Identification and genetic effect of a variable duplication in the promoter region of the cattle ADIPOQ gene.

doi: 10.1111/age.12112

Identification and genetic effect of a variable duplication in the promoter region of the cattle ADIPOQ gene L. Zhang*, M. Yang*, C. Li†, Y. Xu*, J. Sun*, C. Lei*, X. Lan*, C. Zhang‡ and H. Chen* *College of Animal Science and Technology, Northwest A & F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi, 712100, China. †United States Department of Agriculture-Agricultural Research Service, Bovine Functional Genomics Laboratory, Beltsville, MD, 20705, USA. ‡Institute of Cellular and Molecular Biology, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China.

Summary

In cattle, the ADIPOQ gene is located in the vicinity of the quantitative trait locus (QTL) affecting marbling, the ribeye muscle area and fat thickness on BTA1. In our study, a novel variable duplication (NW_003103812.1:g.9232067_9232133dup) in the bovine ADIPOQ promoter region was identified and genotyped in seven Chinese cattle breeds. Using a reporter assay, we demonstrated that g.9232067_9232133dup decreased the basal transcriptional activity of the ADIPOQ gene in the 3T3-L1 and C2C12 cells. Furthermore, g.9232067_9232133dup suppressed the mRNA expression of the gene in adipose and muscle tissues. An association analysis indicated that the incremental variable duplication was associated with body measurements. Keywords Adiponectin, bovine, growth traits, mRNA expression, promoter activity, variation

Adiponectin, an adipokine, was identified in 1995 (Scherer et al. 1995). It modifies glucose and lipid metabolism, insulin sensitivity, food intake, inflammatory processes and cardiovascular function (Lafontan & Viguerie 2006). As a member of the adipocytokine family, adiponectin is secreted mainly by adipocytes; however, it also can be produced by other organs such as bone marrow, foetal tissue and myocytes (Yokota et al. 2002; Corbetta et al. 2005; Pineiro et al. 2005). In contrast to most adipocyte hormones, adiponectin levels are decreased in obesity and increased in response to weight loss (Kubota et al. 2002). Because the gene encoding for adiponectin is located on chromosome 3q27, a region covering an identified diabetes susceptibility locus in humans (Takahashi et al. 2000; Hsueh et al. 2003), a link between the ADIPOQ gene polymorphism and the metabolic phenotype has been demonstrated (Bouatia-Naji et al. 2006; Tso et al. 2006; Hivert et al. 2008; Szopa et al. 2009; Rasmussen-Torvik et al. 2009; Melistas et al. 2009). Consistent with the coding region association, the polymorphism of the promoter region also has been scanned to define the correlation

between the variation and phenotypes (Petrone et al. 2006; Schwarz et al. 2006). A genome-wide association analysis of adiposity also revealed that a SNP of ADIPOQ is related to waist circumference (Fox et al. 2007). In addition, adipose tissue has been reported to be associated with bone metabolism (Biver et al. 2011; Ealey et al. 2008). In cattle, the ADIPOQ gene is located on BTA1 near the QTL affecting marbling, the ribeye muscle area and fat thickness (Morsci et al. 2006). To date, several mutations in the ADIPOQ gene promoter region have been reported in Angus and Chinese cattle (Morsci et al. 2006; Zhang et al. 2009). Among the polymorphisms, variable copy duplication has been demonstrated to be under direct selection for the minor allele in Angus, but the results excluded ADIPOQ as underlying the marbling score QTL (Morsci et al. 2006). However, the effect of the possible variation on other growth traits (such as body measurements and average daily gain) is still unknown, and functional studies are needed to elucidate the action of a putative causal mutation. Thus, the objective of this study was to investigate the variation in the ADIPOQ promoter in Chinese breeds, as well as to examine the function of the polymorphism and evaluate the association between genotypes and growth traits.

