2.1. Molecular Cloning and Sequence of the Bovine SIRT2 Gene
Based on sequencing of a PCR product that encodes the SIRT2 cDNA of Qinchuan cattle, comparison of the SIRT2 amino acid sequence that we obtained with seven other animal species from GenBank revealed the following similarities (
Table 1).
Sus scrofa (93.30%),
Homo sapiens (88.92%),
Rattus norvegicus (88.00%),
Mus musculus (85.27%),
Gallus gallus (68.63%),
Danio rerio (61.93%) and
Drosophila melanogaster (49.86%). The relatively high amino acid similarity observed among mammalians (85.27%–93.30%) suggested that the SIRT2 gene was more conserved within this group.
Table 1.
Comparative analysis of SIRT2 gene amino acid sequence of different animals.
Table 1.
Comparative analysis of SIRT2 gene amino acid sequence of different animals.
Species | GenBank Accession | Similarity |
---|
Sus scrofa | NP_001107743.1 | 93.30% |
Homo sapiens | NP_036369.2 | 88.92% |
Mus musculus | NP_001116237.1 | 85.27% |
Rattus norvegicus | NP_001008369.1 | 88.00% |
Gallus gallus | NP_001088636.1 | 68.63% |
Drosophila melanogaster | NP_001287422.1 | 49.86% |
Danio rerio | NP_955890.1 | 61.93% |
To better understand the relationship between bovine SIRT2 and the potential evolutional process, we constructed the phylogenetic tree based on amino acid sequence of SIRT2 (
Figure 1). It showed that the bovine SIRT2 is phylogenetically closest to pig SIRT2 and then to human, with the mouse and rat forming a separate group, while the non-mammalian species formed an even more distant group.
Figure 1.
Phylogenetic tree of the SIRT2 gene in different species.
Figure 1.
Phylogenetic tree of the SIRT2 gene in different species.
2.2. Ontogenic Expression of SIRT2 in Qinchuan Cattle
Tissue distribution analysis in pigs has indicated that sirtuin genes are expressed ubiquitously and with the highest abundance in brain, spinal cord and genital tissue [
13]. In a calorie-restriction model for rats, SIRT2 was expressed predominantly in white adipose and kidney tissue [
14]. Human SIRT2 is expressed in a variety of tissues, with high levels of expression in skeletal muscle and brain tissue [
15]. To date, there have been no studies of the expression pattern of bovine SIRT2. We performed RT-PCR to determine the expression of SIRT2 in different tissues. The relative expression results were obtained using the 2
−ΔΔCt method, which was first normalized to the geometric mean of β-actin, RPS9 and GAPDH. As shown in
Figure 2, SIRT2 was widely expressed in Qinchuan cattle. Moreover, the comparison of SIRT2 gene expression in diverse tissues demonstrated that the bovine SIRT2 gene was highly expressed in kidney, subcutaneous fat, and lung tissue, moderately expressed in rumen, spleen and abomasum tissue, and only slightly expressed in muscle, large and small intestine, heart, omasum, liver and reticulum tissue. There seem to be clear connections (directly or indirectly) between body size traits and modulation by SIRT2 genes in Qinchuan cattle, which fits to what we know about the important functions of SIRT2 genes in metabolism, especially in gluconeogenesis and lipid oxidation [
16].
Figure 2.
Tissue expression analysis of SIRT2 mRNA in Qinchuan cattle. Different lowercase letters above the bars (a–f) indicate significant difference between tissues (p < 0.05).
Figure 2.
Tissue expression analysis of SIRT2 mRNA in Qinchuan cattle. Different lowercase letters above the bars (a–f) indicate significant difference between tissues (p < 0.05).
2.3. Genetic Polymorphism of Qinchuan Cattle SIRT2 and χ2 Test
Sequence analysis of the SIRT2 gene revealed two C > T mutations in the 3'UTR region at 19,501 bp (
Figure 3) and 19,518 bp (
Figure 4). At the g.19501 C > T locus digestion of the 549 bp PCR fragment of SIRT2 3'UTR with BstX1 resulted in fragment lengths of 549, 459, and 90 bp for genotype CT, and 549 bp for genotype CC. The frequency of allele C was dominant in Qinchuan cattle, and genotype CC was more frequent than CT. At the g.19518 C > T locus, digestion of the 138 bp PCR fragment of SIRT2 3'UTR with Xba1 resulted in fragment lengths of 138 bp for genotype CC; 138, 108, and 30 bp for CT, and 108, 30 bp for TT. The frequency of allele C was dominant in Qinchuan cattle and genotype CT was more frequent than other genotypes.
Figure 3.
