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Genetic variants in the TORC2 gene promoter and their association with body measurement and carcass quality traits in Qinchuan cattle

The TORC2 gene is responsible for nutrient metabolism, gluconeogenesis, myogenesis and adipogenesis through the PI3K-Akt, AMPK, glucagon and insulin resistance signaling pathways. Sequencing of PCR amplicons explored three novel SNPs at loci g.16534694G>A, g.16535011C>T, and g.16535044A>T in the promoter region of the TORC2 gene in the Qinchuan breed of cattle. Allelic and genotypic frequencies of these SNPs deviated from Hardy-Weinberg equilibrium (HWE) (P < 0.05). SNP1 genotype GG, SNP2 genotype CT and SNP3 genotype AT showed significantly (P <0.05) larger body measurement and improved carcass quality traits. Haplotype H1 (GCA) showed significantly (p<0.01) higher transcriptional activity (51.44%) followed by H4 (ATT) (34.13%) in bovine preadipocytes. The diplotypes HI-H3 (GG-CC-AT), H1-H2 (GG-CT-AT) and H3-H4 (GA-CT-TT) showed significant (P<0.01) associations with body measurement and improved carcass quality traits. Analysis of the relative mRNA expression level of the TORC2 gene in different tissues within two different age groups revealed a significant increase (P<0.01) in liver, small intestine, muscle and fat tissues with growth from calf stage to adult stage. We can conclude that variants mapped within TORC2 can be used in marker-assisted selection for carcass quality and body measurement traits in breed improvement programs of Qinchuan cattle.


Introduction
There are three members of TORC gene family: TORC1, TORC2 and TORC3, which is also known as CRTC [CREB (cAMP response element binding protein)-regulated transcription a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 coactivator]. The TORC2 gene is responsible for nutrient metabolism, gluconeogenesis, myogenesis and adipogenesis through the PI3K-Akt, AMPK, glucagon and insulin resistance signaling pathways via promotion of the anabolic and inhibiting catabolic processes within the cells [1]. Moreover, TORC2 is a coactivator gene that plays a key role in glucagon-mediated activation of gluconeogenesis through a synchronized mechanism of glucocorticoid receptor and glucagon-CREB pathways coordinated with PEPCK and G6P genes [2][3][4][5][6]. Additionally, through the CREB pathway in coordination with peroxisome proliferator-activated receptor γ (PPARγ), coactivator 1α (PGC1α) and NR4A (nuclear receptor subfamily 4 group A), CRTC2 promotes gluconeogenesis, adipogenesis [7] and myogenesis [8]. In adipose tissue, TORC2 plays a role in the anabolic processes of adipocytes in lipid metabolism, including lipogenesis, adipogenesis and lipid esterification [9,10]. TORC2 is one of the main inhibitors of lipolysis by its regulation of PKA and HSL activities [11]. Moreover, all members of the TORC gene family regulated cell proliferation through PII activity in human preadipocytes [12]. TORC2 modulates triglyceride synthesis and lipogenesis through regulation of SREBP1, which in turn regulates other fat-related genes [13]. Additionally, the CREB pathway also performs an important function in skeletal muscle. Transgenic mice with A-CREB negative gene exhibited skeletal muscle dystrophy characterized by muscular wasting, muscular inflammation and myonecrosis [14]. The above findings confirmed the role of TORC2 in adipocyte and myocyte proliferation and differentiation. Therefore, investigation of TORC2 genetic polymorphisms might contribute to breed improvement programs for carcass quality and body measurement traits in beef cattle. Body measurement and carcass quality traits are used for the assessment of animals' production. The loin area muscle and intramuscular fat contents are the key indicators of meat quality grading. These traits are mostly affected by the age of the animals, management conditions such as nutrition and by the genetics of the animals [15,16]. To achieve sustainable improvement in these traits of economic importance, selective breeding is an effective strategy, but it takes a very long time to obtain efficient genetic gain due to the longer generation interval in cattle. Genomic selection increases the rate of genetic improvement and reduces cost of progeny testing by allowing breeders to preselect animals that inherited chromosome segments of greater merit [17,18]. Single nucleotide polymorphism (SNP) markers can now cover the genome with high density and are inexpensive to obtain. Evaluations based on SNP genotypes can be computed as soon as DNA can be obtained, which allows selection in both sexes early in life [19,20]. Causal variants identification increase the accuracy of genomic selection, which is considered an efficient way of analyzing the association between genetic polymorphism and traits of economic importance [21].
Polymorphism in the promoter region is of great importance because the promoter region contains cis-acting elements that bind with transcription factors and regulate the respective gene [22][23][24][25] genes promoter regions impacted body measurement and meat quality traits in the Qinchuan cattle breed. Polymorphisms in the promoter region cause the addition or loss of important transcription factor binding sites, which in turn leads to the suppression or activation of target genes. We hypothesized that variants in the promoter region of TORC2 gene may influence phenotype, especially body measurement and carcass quality traits in Qinchuan cattle. Therefore, this study was designed with the objective of exploring SNPs in the TORC2 gene promoter region, the impact of these SNPs on the sequences of transcription factor binding sites and their association with body measurement and carcass quality traits.
Hence, the findings of the present study will enhance our understanding of the TORC2 gene regulation pattern, and the genetic variants may contribute to marker-assisted selection for breed improvement programs of Qinchuan cattle.

