KLF5 regulates chicken skeletal muscle atrophy via the canonical Wnt/ß-catenin signaling pathway.

Recent studies in mice suggested that KLF5 (Kruppel like factor 5), a zinc-finger transcription factor, plays an important role in skeletal muscle development and regeneration. As an important factor in the process of muscle development, KLF5 participates in the regulation of the cell cycle, cell survival, and cell dryness under different environmental conditions, but it is not clear whether KLF5 participates in muscle atrophy. Therefore, we investigated whether KLF5 can regulate the atrophy of chicken satellite cells in vitro and examined its mechanism of action. qPCR showed that KLF5 gene knockdown promoted the expression of key genes in muscle atrophy. Subsequently, we sequenced and analyzed the transcriptomes of KLF5 silenced and control cells, and we showed that the differentially expressed genes were mainly enriched in 10 signaling pathways (P<0.05), with differential gene and enrichment analyses indicating that the Wnt signaling pathways are extremely important. In conclusion, our results indicate that KLF5 may regulate the atrophy of chicken skeletal muscle through the Wnt/ß-catenin signaling pathway.

Polymorphism in MC1R, TYR and ASIP genes in different colored feather chickens.

Coat color genetics successfully adapted and applied to different animal species, which provides a good demonstration of the concept of comparative genetics. In this study, we sequenced 945 bp fragments of melanocortin 1 receptor (MC1R) gene, 421 bp fragments of exon 1 of tyrosinase (TYR) gene and 266 bp fragments of exon 3 of agouti signaling protein (ASIP) gene for 250 individuals with five plumage color patterns. We detected a total of three SNPs (T398A, T637C, and G920C) in MC1R and built six haplotypes (H1-H6) based on the three SNPs. H5 and H6 haplotypes were mainly concentrated in white and grey chicken. And diplotypes H2H3 occurred in white feather and black-speckle feather with the same frequency. Moreover, a total of three SNPs (C47G, T120C, and T172C) in TYR were found and built six haplotypes (P1-P6) based on the three SNPs. Among them, haplotype P2, P3 and P6 were not occurred in black chicken, the diplotypes P1P6 and P4P6 were only distributed in white, gray and black-speckled feather. We only detected one SNP (T168C) in ASIP gene and found that genotype TT was advantage genotype in the different plumage color groups of chickens. Collectively, our study suggested an association between plumage color and genetic variation of MC1R, TYR and ASIP in chicken.

Genotypes of IFIH1 and IFIT5 in seven chicken breeds indicated artificial selection for commercial traits influenced antiviral genes.

Innate immunity is the first line against the invasion of pathogenic microorganisms. Previous reports only demonstrated production traits of commercial importance were often negatively correlated with innate disease resistance. However, whether different purpose of artificial selection influences innate immunity have not been understood. In this study, we cloned exon1, exon6 of IFIH1 and exon2 of IFIT5 by molecular biology techniques in seven different chicken breeds to detect the potential effect of artificial selection for commercial traits on disease resistance for the first time. In total, 8 single nucleotide polymorphisms(SNPs) of IFIH1 gene exon1 and exon6, 19 SNPs of IFIT5 gene exon2 were detected. We found all native chicken breeds had a relatively close relationship to broiler breeds but a remote relationship to layer breed. A great difference between CB and LLH with different selected purpose were observed. The allele frequencies of these two positive antiviral genes were associated with different purpose of artificial selection. Our experiment constituted the foundation for the interaction between commercial traits and immune trait.

Characterization of the expression profiles of calpastatin (CAST) gene in chicken.

The calpain system, a Ca(2+)-activated protease family, plays an important role in postmortem tenderization of skeletal muscle due to its involvement in the degradation of important myofibrillar and associated proteins, as well as in cytoskeletal remodeling and regulation of muscle growth. In this study, we quantified the expression of calpastatin (CAST) in two Chinese chicken breeds (mountainous black-bone chicken breed (MB) and a commercial meat type chicken breed (S01)), to discern the tissue and age-related specific expression pattern and its potential role on muscle tissue metabolism. Real-time quantitative PCR (RT-qPCR) assay was developed for accurate measurement of CAST mRNA levels in various tissues from chicken with different ages (0, 2, 4, 6, 8, 10, and 12 week). CAST mRNA was detected in collected organs. The heart and leg muscle tissues had the highest expression of CAST than other tissues from the same chicken (P < 0.01). Age-related expression pattern of CAST gene was evident in breast muscle, liver, and brain tissues (P < 0.05), but not in heart and leg muscle tissues (P > 0.05). Overall, the CAST mRNA level exhibited a "rise-decline-rise-decline" developmental change in breast muscle and liver, with the highest expression at 2 weeks and the lowest expression at 8 weeks. The S01 chicken had significantly higher expression of CAST in breast muscle and heart than the MB chicken (P < 0.05) at 10 weeks. Our results suggested the CAST expression may be related to muscle fiber development.