(A) PCR-RFLP detection results of SIRT2 gene PCR product (19,501 bp locus); (B,C) The sequencing maps of the novel SNP of SIRT2 gene (19,501 bp locus).
Figure 3.
(A) PCR-RFLP detection results of SIRT2 gene PCR product (19,501 bp locus); (B,C) The sequencing maps of the novel SNP of SIRT2 gene (19,501 bp locus).
Figure 4.
(A) PCR-RFLP detection results of SIRT2 gene PCR product (19,518 bp locus); (B–D) The sequencing maps of the novel SNP of SIRT2 gene (19,518 bp locus).
Figure 4.
(A) PCR-RFLP detection results of SIRT2 gene PCR product (19,518 bp locus); (B–D) The sequencing maps of the novel SNP of SIRT2 gene (19,518 bp locus).
We found that the g.19501 C > T locus had 2 genotypes, and the genotype TT was not observed in the sampled animals. The absence of that genotype in this population of Qinchuan cattle might mean that it does not exist in the population, or that the size of the experimental population was too small to capture its full genetic variation.
Based on analysis of genotype and allele frequencies (
Table 2), we found that for the g.19501 C > T mutation, the CT genotype (20.09%) was less frequent than the wild allele CC (79.91%). Allele frequencies, gene heterozygosity (He), effective allele numbers (Ne) and polymorphism information content (PIC) at the current locus were 0.8996 (C), 0.1004 (T), 0.1807, 1.2205 and 0.1644, respectively. For the g.19518 C > T mutation, the CT genotype was the most prevalent (43.16%) followed by CC (41.03%) and TT (15.81%). The values of He, Ne, and PIC at the current locus were 0.6261 (C), 0.3739 (T), 0.4682, 1.8805 and 0.3586, respectively.
Table 2.
Genotype frequencies (%) of the SIRT2 gene for the SNPs in the Qinchuan cattle populations. Note: HWE, Hardy-Weinberg equilibrium; χ0.052 = 5.991, χ0.012 = 9.21.
Table 2.
Genotype frequencies (%) of the SIRT2 gene for the SNPs in the Qinchuan cattle populations. Note: HWE, Hardy-Weinberg equilibrium; χ0.052 = 5.991, χ0.012 = 9.21.
Locus | Genotypic Frequencies (N) | Total | Allelic Frequencies | χ2 (HWE) | PIC | He | Ne |
---|
CC | CT | TT | C | T |
---|
g.19501 C > T | 0.7991 | 0.2009 | 0 | 468 | 0.8996 | 0.1004 | 5.8328 | 0.1644 | 0.1807 | 1.2205 |
g.19518 C > T | 0.4103 | 0.4316 | 0.1581 | 468 | 0.6261 | 0.3739 | 2.8581 | 0.3586 | 0.4682 | 1.8805 |
According to the conventions for PIC classification (PIC value <0.25 is considered low polymorphism, 0.25–0.50 is intermediate polymorphism, and >0.50 is high polymorphism), our data showed that g.19501 C > T had an intermediate level of polymorphism, while g.19518 C > T locus had low polymorphism. The genotypic distributions of these two mutations were in Hardy-Weinberg disequilibrium (chi-square test, χ2 < χ0.052), indicating that the genotypic frequencies had been affected by selection, mutation or migration.
2.4. Effects of Single Marker on Body Size Traits
Analysis of the SIRT2 gene in five main breeds of Chinese cattle demonstrated that the polymorphism of g.4140A > G was significantly related to body weight in Nanyang cattle [
17]. Here, two novel SNPs (g.19501 C > T and g.19518 C > T) were found in the SIRT2 gene. Association results of single markers with nine economic traits in the Qinchuan population are shown in
Table 3. The g.19501 C > T was significantly associated with body length. Individuals with genotype CC had significantly greater body length than those with genotype CT (
p = 0.0022). For g.19518 C > T, individuals with genotype TT had significantly greater rump length compared with genotype CC (
p = 0.0025), indicating that allele T might be associated with an increase in rump length in Qinchuan cattle. Such associations remained significant even after Bonferroni correction (
p < 2.78 × 10
−3).
Table 3.
Association of different genotypes of single nucleotide polymorphisms (SNPs) in SIRT2 with body size traits in Qinchuan cattle. Note: Values are shown as the least squares means ± standard error. a,b Means with different superscripts are significantly different (p < 2.78 × 10−3) after Bonferroni correction. Body length (BL), withers height (WH), hip height (HH), rump length (RL), hip width (HW), chest depth (CD), chest circumference (CC), and pin bone width (PBW), fat thickness (BF) and ultrasound loin muscle area (ULA).
Table 3.
