Single nucleotide polymorphisms in an intron of the ovine calpastatin gene

Calpastatin is the specific inhibitor of the ubiquitous calcium-dependent proteases mu-calpain and m-calpain. Enzyme assay data from sheep and cattle inversely correlates post-mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three-allele system detected by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR-RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.     

Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.     

Effect of single nucleotide polymorphisms of CAPN1 and CAST genes on meat traits in Nellore beef cattle (Bos indicus) and in their crosses with Bos taurus

The objectives of this work were to study the segregation of single nucleotide polymorphisms of the calpain 1, large subunit ( CAPN1 ) and calpastatin ( CAST ) genes in Nellore ( Bos indicus ) and Nellore × Bos taurus beef cattle, as well as to evaluate their effects on meat traits. For this, 300 animals, including 114 Nellore, 67 Angus × Nellore, 44 Rubia Gallega × Nellore, 41 Canchim, 19 Brangus three-way crosses and 15 Braunvieh three-way crosses, were genotyped for the CAPN4751 [AF_248054.2:g.6545C>T (GenBank accession AF248054.2 )] and CAST/DdeI [AF_159246.1:g.2959A>G (GenBank accession AF159246.1 )] polymorphisms and phenotyped for Ribeye Area, Backfat Thickness, Intramuscular Fat, Shear Force (SF) and Myofibrillar Fragmentation Index (MFI). In relation to the CAPN4751 polymorphism, a frequency of 10.5% was observed for the C allele in the Nellore breed. In the total sample of studied animals, a significant association was found between genotypes and meat tenderness, assessed by SF ( P  = 0.005) and MFI ( P  = 0.008), with genotype CT being more favourable than TT. For the CAST/DdeI polymorphism, a frequency of 55.7% was found for the A allele in the Nellore breed. In the total sample, a significant association was observed between genotypes and meat tenderness – SF ( P  = 0.004) and MFI ( P  = 0.006), with genotype AA being more favourable than AG. The relationship between genotypes and aged meat tenderness in confluence with the distribution of favourable alleles shows great potential for application of the CAPN4751 and CAST/DdeI polymorphisms in the genetic improvement of the Nellore breed, whilst contributing to the validation, in this breed and in its crosses with B. taurus , of the association results previously described in the literature.     

Haplotypic Diversity Within the Ovine Calpastatin <Emphasis Type="Italic">(CAST)</Emphasis> Gene

Calpastatin (CAST) is a protein inhibitor that acts specifically on calpains and plays a regulatory role in postmortem beef tenderization and muscle proteolysis. Polymorphisms in the bovine CAST gene have been associated with meat tenderness, but little is known about how the ovine CAST gene may affect sheep meat quality traits. In this study, we selected two parts of the ovine CAST gene that have been previously reported to be polymorphic (region 1—part of intron 5 and exon 6, and region 2—part of intron 12), to investigate haplotype diversity across an extended region of the ovine gene. First, we developed a simple and efficient polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method for genotyping region 2, which allowed the detection of a novel allele as well as the three previously reported alleles. Next, we genotyped both regions 1 and 2 of the ovine CAST gene from a large number of sheep to determine the haplotypes present. Nine different haplotypes were found across this extended region of the ovine CAST gene and four haplotypes were identified that suggested historical recombination events within this gene. Haplotypes are typically more informative than single nucleotide polymorphisms (SNPs) for analyzing associations between genes and complex production traits, such as meat tenderness, but the potential for intragenic recombination within the ovine CAST may make finding associations challenging.     