Ethical statement
The China Council on Animal Care guidelines were used when dealing with animals throughout the experiments. Approval was also granted for all the experimental protocols by the Experimental Animal Management Committee (EAMC) of Northwest Agriculture and Forestry University, Yangling, China. For the approved protocols that were used when dealing with animals notified vide Notification No. EAMC/20-23 dated 20.04.2013.

Phenotypic data and DNA sample collection
In total, 428 female cows aged 18 to 24 months were randomly selected from the National Beef Cattle Improvement Center's experimental farm (Yangling, China). The subject animals were fed a total mixed ration (TMR) containing 25% concentrate and 75% roughages of dry straw and corn silage, and water was offered ad libitum. The feeding was offered based on NRC standards (Nutrient Requirement of Beef Cattle) and in the similar rearing environment (similar temperature, humidly etc.) [29]. Carcass quality traits, including IF% and ULA, were estimated as per standard procedure [26, 30] using ultrasound technology (Sono-grader ultrasound machine, Renco, USA). The carcass quality traits were estimated in live animals according to the manufacturer's instructions. The ultrasonic probe was placed between the 12 and 13th rib area, and the carcass quality traits including ultrasound loin area (ULA) and intramuscular fat percentage (IF%) were recorded. The blood samples were also collected from these animals and transferred to the molecular laboratory of National Beef Cattle Improvement and Research for DNA extraction. The phenol chloroform protocol was used for the extraction of genomic DNA from the collected blood samples [31].

PCR amplification and genotyping
Primers (Table 1) were designed using Primer Premier 5 software (PREMIER Biosoft International, CA, USA) for the amplification of the -630 bp promoter region, upstream of the transcription start site of the bovine TORC2 gene GenBank NC_037330.1. The KOD plus Neo Enzyme Kit (TOYOBA, Japan) was used for PCR amplification according to the manufacturer's instructions. Genomic DNA from 428 Qinchuan cattle was used as a template for PCR amplification. Thermocycling (PCR) was performed using 3-step cycle conditions with predenaturation temperature at 94.0˚C for 5 minutes followed by 34 cycles of denaturation temperature at 97.0˚C for 30 seconds, annealing Tm of the primers used (see Table 1)˚C for 30 seconds and final extension temperature at 72.0˚C for 45 seconds. The PCR products were sequenced through Sangon (Shanghai, China) to screen for polymorphisms. All sequences were checked using Seq Man (DNASTAR, Inc., USA) software, and the SNPs were identified. TORC2 -a candidate gene for beef production and quality

4 Potential cis-acting element identification
Genomatix Mathinspector software (http://www.genomatrix.com) was used for the identification of the transcription factor (TF) binding sites in the promoter region of the bovine TORC2 gene with the cut off value of 90%. The mutations in the sequences of respective transcription factor binding sites due to SNPs were also checked through the same Genomatix online suite for their specific position and significance (Ci value), and the important transcription factor binding sites that were altered due to respective SNPs were identified. The ci-vector (consensus index vector) value of the respective cis-acting element indicate significance of the mutated nucleotides (SNPs) in the sequences of the respective transcription factor binding sites [32].