Association of different genotypes of single nucleotide polymorphisms (SNPs) in SIRT2 with body size traits in Qinchuan cattle. Note: Values are shown as the least squares means ± standard error. a,b Means with different superscripts are significantly different (p < 2.78 × 10−3) after Bonferroni correction. Body length (BL), withers height (WH), hip height (HH), rump length (RL), hip width (HW), chest depth (CD), chest circumference (CC), and pin bone width (PBW), fat thickness (BF) and ultrasound loin muscle area (ULA).
Locus | Genotypes | BL (cm) | WH (cm) | RL (cm) | HW (cm) | CD (cm) | CC (cm) | PBW (cm) | BF (cm) | ULA (cm2) |
---|
g.19501 C > T | CC (374) | 132.608 ± 0.478 a | 119.222 ± 0.481 | 41.664 ± 0.201 | 38.286 ± 0.258 | 58.345 ± 0.315 | 161.993 ± 0.734 | 18.409 ± 0.142 | 0.873 ± 0.014 | 45.188 ± 0.673 |
CT (94) | 129.210 ± 0.721 b | 117.329 ± 0.770 | 41.287 ± 0.351 | 36.968 ± 0.324 | 57.159 ± 0.447 | 158.596 ± 0.959 | 17.776 ± 0283 | 0.860 ± 0.023 | 42.343 ± 0.962 |
P-value | 0.0022 | 0.0593 | 0.3071 | 0.0204 | 0.1051 | 0.0193 | 0.0387 | 0.3685 | 0.0367 |
g.19518 C > T | CC (192) | 130.844 ± 0.672 | 118.612 ± 0.671 | 41.099 ± 0.278 b | 37.177 ± 0.359 | 57.419 ± 0.440 | 159.276 ± 1.023 | 17.849 ± 0.197 | 0.850 ± 0.019 | 44.600 ± 2.162 |
CT (202) | 132.810 ± 0.655 | 118.381 ± 0.654 | 41.683 ± 0.271 | 38.649 ± 0.350 | 58.369 ± 0.429 | 162.552 ± 0.998 | 18.629 ± 0.193 | 0.883 ± 0.021 | 45.017 ± 2.246 |
TT (74) | 133.682 ± 1.082 | 120.696 ± 0.882 | 42.608 ± 0.447 a | 38.500 ± 0.578 | 59.176 ± 0.522 | 163.203 ± 1.648 | 18.459 ± 0.318 | 0.890 ± 0.031 | 43.566 ± 1.554 |
P-value | 0.0175 | 0.1004 | 0.0025 | 0.0031 | 0.0267 | 0.0127 | 0.0380 | 0.2007 | 0.2068 |
Sequence alignment demonstrated that these two SNPs located in the 3'UTR did not change the structure of their encoded proteins. However, recent research has provided evidence that mutation in the 3'UTR could affect protein expression and phenotype by altering the stability of mRNA [
18]. In Texel sheep, Clop
et al. (2006) [
19] reported a significant association between a G/A polymorphism in the 3'UTR of the MSTN gene and the phenotype of muscular hypertrophy. Ren
et al. (2010) [
20] identified a
HeaⅢ polymorphism in the 3'UTR of the LHX4 gene that was associated with body mass and length in Nanyang cattle. Therefore, we hypothesized that 3'UTR mutation g.19501C > T and g.19518 C > T of SIRT2 may play significant roles in modifying gene expression patterns, which would therefore affect body size traits in cattle.
2.5. Linkage Disequilibrium (LD) and Haplotype Analysis
In regression analysis,
r2 values above 0.33 might imply LD that is strong enough to be used for mapping [
21]. In our study, the pair-wise D' and
r2 values of the SNPs were 0.918 and 0.158, respectively. The
r2 values in SIRT2 were lower than 0.33, indicating that those SNPs had little LD. The rate of recombination may therefore be high, and LD would hence be low in genovariation-dense regions [
22].
In order to perform haplotype-based association analysis, three different haplotypes were constructed in SIRT2. The three most frequent haplotypes had a summed probability of 0.995, and those of frequency less than 0.05 were ignored (
Table 4). Hap
1 (-CC-) had the highest haplotype frequencies (62.10%), followed by Hap
2 (-CT-), and Hap
3 (-TT-). The haplotypes with high frequency have probably been present in the population for a long time, which may be directly or indirectly regulated by different rearing environments [
23].
Table 4.
Haplotypes of SIRT2 gene and their frequencies in Qinchuan cattle.
Table 4.
Haplotypes of SIRT2 gene and their frequencies in Qinchuan cattle.