Association of polymorphisms in calpain 1, (mu/I) large subunit,calpastatin, and cathepsin D genes with meat quality traits in double‐muscled Piemontese cattle

Five single‐nucleotide polymorphisms (SNPs) located in the calpain 1, (mu/I) large subunit (CAPN1), calpastatin (CAST), and cathepsin D (CTSD) genes were analyzed in a large sample of Piemontese cattle. The aim of this study was to evaluate allele and genotype frequencies of these SNPs and to investigate associations of CAPN1, CAST, and CTSD gene variants with meat quality traits. Minor allele frequencies ranged from 30 to 48%. The presence of the A allele at CAPN530 increased yellowness and drip loss. The CAST282 G allele was associated with an increased drip loss compared to the C allele, and the CAST2959 A allele decreased redness compared to the G allele.     

Association of CAST gene polymorphisms with carcass and meat quality traits in Yanbian cattle of China

Bovine calpastatin (CAST) gene polymorphisms have been associated with meat tenderness traits; however, little is known about how the CAST gene affects beef quality traits. In this study, 25 single nucleotide polymorphisms were detected in the CAST gene using polymerase chain reaction with single-strand conformation polymorphism and gene sequencing. Different preponderant genotypes were found at the corresponding gene locus. The E1-1, E1-2, and C3-1 loci were correlated with meat tenderness height and highly correlated with the fatty acid content and the amino acid content. The E4-2 locus was not correlated with meat tenderness, but it was correlated with cooking loss, brightness, and yellowness, among others. The CAST gene is a potential marker for these meat quality traits, but further research is required.     

Genotypic effects of calpain 1 and calpastatin on the tenderness of cooked M. longissimus dorsi steaks from Jersey x Limousin, Angus and Hereford-cross cattle

Single nucleotide polymorphisms (SNPs) in the calpain 1 (CAPN1) and calpastatin (CAST) genes were studied to determine their effects on meat tenderness in Bos taurus cattle. Strip loins (M. longissimus dorsi) were removed from cattle in four resource populations after slaughter (n = 1042), aged under controlled conditions until fixed times after rigor mortis, cooked and measured using a tenderometer. Animals were genotyped for the CAPN1 SNP c.947C>G (p.Ala316Gly; AF252504) and for the CAST SNP c.2959A>G (AF159246). Frequencies of CAPN1 C alleles ranged from 23% to 68%, and CAST A alleles from 84% to 99.5%. From all data combined, the CAPN1 CC genotype (compared with the GG genotype) was associated with a 20.1 +/- 1.7% reduced average shear force at intermediate stages of ageing (P < 0.001) and with a 9.5 +/- 1.3% reduction near ultimate tenderness (P < 0.001). The heterozygote was intermediate. For CAST, corresponding values for AA compared with AG genotypes were reductions of 8.6 +/- 2.0% and 5.1 +/- 1.6% respectively (both P < 0.001), but there were too few GG genotypes for comparison. There were small interactions between the CAPN1 and CAST genotypes. For the CAPN1 and CAST genotypes combined, the maximal genotype effect in average shear force was 25.7 +/- 5.5% (P < 0.001) at intermediate stages and 15.2 +/- 4.8% near ultimate tenderness (P < 0.01).     

Single nucleotide polymorphisms in caprine calpastatin gene

The calpains and calpastatin (CAST) make up a major cytosolic proteolytic system, the calpain-calpastatin system, found in mammalian tissues. The relative levels of the components of the calpain-calpastatin system determine the extent of meat tenderization during postmortem storage. Calpastatin (CAST) is a protein inhibitor of the ubiquitous calcium-dependent proteases, μ-calpain, and m-calpain. Polymorphisms in the bovine, ovine and pig CAST gene have been associated with meat tenderness but little is known about how caprine CAST gene may affect goat meat quality traits. In this study we selected different parts of the CAST gene: (1) that have been previously reported to be polymorphic, intron 5 and 12 and 3’UTR; (2) first time explored (exon 3, 7 and 8 and part of intron 7 and 8) to investigate polymorphic status of caprine CAST gene. Using comparative sequencing ten novel SNPs located in exon 3 and intron 5, 7 and 8 were identified. Previously reported SNPs in intron 5, 3’UTR and intron 12 were absent. Sequence analysis revealed a non synonymous amino acid variation in exon 3, which would result in Lys/Arg substitution in the corresponding protein sequence. Considerable variation was detected in intronic regions. Twenty-four InDel were also recognized in intronic regions (15) and 3’UTR (9). All the sequences shared high homology with published bovine and ovine sequences. Three PCR-RFLP loci have been established for further analyzing genetic polymorphism in indigenous goats.     