Address for correspondence

Materials and methods

Introduction

H. Chen, College of Animal Science and Technology, Northwest A & F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, China. E-mail: [email protected] Accepted for publication 21 October 2013

Samples Blood samples were obtained from seven representative cattle breeds including Nanyang (n = 224), Jiaxian

© 2013 Stichting International Foundation for Animal Genetics, 45, 171–179

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Zhang et al. (n = 142), Qinchuan (n = 318), Luxi (n = 142), Jinnan (n = 72), Chinese Red Steppe cattle (n = 216) and Chinese Holstein (n = 97). Records of the growth traits of the Qinchuan, Jiaxian and Nanyang cattle were collected for statistical analysis. Growth traits, including body height, body length, hip width, heart girth, hucklebone width and body weight of the adult individuals (older than 2 years old) were collected from Qinchuan and Jiaxian cattle. Body sizes for various growth periods (at 6 months, 12 months, 18 months and 24 months of age) in the Nanyang breed also were collected. Adipose and muscle tissues were collected from Qinchuan cattle (n = 60) at the Kingbull Company slaughterhouse. DNA samples were extracted from whole blood and tissues according to procedures described by Sambrook & Russell (2002). Total RNA was isolated from flash-frozen adipose and muscle tissues. First-strand cDNA was synthesised from 500 ng of total RNA with the Prime Script RT Reagent Kit (TaKaRa) according to the manufacturer’s instructions.

PCR amplification and genotyping Based on the bovine genome sequences (GenBank accession no. NW_003103812.1), a pair of primers (F: 5′-AGAAA TGTTCCCTCACCTCAGT-3′ and R: 5′-CTCGGTACTCATGGG GAC-3′) was designed to amplify the promoter region of the ADIPOQ gene. The 25-ll PCR amplification mix contained 50 ng genomic DNA, 0.5 lM of each primer, 19 buffer (including 1.5 mM MgCl2), 200 lM dNTPs and 0.625 units of Taq DNA polymerase (MBI). The PCR protocol was 5 min at 95 °C, followed by 35 cycles of 94 °C for 30 s, annealing at 56 °C for 30 s, 72 °C for 40 s and a final extension at 72 °C for 10 min. Because of its high accuracy and sensitivity, PCR-SSCP was used to investigate the possible polymorphism within the amplification fragment. In the PCR-SSCP analysis, the homologous sequences showed a 67-bp indel polymorphism in the promoter region of the cattle ADIPOQ gene. Comparisons between the reference and tested sequences revealed that the polymorphism resulted in a variable duplication. We named it NW_003103812.1:g.9232067_ 9232133dup. Considering the better convenience and accuracy of the agarose electrophoresis method, the above-described PCR products of different individuals were genotyped visually with direct agarose gel electrophoresis. The PCR products, 471 bp (1D allele) or 538 bp (2D allele), were used to distinguish the mutation. Finally, samples of different genotypes were randomly selected and confirmed with sequencing, which were identical to the PCR-SSCP results.

Reporter gene constructs First, a 1.2-kb fragment of the proximal promoter region of the ADIPOQ gene was cloned and analysed to identify the

promoter region required for basal transcriptional activity. The 5′ product of the cattle adiponectin, spanning 1219 bp of the genomic sequence, was amplified from genomic DNA with PCR and the following primers: F: 5′-ACTGGTACCGA ATTTGTGGCTTGCAGGAA-3′ and R: 5′-GAAGATCTTCTC GGTACTCATGGGGAC-3′, which included the KpnI and NheI restriction sites (the attached protective nucleotides are in bold, and the restriction sites are underlined). Then, the PCR products were digested with the corresponding restriction enzymes and ligated into the pGL3-basic vector (Promega). The resulting luciferase reporter construct was designated pAdp-1230-LUC. The truncated promoter fragments were amplified with PCR and the KpnI-containing sense primers, F1: 5′-CTAGGTACCGTGCTAAATTGCTTC AGTTGTGTC-3′, F2: 5′-AGAGGTACCAGAAATGTTCCCTCA CCTCAGT-3′ and F3: 5′-GGGGTACCCCGTAAGAGGCAAAG ATAAA-3′ (the attached protective nucleotides are in bold, and restriction sites are underlined), and the same antisense primer. All fragments were cloned into the pGL3-Basic vector. After cloning, all vectors were sequenced to confirm the orientation and integrity of each construct. The resulting plasmids were designated as pAdp-830-LUC, pAdp-483-LUC and pAdp-237-LUC. These deletion constructs are depicted in Fig. 1. Through the experiment, we determined that g.9232067_9232133dup is in the transcription activity core region. Second, based on genotyping results, the 2D and 1D allelic reporter constructs were prepared by amplifying the target ADIPOQ promoter region, which included the different duplications from individual homozygous 2D/2D or 1D/ 1D genotypes. The amplified fragments also included the core promoter region, which was identified by the experiment (Fig. 2a). Both amplified fragments were sequenced to confirm that there were no mismatching nucleotides and that they encompassed the 2D or 1D alleles. Next, both fragments were cloned into the pGL3-basic vector. After cloning, all vectors were sequenced to confirm the orientation and integrity of each construct.