Construction of plasmid, isolation, culture and transfection of preadipocyte cells for luciferase reporter assay
The roles of TF binding sites, which are affected by the SNPs in the TORC2 gene promoter, were assessed through a dual luciferase activity assay. The four selected haplotypes were amplified using specific primers with added enzyme site sequences of Sac I and Hind III enzymes to forward and reverse primers, respectively ( Table 1). The PCR amplicons were cloned into pMD-19 T-Vector (Takara, Japan) and digested with Sac I and Hind III restriction enzymes (Takara, Japan). The haplotype DNA was then extracted from the gel through the E.Z.N.A gel extraction kit (Omega, Biotek, Inc, USA) and ligated through T4 ligation (Takara, Japan) into pGL3 basic (luciferase reporter vector), which was also digested with Sac I and Hind III (Takara, Japan) restriction enzymes. The bovine preadipocyte cells were collected from healthy newborn calves (5 days old) of Qinchuan cattle breed at the experimental farm of National Beef Cattle Improvement Center of Northwest A&F University, located in Yangling, Shaanxi, China [33]. The cells were cultured and maintained in HyClone™ Dulbecco's Modified Eagle's Medium (DMEM)/F12 1:1 cell culture media (ThermoFisher Scientific, Inc. USA), supplemented with 10% FBS (fetal bovine serum) and 1% antibiotic (penicillin 100 IU/mL and streptomycin 100 μg/mL) in an atmospheric incubator at 37˚C and 5% CO 2 . The cells were plated in 24-well plates and transiently transfected at 70-90% confluence with Lipofectamine 3000 (Invitrogen, USA), and 10 ng of pRL-TK was used as a normalizing reporter vector along with 500 ng of luciferase reporter vector (pGL3-basic) harboring DNA of selected haplotypes. Forty-eight hours post-transfection, the cells were lysed, and both firefly luciferase and Renilla luciferase activities were measured as per the standard protocol of the dual luciferase reporter assay (Promega, USA) using a Nano Quant Plate TM (TECAN, Infinite, M200 PRO System).

Estimates of conservation and biological evolution
The TORC2 gene is located on chromosome 3 of the bovine genome. The total length of TORC2 is 9,554 bp, comprising the genomic coordinates starting from 16

Tissue collection, RNA extraction, preparation of cDNA and real-time PCR
For tissue collection, three samples from Qinchuan calves (7 days old) and three samples from mature Qinchuan cattle (two years old) were selected. The animals from the two groups (calf and mature) were nonrelatives for at least three generations. The animals were dressed in a local abattoir under standard procedure of animal stunning, exsanguination and skinning. To measure the relative expression of the TORC2 gene, eight tissues, including dorsal muscle, fat, heart, kidney, lung, liver, rumen and small intestine, were collected from both the calf and mature Qinchuan groups. After collection from the carcass, the tissue samples were preserved in liquid nitrogen and were transferred immediately in frozen form to the molecular laboratory for the extraction of total RNA. Total RNA was extracted from the tissue using TRIzol™ reagent (Invitrogen, ThermoFisher Scientific, Inc. USA) and subjected to reverse transcription for synthesis of cDNA using the PrimeScriptTM RT Reagent Kit with gDNA eraser (Perfect Real Time, Takara). Quantitative real time (RT-PCR) was performed using the Sybr Premix EX Taq Kit (Takara, Dalian, China). The prepared cDNA of each tissue was used as a template, and gene-specific primers (Table 1) were used in a 20 μL reaction mixture. Two bovine genes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and β-actin, from NCBI website, with mRNA GenBank accession numbers NM_001034034.2 and NM_173979.3, respectively, were used as endogenous control genes. The thermocyclic reaction was performed using a thermocycler 7500 system SDS V 1.4.0 (Applied Biosystems, USA), with cyclic reaction conditions of preheating at 95˚C for 5 minutes, a total of 34 cycles of denaturation temperature at 95˚C for 30 seconds, annealing temperature at 60˚C for 30 seconds and extension temperature at 72˚C for 30 seconds. The thermocylic reactions were run in triplicate for each tissue sample, and the mRNA relative expression levels were calculated using the 2 −ΔΔCt method [38].