Relationships among calpastatin single nucleotide polymorphisms,calpastatin expression and tenderness in pork longissimus1

Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphisms (SNP) in calpastatin were identified and used to genotype a population (n = 1042) of Duroc–Landrace–Yorkshire swine for association with longissimus lumborum slice shear force (SSF) measured at days 7 and 14 postmortem. Three genetic markers residing in the calpastatin gene were significantly associated with SSF (P < 0.0005). Haplotypes constructed from markers in the calpastatin gene were significantly associated with SSF (F‐ratio = 3.93; P‐value = 0.002). The levels of normalized mRNA expression of calpastatin in the longissimus lumborum of 162 animals also were evaluated by real‐time RT‐PCR and were associated with the genotype of the most significant marker for SSF (P < 0.02). This evidence suggests that the causative variation alters expression of calpastatin, thus affecting tenderness. In summary, these data provide evidence of several significant, publicly available SNP markers associated with SSF that may be useful to the swine industry for marker assisted selection of animals that have more tender meat.     

Calpastatin Gene (CAST) Is Not Associated with Late Onset Sporadic Parkinson’s Disease in the Han Chinese Population

Recent studies point to an association between the late-onset sporadic Parkinson’s disease (PD) and single nucleotide polymorphisms (SNPs) rs1559085 and rs27852 in Ca²⁺-dependent protease calpain inhibitor calpastatin (CAST) gene. This finding is of interest since loss of CAST activity could result in over activated calpain, potentially leading to Ca²⁺ dysregulation and loss of substantia nigra neurons in PD. We explored the association between CAST SNPs and late-onset sporadic PD in the Han Chinese population. The study included 615 evaluable patients (363 male, 252 female) with PD and 636 neurologically healthy controls (380 male, 256 female) matched for age, gender, ethnicity, and area of residence. PD cases were identified from the PD cohort of the Chinese National Consortium on Neurodegenerative Diseases (www.chinapd.cn). A total of 24 tag-SNPs were genotyped capturing 95% of the genetic variation across the CAST gene. There was no association found between any of the polymorphisms and PD in all models tested (co-dominant, dominant-effect and recessive-effect). Similarly, none of the common haplotypes was associated with a risk for PD. Our data do not support a significant association between the CAST gene polymorphisms and late onset sporadic PD in the Han Chinese population.     

The polymorphisms of LYRM1 gene and their association with body measurement and ultrasound traits of Qinchuan cattle

Body measurement and meat quality traits which play important roles in the assessment of productivity and economy in cattle were influenced by genes and environmental factors. Latest studies showed that LYR motif containing 1 (LYRM1) may be involved in influencing fatness deposition in animals. The objective of this study was to detect bovine LYRM1 gene polymorphism and analyze its association with body measurement and meat quality traits of cattle. Blood samples were taken from a total of 404 Qinchuan cattle aged from 18–24 months. Created restriction site-polymerase chain reaction-restriction fragment length polymorphism (CRS-PCR–RFLP) and DNA sequencing were used to find out LYRM1 single polymorphism nucleotide (SNPs). Sequence analysis of LYRM1 gene revealed two SNPs (g.165 C > A, g.193 A > G) in 3′ untranslated region (3′UTR) of exon 3. And g.165 C > A showed two genotypes namely AC and CC while g.193 A > G showed three genotypes: AA, AG and GG. Analysis results showed that there were significant associations between polymorphism of these two and body measurement and meat quality traits in Qinchuan cattle population. Based on the results obtained from this study, it is inferred that LYRM1 gene may have potential effects on body measurement and meat quality traits in Qinchuan cattle population and could be used for marker-assisted selection.     