Cell culture and dual-luciferase reporter assays 3T3-L1 pre-adipocytes and C2C12 myotubes were obtained from the Chinese Academy of Sciences and cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 100 units/ml of penicillin, 100 lg/ml of streptomycin and 10% foetal bovine serum (FBS). The cells were grown at 37 °C with 5% CO2 in a humidified incubator. When cells reached about 80% (3T3-L1 reached about 85%, C2C12 reached about 75%) confluence, they were collected, centrifuged, diluted in growth medium prepared with DMEM and split onto 48-well plates at a density of about 1.5 or 2 9 105 cells per well (3T3-L1 was 2 9 105, C2C12 was 1.5 9 105). The following day (the cells reached about 80% confluence) cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s


Duplication of ADIPOQ and its effect in cattle

Figure 1 Promoter activity analysis of the cattle ADIPOQ gene. Serial deletion plasmids containing the 5′ promoter region of the ADIPOQ gene (schematically shown on the left) were constructed and transfected into 3T3-L1 and C2C12 cells. Luciferase activity was normalised by each internal control activity (pRL-TK). The mean expression levels and standard deviations were obtained from three independent experiments (shown on the right).

(a)

(b)

Figure 2 Effect of g.9232067_9232133dup on ADIPOQ transcriptional activity in the 3T3-L1 and C2C12 cells. (a) Schematic representation of the reporter plasmids containing one or two element repeats (1D and 2D) in the region required for basal transcriptional activity, which was inserted upstream of the luciferase reporter gene in the pGL3 basic plasmid. (b) The two constructs and the negative control pGL3 basic plasmid were transiently transfected into 3T3-L1 and C2C12 cells. The luciferase activity of each construct was normalised against the activity of Renilla luciferase. The fold increase was determined relative to the activity of the empty pGL3 basic plasmid. The mean activity levels and standard deviations were obtained from three independent experiments (*P < 0.05, compared with the 1D construct).

instructions. Briefly, for each well, 1.0 ll of Lipofectamine 2000 and 0.4 ll of plasmid were mixed in 50 ll of FBS-free and pen/strep-free DMEM medium for 20 min. To normalise the transfection efficiency, all plasmids were cotransfected with pRL-TK (a ratio of test vector DNA: internal con-

trol = 20:1 and 25:1 in the 3T3-L1 cell and C2C12 cell respectively) as an internal standard, which contains the Renilla luciferase gene. Luciferase activity was measured with a dual-Luciferase reporter assay system (Promega). Cells were collected 48 h


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Zhang et al. after transfection, and cell lysates were prepared according to Promega’s instruction manual. The activity of the firefly luciferase gene was normalised by the activity of the Renilla luciferase gene. Independent triplicate experiments were performed for each plasmid.

Real-time PCR assays ADIPOQ mRNA expression in adipose and muscle tissues was determined with the CFX-96 Real-Time PCR Detection System (Bio-Rad), using ADIPOQ mRNA-specific primers (F: 5′-GGCATTCCAGGGCATCCT-3′ and R: 5′-TTCCAGTTTCA CCAGTGTCAC-3′). GAPDH transcripts were amplified for normalisation within each sample. The reaction was performed in 25 ll, containing 12.5 ll SYBR Premix Ex Taq II (TaKaRa), 1 ll of each primer (10 lM), 2 ll cDNA (2.5 ng/ll) and 9.5 ll H2O. The relative fold change was calculated using the 2 DDCt calculation (Schmittgen & Livak 2008). To better reveal the effects of different genotypes, the expression level of the homozygous 1D was considered as 1 in different tissues. The mean expression levels and standard deviations were obtained from three independent experiments.