Data analyses
The general linear model (GLM) using SPSS 20.0 software (SPSS, Inc., Chicago, USA) was used for the association analysis between SNPs and selected traits of carcass quality. The linear TORC2 -a candidate gene for beef production and quality model was the same as that used in previous publications of our research group [23, 39,40]. The statistical linear model was Y ijkm = u+G i = A j +A k +S m +E ijkm , where Y ijkm are the traits measured on each animal; u is the overall mean for each trait; G i is the fixed effect associated with jth genotype; A j is the fixed effect of the jth age; A k is the fixed effect due to the age of the dam; S m is the random effect with the mth sire; and E ijkm is the standard error.
A similar statistical linear model to the model above with slight modifications was used for the association of combined genotypes; in this analysis, G j represents the fixed effect associated with the jth combined genotypes. The Bonferroni correction was made for the adjustment of the p values.
The allelic and genotypic frequencies for all three SNPs were calculated. Estimation of Hardy-Weinberg equilibrium (HWE) was measured through the chi square test in Pop Gene software version 3.2 [41]. Population genetic indicators, such as gene heterozygosity (He) and polymorphism information content (PIC), were measured through established methods [42]. The haplotypes and the D' and r 2 linkage disequilibrium (LD) were determined through Haploview (http://analysis.bio-.cn/myAnalysis.php) [43]. GraphPad Prism (6.0) was used to perform Dunnett's multiple comparisons test for the analysis of dual luciferase reporter transcriptional activities of selected haplotypes normalized against the activity of blank luciferase reporter vector.
The TORC2 gene mRNA relative expression levels were calculated using the 2 −ΔΔCt method [38]. The data were expressed as the means ± SE, and p<0.05 was considered an accepted value for statistical significance.

SNP identification
Three SNPS at loci g.16534694G>A, g.16535011C>T and g.16535044A>T in the promoter region of the TORC2 gene were identified. SNP1 yielded two genotypes, including GA and GG, while SNP2 produced CC, CT and TT. The genotypes produced by SNP3 were AA, AT and TT (Table 2). Allelic and genotypic frequencies analysis exhibited that all three SNPs deviated from the Hardy-Weinberg equilibrium (HWE) ( Table 2, p < 0.05). In the present study, SNP1 showed a low PIC value, while the PIC classification of SNP2 and SNP3 were found to be moderately polymorphic (0.25 < PIC < 0.50) [44].

Linkage disequilibrium and haplotype identification of the bovine TORC2 gene
The LD (D'/γ 2 ) was highest between SNP2 and SNP3 (0.856/0.399). The LD between SNP1 and SNP2 was 0.748/0.136, while SNP1 and SNP3 was 0.843/0.095. Eight haplotypes were TORC2 -a candidate gene for beef production and quality found, but four haplotypes with a frequency of <5% were excluded, and the frequencies of the remaining (four) haplotypes are presented in Table 3.

Association of genotype and diplotype with physical measurements and carcass quality traits
Three SNPs located in the promoter region of the bovine TORC2 gene exhibited associations with body measurement and carcass quality traits of economic importance in the Qinchuan breed of cattle (Table 4). In SNP1, the cattle with genotype GG showed significantly (P <0.01 and P <0.05) larger body length (BL), hip height (HH), chest depth (CD), chest circumference (CC), ultrasound loin area (ULA) and intramuscular fat percentage (IF%) than genotype GA, while no significant variation was found in the hip width (HW) body measurement trait in both genotypes of SNP1. In SNP2, the cattle with genotype CT exhibited significantly (P< 0.01 and 0.05) larger body length (BL), hip width (HW), chest depth (CD), chest circumference (CC) and intramuscular fat percentage (IF%) than genotypes CC and TT, while no significant variation was found in hip height (HH) and ultrasound loin area (ULA) traits in all three genotypes of SNP2. In SNP3, the cattle with genotype AT showed significantly (P< 0.01 and P< 0.05) larger body length (BL), chest depth (CD) and ultrasound loin area (ULA) than genotypes AA and TT. These four haplotypes produced five diplotypes (haplotype combinations), which showed significant (P< 0.01 and P< 0.05) associations with body measurement and carcass quality traits (Table 5). Qinchuan cattle with diplotype HI-H3 (GG-CC-AT) showed significantly