奶牛TLR4基因的多态性与乳房炎的相关性研究

TLR4通过识别病原体而激活免疫细胞,在先天免疫和适应性免疫防御中起着重要作用。以中国荷斯坦奶牛、三河牛和中国西门塔尔牛为研究对象,以TLR4为乳房炎抗性的候选基因,分别扩增316bp和382bp2个片段,分别采用SSCP和RFLP-AluⅠ方法来检测TLR4基因的多态性。结合测序发现:在intron1的第4,525bp处的A→G的突变,和exon3的第1,397bp处的T→C突变,使得产生多态。2个位点的A、B等位基因在3个群体中都有分布,且处于中度多态。χ2适合性检验表明,3个群体在这2个位点的突变达到Hardy-Weinberg平衡状态(P>0.05)。运用SAS8.0软件采用最小二乘法拟合线性模型,将基因座不同基因型与奶牛乳房炎进行了关联分析,结果表明:T4CRBR1的AA基因型为乳房炎抗性基因型(P<0.05),A等位基因为乳房炎抗性的有利基因,T4CRBR2的各基因型个体间的体细胞评分差异不显著(P>0.05)。     

Bovine mu-calpain (CAPN1) gene: new SNP within intron 14

The calpain system originally comprised molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. This proteolytic system plays a key role in the tenderisation process that occurs during post-mortem storage of meat under refrigerated conditioning. Their polymorphism is examined from the point of view of their effect on corresponding production traits. The calpain genes are investigated as potential candidate genes for a quantitative trait locus (QTL) affecting meat tenderness. In this study a new single nucleotide polymorphism (SNP) was found within intron 14 of the bovine CAPN1 gene, being transition C --> T at position 4685 nt (consensus sequence - GenBank No. AF 248054), as this mutation creates a new FokI restriction site detected with PCR-RFLP analysis. This sequence fragment of the SNP position has already been deposited in the GenBank database under accession No. AY639597. The RFLP-FokI polymorphism was studied in 141 bulls of seven breeds, including the native Polish Red (PR, preserved), and Polish Black-and White (BW) breed. The frequency of alleles T and C varied between the breeds considered, the mean reaching 0.38 and 0.62, respectively. Associations between CAPN1/FokI gene polymorphism and meat production traits were studied in BW (n = 84) young bulls. In the animals of the TT genotype the lean share in valuable cuts (%) was found more favourable than in CC animals.     

Association of the expression levels in the skeletal muscle and a SNP in the CDC10 gene with growth‐related traits in Japanese Black beef cattle

Growth performance, as well as marbling, is the main breeding objective in Japanese Black (JB) cattle, the major beef breed in Japan. The septin 7 (CDC10) gene, involved in cellular proliferation, is located within a genomic region of a quantitative trait locus for growth‐related traits. In this study, we first showed that the expression levels of the CDC10 gene in the skeletal muscle were higher in JB steers with extremely high growth performance than in JB steers with extremely low growth, using real‐time PCR. Further, a single nucleotide polymorphism (SNP), NC_007302.5:g.63264949G>C, was detected in the promoter region of the CDC10 gene and genotyped in three Japanese cattle breeds (known as ‘Wagyu’ in Japan) and the Brown Swiss dairy cattle breed. All four cattle populations showed a moderate genetic diversity at the SNP of the CDC10 gene. An association analysis indicated that the SNP was associated with growth‐related traits in JB cattle. These findings suggest possible effects of the expression levels in the skeletal muscle and the SNP of the CDC10 gene on growth‐related traits in JB cattle. The CDC10 SNP may be useful for effective marker‐assisted selection to increase beef productivity in JB beef cattle.     