Data analyses Genotypic frequencies, allelic frequencies and Hardy–Weinberg equilibriums were calculated. A chi-square test was applied to assess statistical significance using SPSS software (version 19.0, SPSS, Inc.). Population genetic indices, gene homozygosity (Ho), gene heterozygosity (He), effective allele numbers (Ne) and polymorphism information content (PIC), were calculated with the Nei methods (Nei & Li 1979; Nei & Roychoudhurg 1974). The associations between the cattle genotypes and body measurements were analysed with SPSS. We adopted two steps for our analysis. First, we analysed all factors which existed in the analysed populations; the factors included fixed effects of marker genotypes, birth year, season of birth (spring versus fall), sire, farm, sex and random effects of the animal. Through the analysis, we selected the factors which have a significant effect on variability of the traits for the reduced model. Second, the adjusted linear model with fixed effects was established and used for the final analysis. In the adjusted model for the Nanyang cattle, the effects of age, genotype as well as the interaction between age and genotypes were included. The adjusted linear model was Yij = l + Ai + Gj + (AG)ij + Eij, where Yij was the trait measured on each of the ij, l was the overall population mean, Ai was the fixed effect due to the ith age, Gj was the fixed effect association with the jth genotype (1D/1D and 1D/2D genotype), (AG)ij was the interaction between the ith age and the jth genotype and Eij was the random error. The effects associated with farm, sex, sire and

season of birth (spring versus fall) were not matched in the linear model, as the preliminary statistical analysis indicated that these effects did not have a significant (P < 0.05) influence on the variability of the traits in the analysed populations. In the following model for the Qinchuan and Jiaxian cattle, the effects of farm, genotype and sex were included. The adjusted linear model was Yijk = l + Fi + Gj + Sk + Eijk, where Yijk was the trait measured on each of the ijk, l was the overall population mean, Fi was fixed effect due to the ith farm, Gj was the fixed effect association with the jth genotype (1D/1D and 1D/2D genotype), Sk was the fixed effect associated with the ith sex and Eijk was the random error. The effects associated with age were not matched in the linear model, as the preliminary statistical analysis indicated that these effects did not have a significant (P < 0.05) influence on the variability of the traits in the analysed populations. Lack of association of the sire and season of birth (spring versus fall) with the variability of the traits indicated that these factors could be excluded from the linear model in the Qinchuan and Jiaxian cattle. The least squares means estimates with standard errors for two genotypes and growth traits were used (Hickford et al. 2010; Huang et al. 2011). Differences in the transcription activity in the dualluciferase reporter assays were determined with a t-test. P values less than 0.05 were considered statistically significant.

Results Genotype and distribution of the g.9232069_ 9232135dup locus A 67-bp duplication, namely g.9232069_9232135dup (EU492456), was detected and confirmed in seven Chinese cattle breeds (Fig. 3a). As shown in Fig. 3b, there were two alleles. One included 134 nucleotides, composed of two 67nucleotide duplication elements (2D, 217 to 84, relative to the initiation of transcription), and the other contained only one 67-nucleotide element (1D, 150 to 84). The variable duplication in our study was shorter (g.1436_1438delTTG and g.1506delG; accession no. DQ156120) than that of Angus (g.1436_1506dup, accession no. DQ156120) (Morsci et al. 2006). The frequencies of the two alleles and the three genotypes in the seven breeds are listed in Table 1, as are the genetic indices (Ho, He, Ne and PIC). The frequencies of the 2D allele varied from 0.08 to 0.26. The tested population was in Hardy–Weinberg equilibrium except for the Qinchuan breed. The major allele was 1D, and the 1D/1D genotype was more frequent than the others. In the tested populations, the 2D/2D genotype was detected only in Red Steppe cattle, a new cultivated breed. Based on the k2 test, statistical differences in the genotypic and allelic frequencies


Duplication of ADIPOQ and its effect in cattle (a)

(b)

Figure 3 Identification and analysis of g.9232067_9232133dup in the promoter region of the cattle ADIPOQ gene. (a) Electrophoretic analysis of g.9232067_9232133dup in Chinese cattle. Representative PCR products from different templates demonstrated three genotypes in 2% agarose gel; 1D/1D (471 bp), representing the wild type, contains one copy; 1D/2D (471 and 538 bp) represents the heterozygote; 2D/2D (538 bp) represents the mutation type, which contains two copies; NC, negative control. (b) A schematic diagram depicting the duplication element. There are two different alleles of g.9232067_9232133dup. One includes 134 nucleotides composed of two 67-nucleotide repeats (2D), and the other is only the 67-nucleotide element (1D). The nucleotide that is different between the repeats (A–G) is in bold. The carbohydrate response element (ChRE) in the repeats is italicised. The transcriptional initiation site is underlined.

