SIRT 1 gene polymorphisms associated with carcass traits in Luxi cattle

SIRT1 is the gene that codes for Sirtuin 1, an NAD (nicotinamide adenine dinucleotide)-dependent class III histone deacetylase. This gene plays a key role in adipose tissue and muscle development in animals. Chinese Luxi cattle (n = 169) were selected to identify SIRT1 SNPs (single nucleotide polymorphisms) and investigate the relationship of these SNPs with carcass traits. Five SNPs (g.-382G>A, g.-274C>G, g.17324T>C, g.17379A>G, and g.17491G>A) were identified by direct sequencing. SNPs g.-382G>A and g.-274C>G were located within the promoter region of this gene. SNP g.-382G>A was significantly associated with dressing percentage, meat percentage, and striploin and ribeye weights, and the g.-274C>G polymorphism had a strong effect on carcass, tenderloin, and high rib weights in Luxi cattle. These findings will provide possible clues for the biological roles of SIRT1 underlying beef cattle carcass traits.

The bovine SIRT1 gene, which includes nine exons on chromosome 28, is highly expressed in the liver and adipose tissue (Ghinis-Hozumi et al., 2011).SIRT1 may play an important role in the development of bovine adipose tissue in vivo.Although SIRT1, forkhead box O1 (FOXO1), and Published by Copernicus Publications on behalf of the Leibniz Institute for Farm Animal Biology.PPAR-γ expression appear to be nonlinear during the stages of preadipocyte differentiation, these genes play an important role during bovine adipocyte development in Lilu cattle (Liu et al., 2014).The study examined the variations of SIRT1 in Luxi beef cattle by identified SNPs, and explored possible associations between SIRT1 variants and carcass traits.
These molecular markers will provide some theoretical basis for improving cattle carcass characteristics.

Animals and genomic DNA isolation
In the Shandong province, 169 Chinese Luxi cattle were reared in same conditions.The animals were slaughtered at the age of 24 months according to Chinese national law (China Administration Rule of Laboratory Animal; Operating Procedure of Cattle Slaughtering GB/T 19477-2004).Carcass traits were recorded and blood samples were collected.Genomic DNA containing nucleotides from leukocytes was isolated from blood samples and stored at −20 • C following standard procedures (QIAamp DNA Blood Mini Kit, Qiagen, Germany).
The g.-382G > A and g.17379A > G polymorphisms were genotyped using the amplification-created restriction site (ACRS) method (Figarska et al., 2013).The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) was carried out to genotype SNPs g.17324T > C and g.17491G > A (Haliassos et al., 1989).The PCR reactions were performed in a total volume of 10 µL, containing 10 pmol of each of the inner primers, 1 pmol of each of the outer primers, 200 mM of each dNTP, 2 mM of MgCl 2 , 1 × PCR buffer, 50 ng of DNA, and 0.2 U of Taq DNA polymerase (MBI, Fermentas, Waltham, MA, USA).To increase the specificity of the reaction, a touchdown profile was followed.

Statistical analysis
DNA sequences were assembled and aligned for mutation analysis with DNASTAR (DNAS Inc., Madison, WI, USA).Allele and genotype frequencies were directly calculated.Heterozygosity, effective number of alleles, and polymorphic information content (PIC) were estimated based on Botstein et al. (1980).A chi-square test assessed conformance with Hardy-Weinberg equilibrium (HWE).Association of genotype with performance traits was analyzed with the general linear model (GLM) procedure of SPSS 16.0.

Identification of SNPs
Five SNPs were detected in the exons, flanking introns, and promoter sequences of SIRT1, including four transi-

The relationship between SNPs and carcass traits
Significant differences between genotypes and carcass traits of beef cattle are shown in Table 3.In g.-382G > A, AA genotypes have a more significant difference (P < 0.05) in dressing percentage, meat percentage, and striploin than the GG and GA genotypes; however, there is no difference in ribeye.In g.-274C > G, AA genotypes have a more significant difference in carcass, tenderloin and high rib weight than GG and GC genotypes.However, no differences between SNPs and carcass traits were found when focusing on 17379A > G and g.17491G > A.
Based on these results, we predicted potential differential transcription factor (TF) binding sites according to the presence of different alleles using MatInspector Release 8.0.At g.-382G > A, a myocyte-specific enhancer factor 2 (MEF2) binding site was generated on substitution to the A allele.At g.-274C > G, in the presence of the C allele, a binding site for a CDE (cell-cycle-dependent element) was generated, whereas the same binding site was abolished in the presence of the G allele.

Discussion
There are several variants associated with body mass index and risk of obesity in human SIRT1 gene (Zillikens et al., 2009).Recent studies have found possibly useful SNPs in the SIRT1 gene and explored the relationships between these SNPs and ultrasound-measured carcass traits in Qinchuan cattle (Gui et al., 2015).We identified five SNPs in bovine SIRT1 and estimated the extent of associations between these SNPs and carcass traits in Chinese Luxi cattle.Association analysis showed that SNP g.17379A > G was significantly associated with tenderloin, striploin, and ribeye and that polymorphisms with g.17324T > C had a strong effect on bone weight (these effects became non-significant following the Bonferroni correction).This SNP did not result in changes in amino acids.Such associations may be a result of linkage disequilibrium between SIRT1 and other genes on the same chromosome that have a significant effect on these carcass traits.It is interesting to note that the SNP g.17379A > G was severely out of HWE.Subsequent sequencing showed that this was not due to technical error.We considered two possible explanations: (1) Luxi cattle have experienced high selection pressure.Artificial selection led to the loss of non-favored alleles.(2) The analyzed breed has an insufficiently large population size.
Five SNPs (g.-382G > A, g.-274C > G, g.17324T > C, g.17379A > G, and g.17491G > A) were identified in the Luxi cattle and are similar to previous research results (Ye et al., 2001;M. Li et al., 2013).The role of SIRT1 as an inhibitor of adipogenesis and the recent demonstration of its involvement in white adipose tissue "browning" (M.X. Li et al., 2013) as well as the roles played by SIRT1 in muscle metabolism (Qiang et al., 2012) have motivated us to further investigate the effects of the identified SNPs on beef cattle carcass traits.Our results showed that SNP g.-382G > A was significantly associated with dressing percentage, meat percentage, and striploin and ribeye weights, and g.-274C > G polymorphism had a strong effect on carcass, tenderloin, and high rib weights in Luxi cattle.
At g.-382G > A, a MEF2 binding site was generated on substitution to the A allele.At g.-274C > G, in the presence of the C allele, a binding site for a CDE was generated, whereas the same binding site was abolished in the presence of the G allele.These indicated that g.-382G > A and g.-274C > G polymorphisms might affect the binding affinity of the surrounding sequences with TF and further influence the activity of the SIRT1 promoter that was associated with growth trait regulation.
Carcass traits are regulated by multiple genes and are influenced by interactions among them; thus, the effects of these SNPs should be further validated before they can be incorporated into beef cattle breeding practices.

Figure 1 .
Figure 1.Schematic representation of the SIRT1 gene with the localization of the five identified SNPs.

Table 1 .
PCR primers and conditions for identification of SNPs in SIRT1 (NM_001192980).purposeful mismatch was introduced in the sequence to create a restriction site.

Table 2 .
Genotypic and allelic frequencies (%), value of χ 2 test, and diversity parameters of the bovine SIRT1 gene.