Comparison of three PCR-based methods to detect a Piedmontese cattle point mutation in the Myostatin gene

Despite many recent advances in single nucleotide polymorphism (SNP) genotyping technologies, there is still the need for methodologies with a reasonable throughput. In this study, we compared three PCR-based methods for SNP detection: (1) a conventional PCR-based allele detection system with fluorescent genotyping technology, (2) a SNaPshot methodology by single nucleotide primer extension and, (3) a real-time PCR-based method by allele-specific minor groove-binder probes. These three methodologies were used to analyze 104 meat samples of a particular Italian cattle breed known for producing excellent quality meat and for a characteristic increased development of muscle mass, caused by a point mutation (C313Y) in the Myostatin gene. The analysis revealed 98 samples to be homozygous (mh/mh) and five to be heterozygous (mh/+) for the mutation whereas one sample resulted to be homozygous for the wild type (+/+). The results obtained with the three different assays were consistent. Overall, all three methodologies proved to be efficient for allelic discrimination studies; however, real-time PCR was faster and allowed to genotype up to 96 samples in a single step, minimizing the number of steps required for samples manipulation.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

Back fat thickness and meat tenderness are associated with a 526 T→A mutation in the exon 1 promoter region of the MyF-5 gene in Chinese Bos taurus

Qualitative trait loci (QTL) for growth and meat quality traits in cattle (Bos taurus) have been previously mapped to three chromosome regions, 0 to 30, 55 to 70, and 70 to 80 cM on chromosome 5. We evaluated the allele frequencies and gene-specific single nucleotide polymorphisms (SNPs) of bovine myogenic factor 5 (MyF-5) in the QTL regions and their associations with live weight and meat characteristics in indigenous Chinese cattle breeds. PCR-SSCP methodology showed a T>A mutation at 526 bp. Least square analysis revealed a significant association of this SNP with backfat thickness and meat tenderness (P < 0.05), while no significant association was found with live weight, loin eye height, loin eye area, rib area, or water holding capacity. Allele frequencies of MyF-5-A/B in the five breeds were 0.760/0.239, 0.752/0.247, 0.629/0.370, 0.715/0.284, and 0.750/0.250, for JiaXian red, Luxi, Nanyang, Qinchuan, and XiaNan crossbreed, respectively. The genotype distributions for these alleles in two of the Chinese cattle breeds (Luxi and Qinchuan) were not in Hardy-Weinberg equilibrium (P < 0.05); while those for the other three breeds (JiaXian red, Nanyang, and XiaNan) were in agreement with Hardy-Weinberg equilibrium (P > 0.05). The genotypic frequencies among all five cattle breeds showed moderate diversity (0.25 < polymorphism information content < 0.5). Based on our findings, we suggest that the MyF-5 gene influences back fat thickness and meat tenderness in Chinese Bos taurus. This SNP could be useful for marker-assisted selection for meat quality traits in these cattle.     

Association of a single nucleotide polymorphism in the akirin 2 gene with economically important traits in Korean native cattle

The akirin 2 gene, located on chromosome 9 in cattle, was previously reported to be associated with nuclear factor‐kappa B (NF‐κB), involved in immune reactions and marbling of meat. To determine whether a single nucleotide polymorphism (SNP) in akirin 2 is associated with economically important traits of Korean native cattle, the c.*188G>A SNP DNA marker in the 3′‐UTR region of akirin 2 was analyzed for its association with carcass weight, longissimus muscle area and marbling. The c.*188G>A SNP was genotyped by polymerase chain reaction restriction fragment length polymorphism, and the frequency of the AA, AG, and GG genotypes were 6.82%, 71.29% and 21.88% respectively. This SNP was significantly associated with longissimus muscle area (Bonferroni corrected P < 0.05), and marbling score (Bonferroni corrected P < 0.01). These results suggest that the c.*188G>A SNP of akirin 2 might be useful as a DNA marker for longissimus muscle area and marbling scores in Korean native cattle.