Characterization of the expression profile of calpain-3 (CAPN3) gene in chicken.

Calpain-3 is a skeletal muscle-specific protease and participates in the regulation of myogenesis. In this study, we quantified the expression of calpain-3 (CAPN3) mRNA in a Chinese local chicken breed (Sichuan Mountainous Black-boned chicken [MB]), to discern the tissue and ontogenic expression pattern. Meanwhile, we compared the CAPN3 mRNA expression pattern in MB chicken at 10 weeks with a commercial meat type chicken line (S01) of the same age to identify the unique expression pattern under different genetic background. A real time quantitative PCR (qRT-PCR) assay was developed for an accurate measurement of its expression in various tissues from chickens at different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Expression of the CAPN3 mRNA was detected in the selected tissues, regardless of age. The breast muscle and leg muscle tissues had a significantly higher expression than the other tissues from the same individual (P < 0.01). Overall, the CAPN3 mRNA level exhibited a "rise-decline" developmental change in detected tissues except for brain. The S01 chicken had a higher expression of the CAPN3 mRNA in detected tissues than the MB chicken at 10 weeks. The present expression data of chicken CAPN3 gene may provide some information to shed light on the tissue and ontogenic expression pattern during chicken development.

Tissue-specific expression of the chicken calpain2 gene.

We quantified chicken calpain 2 (CAPN2) expression in two Chinese chicken breeds (mountainous black-bone chicken breed [MB] and a commercial meat type chicken breed [S01]) to discern the tissue and ontogenic expression pattern and its effect on muscle metabolism. Real-time quantitative PCR assay was developed for accurate measurement of the CAPN2 mRNA expression in various tissues from chickens of different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Results showed that the breast muscle and leg muscle tissues had the highest expression of CAPN2 compared to the other tissues from the same individual (P < .05). Overall, the CAPN2 mRNA level exhibited a "rise" developmental change in all tissues. The S01 chicken had a higher expression of the CAPN2 mRNA in all tissues than the MB chicken. Our results suggest that chicken CAPN2 expression may be related to chicken breeds and tissues.

Identification and association of the single nucleotide polymorphisms in calpain3 (CAPN3) gene with carcass traits in chickens.

BACKGROUND: The aim of this study is to screen single nucleotide polymorphisms (SNP) of chicken Calpain3 (CAPN3) gene and to analyze the potential association between CAPN3 gene polymorphisms and carcass traits in chickens. We screened CAPN3 single nucleotide polymorphisms (SNP) in 307 meat-type quality chicken from 5 commercial pure lines (S01, S02, S03, S05, and D99) and 4 native breeds from Guangdong Province (Huiyang Huxu chicken and Qingyuan Ma chicken) and Sichuan Province (Caoke chicken and Shandi Black-bone chicken), China. RESULTS: Two SNPs (11818T>A and 12814T>G) were detected by single strand conformation polymorphism (SSCP) method and were verified by DNA sequencing. Association analysis showed that the 12814T>G genotypes were significantly associated with body weight (BW), carcass weight (CW), breast muscle weight (BMW), and leg muscle weight (LMW). Haplotypes constructed on the two SNPs (H1, TG; H2, TT; H3, AG; and H4, AT) were associated with BW, CW (P < 0.05), eviscerated percentage (EP), semi-eviscerated percentage (SEP), breast muscle percentage (BMP), and leg muscle percentage (LMP) (P < 0.01). Diplotype H1H2 was dominant for BW, CW, and LMP, and H2H2 was dominant for EP, SEP, and BMP. CONCLUSION: We speculated that the CAPN3 gene was a major gene affecting chicken muscle growth and carcass traits or it was linked with the major gene(s). Diplotypes H1H2 and H2H2 might be advantageous for carcass traits.

[Associations between SNP of chicken PRKAB2 gene and slaughter and meat quality traits].

SSCP (Single-strand conformation polymorphism, SSCP) technique was applied in this study to analyze the population genetic information about genetic distribution, variation and heterozygosity of PRKAB2(Protein kinase, AMP-activated, beta 2 non-catalytic subunit) gene in five purelines and three crossbreds. The genetic effects of PRKAB2 gene on fiber density and partial carcass traits were also analyzed. Only one point mutation T-->C at base position 406 located among the first exon of the whole CDS was found in the whole CDS.The wild-type allele A had significant positive effects on live-weight, carcass-weight, leg-muscle-weight and abdomen-fat-weight (P<0.05), and had positive effects on meat tenderness.

