The obvious difference in muscle growth and meat quality traits exists between Chinese indigenous pig and exotic pigs. In order to study the reason of these phenotypic differences and search the potential gene related to growth and meat quality traits, silver-stained mRNA differential display technique was used to detect the difference with mRNA of loin-eye muscle tissue from maturity pigs of Lantang in Guangdong Province and Large Yorkshire. One of the newly discovered expressed sequence tag (ESTsp3) was analyzed by using bioinformatic technique. The results showed: (1) nearly 2000 cDNA fragments were detected with 30 primer pairs, and 6 differentially expressed ESTs in the Ioin-eye muscle tissues from the two breeds were isolated and obtained. The differential fragments were cloned and sequenced. The all sequences were recorded in the GenBank. (2) The 786 bp fragment of ESTsp3 was obtained with in silico elongation system, the ORF analysis revealed that it existed as an 83 aa complete open reading frame, and the elongation sequences were verified by RT-PCR. The analysis of in silico expression profile showed that ESTsp3 is expressed in various growth stages and in most tissues and organs, such as soft tissue, skin, skeletal muscle and kidney, but with variant expression quantity.