Transcription factor binding site prediction
In silico analysis showed that mutations due to SNPs caused gains and loss of transcription factor binding sites (Table 6 and Table 7). The G> A in SNP1 caused the loss of three important transcription factor binding sites, E2F7, PRDI and ARP1. In SNP2, the T>C also caused the loss of three important transcription factor binding sites (AREB6, FOXP1, and NFAT). However, none of the cis-acting element binding sites were changed due to SNP3 (Table 7). Similarly, with G>A in SNP1, six (6) new potential cis-acting element binding sites (KLF6, KLF1, MAZ, HSF2, MZF1, and SPI1) were gained, while in SNP2, the C>T causes a gain of one transcription factor binding site KLF2 (Table 7).

Luciferase reporter assay
To determine the transcriptional activities of different haplotypes, a dual luciferase reporter assay was conducted. The results analyzed with Dunnett's multiple comparisons test showed significant (p<0.01) variation in the transcription activities of these selected haplotypes ( Fig  2). The mean difference in the transcriptional activity of H1 was 51.44 (p<0.01) followed by H4 (34.13) (p<0.01) compared to the transcription activity of the pGL3-Basic vector.

Bioinformatics study of the TORC2 gene
The multiple sequence alignment of the TORC2 protein was performed for 10 species (S1 Fig).
The protein structure was highly conserved among the 7 mammalian species but was up to 40% different in Mus musculus, Canis lupus familiaris, and Gallus gallus. A phylogenetic tree Table 5. Association of diplotype combination of TORC2 gene with body measurement and carcass quality traits of Qinchuan cattle. was constructed using MEGA 7 software (Fig 3A). The MEME online suit was used to find common significant motifs in the super secondary protein structure of the TORC2 gene in 10 target species (Fig 3B).  (Fig 3C). The two domains hits (TORC_N and TORC_N super family) were not conserved in Mus musculus. For the rest of the species, all domains hits were conserved. A total of 20 significant motifs were found among 10 species (Fig 4), which indicated functional similarity among the selected species at the protein super secondary structure level.

Relative mRNA expression of the TORC2 gene at different ages
Relative mRNA expression was identified in eight different tissues (muscle, fat, liver, lung, heart, rumen, small intestine, and kidney) and within two different age groups (calf and mature) of Qinchuan cattle (Fig 5A and 5B). In the calf group, the highest mRNA expression was found in lung, followed by kidney, rumen, muscle, small intestine, heart, and fat, and the lowest expression level was found in liver. In the adult age group, the highest expression was found in the liver, muscle, small intestine, fat, lung, heart, kidney and rumen tissues,  TORC2 -a candidate gene for beef production and quality respectively. The heat map shows that in the calf group, the expression level of the TORC2 gene was low in all tissues except in the lung, which exhibited moderate expression levels. In mature Qinchuan cattle, the expression levels were high in the small intestine, liver, fat, and muscle; moderate in the lung; and low in the rumen. Significant increases (p<0.01 and p<0.05) in the relative mRNA expression level of TORC2 in liver, small intestine, muscle, fat, and kidney tissues were found with growth from the calf stage to the adult stage group. No statistically significant variation was found in the expression of the TORC2 gene in lung and rumen tissues during growth from calf to adult stage. The results obtained in the present study suggest the role of TORC2 in growth and fat deposition traits in Qinchuan cattle.