Table 1 Genotypic, allelic frequencies and diversity parameters of the g.9232067_9232133dup locus in the cattle ADIPOQ gene. Genotypic frequency

Allelic frequency

Diversity parameters

Breed

1D/1D

1D/2D

2D/2D

1D

2D

Ho

He

Ne

PIC

HWE1

QC JX JN LX NY CRS CH

0.7484 0.8169 0.8889 0.8944 0.9241 0.8194 1

0.2516 0.1831 0.1111 0.1056 0.0759 0.1713 0

0 0 0 0 0 0.0093 0

0.8742 0.9085 0.9444 0.9472 0.9621 0.9051 1

0.1258 0.0915 0.0556 0.0528 0.0379 0.0949 0

0.7801 0.8337 0.8951 0.9000 0.9270 0.8282 1

0.2199 0.1663 0.1049 0.1001 0.0730 0.1718 0

1.2819 1.1995 1.1172 1.1111 1.0787 1.2074 1

0.1957 0.1525 0.0994 0.0951 0.0704 0.1570 0

P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 –

Ho, gene homozygosity; He, gene heterozygosity; Ne, effective allele numbers; PIC, polymorphism information content; QC, Qinchuan; JX, Jiaxian; JN, Jinnan; LX, Luxi; NY, Nanyang; CRS, Red Steppe cattle; CH, Chinese Holstein. 1 HWE: Hardy–Weinberg equilibrium was determined by a chi-square test. P-value greater than 0.05 meant the population was in Hardy–Weinberg equilibrium; P-value less than 0.05 meant the population was not in Hardy–Weinberg disequilibrium.

of the ADIPOQ gene between the tested breeds were observed (Table 2). The genotypic frequency distribution had significant differences among the breeds (k2 = 66.11, df = 18, P < 0.01), especially between the Chinese Holstein breed and the other breeds (P < 0.001).

The effects of the g.9232069_9232135dup on ADIPOQ basal transcriptional activity To determine whether g.9232069_9232135dup affected the basal transcriptional activity of the ADIPOQ gene, we first analysed the promoter activity of ADIPOQ gene with a

dual-luciferase reporter assay system and identified the promoter region required for basal activity of the gene in the 3T3-L1 and C2C12 cells. We generated a series of plasmid constructs, which contained 5′ serial deletions, and transfected them into 3T3L1 pre-adipocytes and C2C12 myotubes. As shown in Fig. 1, the activity of pAdp-1230-LUC, pAdp-830-LUC and pAdp-483-LUC in the 3T3-L1 cells changed slightly, whereas the activity of pAdp-237 was 13% lower compared with pAdp-483-LUC. The data indicated that the region from 483 to 12 was sufficient for promoter activity in 3T3-L1 cells.


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Zhang et al. Table 2 The chi-square test of genotypic and allelic frequencies of the g.9232067_9232133dup locus among Chinese cattle breeds.

Breed

Qinchuan (QC)

Qinchuan (QC) Nanyang (NY) Jiaxian (JX) Jinnan (JN) Luxi (LX) Red Steppe cattle (CRS) Chinese Holstein (CH)

27.61** 2.60 6.63* 11.47** 7.56 30.23**

Nanyang (NY)

Jiaxian (JX)

0.03 9.63** 0.87 1.44 11.61** 7.76*

0.82 2.06

Jinnan (JN) 2.85 0.01 0.65

1.85 2.79 1.38 19.93**

0.00 2.23 11.31**

Luxi (LX) 3.91* 0.05 1.23 0.01 3.77 19.93**

Red Steppe cattle (CRS) 0.51 2.06 0.00 0.65 1.23

Chinese Holstein (CH) 13.90** 2.30 7.45** 4.30* 3.28 7.45**

50.66**

Chi-square values (k2) for differences in genotypic frequencies between two breeds are below the diagonal; chi-square values (k2) for differences in allelic frequencies between two breeds are above the diagonal. *P < 0.05, **P < 0.01.