Haplotype | g.19501 C > T | g.19518 C > T | Frequency |
---|
Hap1 | C | C | 0.621 |
Hap2 | C | T | 0.279 |
Hap3 | T | T | 0.095 |
Hap4 | T | C | 0.005 |
2.6. Effects of Haplotype Combinations on Body Size Traits
We inferred that the effects of variation of a gene could be demonstrated more readily by integrating analysis of the haplotype combinations with the single locus effects. Then the effects of the combinations of the two SNPs were evaluated, and a total of five haplotype combinations were identified for further analysis. Compared with the combination results, individuals with H
2H
2 diplotypes performed better in terms of their body traits (
Table 5). Specifically, the H
2H
2 diplotypes had significantly greater body length (
p = 0.0004), withers height (
p = 0.0005), hip width (
p = 0.0024), chest depth (
p = 0.0023) and pin bone width (
p = 0.0019), than H
1H
3 diplotypes; such associations remained significant even after Bonferroni correction for multiple testing (
p < 2.78 × 10
−3). Based on our findings, we infer that the H
2H
2 diplotypes could be used as a molecular marker of combined genotypes for future selection of body size traits in Qinchuan cattle.
Several studies have reported that marker-assisted selection (MAS), which selects particularly for beneficial traits that have low heritability, can accelerate genetic gains dramatically compared to conventional breeding [
24]. The use of MAS technology has demonstrated that many genes are related to growth [
25], production [
26] and meat quality traits [
27] in livestock. Our previous study revealed significant associations between polymorphisms in SIRT1 and body size in Qinchuan cattle [
28]. Functional studies of SIRT1 and SIRT2 showed that they share a conserved central deacetylase domain [
29] and that both proteins inhibit proliferation and differentiation in adipocytes [
8,
30]. Those similar genetic effects can be attributed to their similar function in metabolism. These findings suggest that the SIRT2 gene may have an important influence on animal body size traits, thus making it useful in MAS in cattle.
Table 5.
Associations of haplotypes with body size traits in Qinchuan cattle. Note: Values are shown as the least squares means ± standard error. a–c Means with different superscripts are significantly different (p < 2.78 × 10−3) after Bonferroni correction. Body length (BL), withers height (WH), hip height (HH), rump length (RL), hip width (HW), chest depth (CD), chest circumference (CC), and pin bone width (PBW), fat thickness (BF) and ultrasound loin muscle area (ULA).
Table 5.
Associations of haplotypes with body size traits in Qinchuan cattle. Note: Values are shown as the least squares means ± standard error. a–c Means with different superscripts are significantly different (p < 2.78 × 10−3) after Bonferroni correction. Body length (BL), withers height (WH), hip height (HH), rump length (RL), hip width (HW), chest depth (CD), chest circumference (CC), and pin bone width (PBW), fat thickness (BF) and ultrasound loin muscle area (ULA).
Combined Genotypes | Body Measurement | Meat Quality Trait |
---|
BL (cm) | WH (cm) | RL (cm) | HW (cm) | CD (cm) | CC (cm) | PBW (cm) | BF (cm) | ULA (cm2) |
---|
Hap1/1 (188) | 131.016 ± 0.658 b | 118.332 ± 0.674 | 41.309 ± 0.281 | 37.319 ± 0.357 | 57.582 ± 0.440 | 159.297 ± 1.016 | 17.899 ± 0.197 b | 0.851 ± 0.020 | 44.054 ± 1.152 |
Hap1/2 (168) | 133.717 ± 0.696 a,b | 119.884 ± 0.713 a | 41.851 ± 0.297 | 39.089 ± 0.377 a | 58.919 ± 0.465 | 164.354 ± 1.075 a | 18.863 ± 0.209 | 0.891 ± 0.021 | 46.341 ± 1.073 |
Hap1/3 (34) | 125.359 ± 1.520 c | 113.779 ± 1.584 b | 40.294 ± 0.660 | 35.735 ± 0.839 b | 55.588 ± 0.881 b | 153.970 ± 2.389 b | 17.088 ± 0.464 b | 0.811 ± 0.046 | 40.579 ± 1.741 |
Hap2/2 (18) | 138.868 ± 1.776 a | 122.333 ± 1.399 a | 43.667 ± 0.907 | 40.726 ± 0.559 a | 60.944 ± 0.824 a | 168.121 ± 2.283 a | 19.500 ± 0.638 a | 0.927 ± 0.063 | 46.266 ± 1.271 |
Hap2/3 (56) | 132.009 ± 1.204 a,b | 119.438 ± 1.234 | 41.857 ± 0.514 | 37.788 ± 0.653 | 58.161 ± 0.806 | 161.464 ± 1.861 | 18.161 ± 0.362 | 0.898 ± 0.036 | 43.347 ± 2.271 |
P-value | 0.0004 | 0.0005 | 0.0028 | 0.0024 | 0.0023 | 0.0084 | 0.0019 | 0.1142 | 0.0192 |