Discussion
The results of the present study showed a significant association of genotypes, haplotypes and diplotypes with body measurement and carcass quality traits. The genotypes GG and TT showed better phenotypes for body measurement and carcass quality traits; similarly, the diplotypes H1-H3 (GG-CC-AT) and H1-H2 (GG-CT-AT) contained the same genotypes GG and AT from SNP1 and SNP3. Moreover, findings of the dual luciferase reporter assay also explored highest transcriptional activity of the haplotype H1 (GCA) in bovine preadipocytes. The probable reason of exhibiting significant association of genotype GG (SNP1), haplotype TORC2 -a candidate gene for beef production and quality GCA (H1) with meat quality and body measurement traits; and highest transcriptional activity in bovine preadipocytes may be the gain of transcription factor binding sites due to genotype GG. As in SNP1, genotype GG caused a gain of six new transcription factor binding sites, including KLP6, KLF2, Myc zinc finger, Myeloid zinc finger 1, Heat shock factor 2 and SPI1. In In silico analysis revealed that SNP3 neither gained nor loss of any new transcription factor binding site in the DNA sequence; however, this locus already contained binding sites for the MZF1, KLF1 and ZNF263 transcription factors, which remained intact as no loss occurred at this position. Moreover, previously, we confirmed the role of ZNF263 transcription factor in the regulation of TORC2 gene as transcriptional activator [10] Furthermore, SNP3 is located in the proximal minimal promoter region; therefore, these transcription factors may have a substantial role in the regulation of the TORC2 gene. In previous experiments conducted by our research group in the same breed of Qinchuan cattle, we demonstrated the role of the KLF transcription factor family and the MZF1 transcription factor in the regulation of genes responsible for adipogenesis and myogenesis [45,46]. The KLFs (Kruppel-like factors) are members of the zinc finger transcription factor group, which binds to the consensus 5'-C(A/T) CCCC-3' motif in the promoter of various genes that regulates adipogenesis and myogenesis [47][48][49][50][51]. Hence, the role of this location may be due to these transcription factor binding sites in the regulation of the TORC2 gene. Transcription factor binding sites are the actual controlling factor of gene function. Binding of transcription factors with their respective binding sites at specific locations in the genome regulates gene function [52]. Therefore, genotypes AT and GG at loci SNP3 (g.16535044) and SNP1 (g.16534694), respectively, were the best variants of the TORC2 gene. Haplotype H1 (GCA) was the most frequent haplotype. The probable cause could be artificial selection in the Qinchuan cattle population, particularly the genomic regions influencing traits of economic importance [53,54]. Moreover, the evolutionary conservation analysis conducted for 10 species exhibited close homology within the protein sequences of common livestock species, which predicts functional similarity of TORC2 gene in these target species. In addition, to further exploit the function of the TORC2 gene in the growth and development of Qinchuan cattle, spatiotemporal mRNA expression was investigated in calf TORC2 -a candidate gene for beef production and quality and adult tissues. High expression in the liver is in line with findings of previous studies [6,55,56], where its core function is glucagon-mediated activation of hepatic gluconeogenesis [3,5,6] to maintain energy balance in vital tissue of the body [57,58]. Second, a significant (p<0.01) increase in the mRNA expression level of the TORC2 gene in the small intestine is in line with the findings of Liuqin et al., 2017. They further concluded that the AMP-activated protein kinase (AMPK) pathway, which is regulated by the TORC2 gene, is mainly responsible for water and ionic homeostasis in the small intestine in pigs [59]. After the small intestine, we found a significant increase in the TORC2 gene mRNA relative expression in muscle and fat tissue. The role of TORC2 through the CREB pathway is responsible for skeletal muscle functioning and myogenesis, glucose homeostasis and lipid metabolism in adipocytes [60,61]. Moreover, the TORC2 gene regulates adipogenesis and glucose homeostasis and monitors insulin sensitivity [62]. Previous literature has shown that the TORC2 gene functions as a nutrient transporter and regulates adipogenesis through regulation of the transcriptional activity of PPARγ [63]. Various findings [2,3,6,61,64,65] confirmed the significant roles of the TORC2 gene in cell growth, nutrient metabolism, gluconeogenesis, myogenesis and adipogenesis. Therefore, we can conclude that variants in TORC2 gene might be good markers for body measures and carcass quality traits in the breed improvement program of Qinchuan cattle. Based upon the findings of the present study, we mapped variants as genotypes GG and AT in SNP1 at locus g.16534694 and SNP3 at locus g.16535044, respectively; haplotypes H1 (GCA) and H4 (ATT); and diplotypes H1-H3 (GG-CC-AT) and H1-H2 (GG-CT-AT) within TORC2 can be used in marker-assisted selection for body measurement and carcass quality traits in breed improvement programs of Qinchuan cattle.