[Genetic diversity and genetic effects of Myf6 gene in chickens].

The population genetic information about genetic distribution, variation and heterozygosity of Myf6 gene in five purelines and three crossbreds of chicken was analyzed using PCR-SSCP. And the genetic effects of Myf6 gene on fiber density and partial carcass traits were investigated. Only one mutation G/A at base position 47 of the whole CDS was found among individuals in each line. The distribution of Myf6 genotype strayed off the Hardy-Weinberg equilibrium and the heterozygosity was low in two purelines but not in three crossbreds. The wild-type allele A had significant positive effects on live-weight, carcass-weight, breast-muscle-weight and leg-muscle-weight (P<0.05), but had negative effects on most of meat quality traits. The A allele had additive effects of increasing 45.54 g of the live-weight, 41.03 g of the carcass-weight, 6 g of the breast-muscle-weight, 9.775 g of the leg-muscle-weight. The A allele had dominant effects of increasing 1.025 mm of the diameter of muscle fiber, -29.99 mm2 of the density of muscle fiber.

[Association between single nucleotide polymorphism of ADFP gene and carcass traits and intramuscular fat content in chicken].

In order to investigate the effect of ADFP gene on the carcass traits and intramuscular fat content of Da-heng high-quality chicken, we analyzed the coding sequence of the ADFP gene by single-strand conformation polymorphism (SSCP) and DNA sequencing in five pure lines and three crossbreds. We found three single nucleotide polymorphism sites, including A/T at base position 4,079 (locus A), C/T at base position 4,843 (locus B) and A/G at base position 7,070 (locus C). GLM analysis for the effect of genotypes on carcass traits and intramuscular fat content demonstrated a significant effect on leg muscle percentage, abdomen fat weight, abdominal fat percentage and intra-muscular fat content in locus A (P < 0.05), a significant association with living body weight and carcass weight for locus B (P < 0.05) and a significant association with breast muscle weight and intramuscular fat content (P < 0.05), and a very significant association with breast muscle percentage for locus C (P < 0.01). It was concluded that ADFP gene may be a major gene or linked to such a gene that affects the fat traits of birds. Polymorphic loci A and C could be used as molecular markers for abdominal fat weight, abdominal fat percentage and intramuscular fat content in birds in future studies.

[Correlation analysis of relationships between polymorphisms of high quality chicken myogenin gene and slaughter and meat quality traits].

PCR-SSCP technique was designed in this study to investigate the effect of MyoG on quality meat chicken (developed by Sichuan Dahen Poultry Breeding Company using local breeds). Four mutations at base position in promoter were detected among individuals in each line, i.e. T/C in locusA and T/A, T/C and A/G in locus B . The least square analysis showed that there were a significant difference between genotypes and breast muscle percentage and some carcass traits (P<0.05for locus A. There were a significant difference (P <0.05) in breast muscle weight between ACAA and AB geno- types; a significant difference (P <0.05) in leg muscle percentage between CC and AC for locus B, and a extremity signifi-cant difference (P <0.01) in the frequency of genotype muscle Fibre Density for Bothlocus A to locus B. There was no sig-nificant difference (P >0.05) in the other triats. It was concluded from the results that MyoG gene is the major gene affect-ing the muscle fiber traits of chicken or it links with the candidate gene, and the mutation could be used as the molecular genetic marker to select the chickens for Meat Quality traits.

[Study on correlation between single nucleotide polymorphism of CAPN1 gene and muscle tenderness and carcass traits in chicken].

In order to investigate the effect of CAPN1 on broiler tenderness traits, its CDS was analyzed with PCR-SSCP and DNA sequencing, and the genotype analyses covered individuals from five purelines and three crossbreds. Primers for exon5 and exon6 of CAPN1 were designed from database of chicken genomic sequence. The frequency of allele and PIC were calculated. A C/T mutation at base position 2546(site A) and a G/A mutation at base position 3535(site B) were found among individuals in each line. The least square analysis showed that three genotypes of birds had significant correlation with fiber density and partial carcass traits in site A and B. It was concluded from the results that CAPN1 gene may be the major gene affecting the tenderness traits of chicken or it links with the major gene, and the the polymorphic site may be used as molecular markers for meat tenderness and chicken quality.