In the C2C12 cells, the activity of pAdp-1230-LUC, pAdp830-LUC and pAdp-483-LUC also changed slightly. However, the activity of pAdp-237 was dramatically lower (0.05%) compared with that of pAdp-483-LUC. The data indicated that the region from 483 to 12 was sufficient and crucial for promoter activity in C2C12 cells. Moreover, the activity of all reporter genes was lower in the C2C12 cells than in the 3T3-L1 cells. These results indicated that the region from 483 to 12 was sufficient for ADIPOQ promoter activity in C2C12 and 3T3-L1 cells. The g.9232069_9232135dup was located precisely in the basal promoter region ( 483 bp to 12 bp) of the gene. This result also revealed that g.9232069_9232135dup maybe had significant effects on the promoter activity. Second, we constructed another plasmid containing different haplotypes (Figs. 2a & 3a) to compare the activities of 1D and 2D in both cell lines. The activity of the reporter plasmid containing 2D was 32%, and 37% were lower in the 3T3-L1 and C2C12 cells respectively compared with the activity of the 1D construct (Fig. 2b). This revealed that the Relative mRNA expression level (normalized to GAPDH)

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1.4

*

*

1D 2D

1.2 1 0.8 0.6 0.4 0.2 0

Adipose tissue

Muscle tissue

Figure 4 Relative mRNA expression levels in adipose and muscle tissues of different genotypes in g.9232067_9232133dup loci. Relative mRNA expression levels in adipose and muscle tissues of homozygous 2D or 1D were analysed by real-time PCR. The mRNA expression level of ADIPOQ was normalised to GAPDH. The expression level in the 1D homozygous was considered as 1. The mean expression levels and standard deviations were obtained from three independent experiments. An asterisk denotes a significant difference by t-test between two groups (P < 0.05).

incremental duplication suppressed the basal transcriptional activity of the ADIPOQ promoter in the 3T3-L1 and C2C12 cells.

The effects of g.9232069_9232135dup on ADIPOQ mRNA expression levels To confirm the hypothesis that the g.9232069_ 9232135dup locus in the ADIPOQ promoter could affect ADIPOQ mRNA expression levels, adipose and muscle tissues from 30 samples (1D/1D = 25, 1D/2D = 5, 2D/2D not detected in our tested population) were analysed with real-time PCR. The mRNA expression level in individuals with the 1D/2D genotype was 43% and 54% lower in adipose and muscle tissues respectively compared with the 1D/1D genotype (Fig. 4). This indicated that the 2D allele inhibited the ADIPOQ mRNA expression levels in adipose and muscle tissues.

Association analysis of g.9232069_9232135dup and growth traits in Chinese cattle The evidence that inhibited expression of ADIPOQ was related to the 2D allele of g.9232069_9232135dup prompted us to investigate the associations between the genotypes of the ADIPOQ gene and body measurements in cattle. The relationships between the genotypes and growth traits of Qinchuan, Jiaxian and Nanyang cattle were tested. Body measurements (including heart girth, hip width, hucklebone width, rump length, chest depth and chest width) were significantly associated with the genotypes (Tables S1–S3). Comparing the association results, the heart girth and hucklebones width were significantly associated with the genotypes among the three breeds. Individuals with the 2D allele had larger heart girth and hucklebone width (P < 0.05) in three Chinese cattle breeds (Table 3). In a previous study, another 5-bp deletion mutation (g.9232974_9232978del) was also detected near the locus (Zhang et al. 2009), so the linkage disequilibrium of the two


Duplication of ADIPOQ and its effect in cattle Table 3 Growth traits significantly associated with genotypes of the g.9232067_9232133dup locus in the ADIPOQ gene in Qinchuan, Jiaxian and Nanyang cattle breeds. Qinchuan (mean SE)

Jiaxian (mean SE)

Nanyang (mean SE)

Growth traits

1D/2D (n = 80)

1D/1D (n = 238)

P-value1

1D/2D (n = 23)

1D/1D (n = 107)

P-value1

1D/2D (n = 8)

1D/1D (n = 88)

P-value1

Heart girth Hucklebone width

188.40 4.38 30.74 0.90

177.60 1.91 28.56 0.39

0.025 0.028

181.15 1.58 27.42 0.90

177.43 0.72 24.74 0.41

0.034 0.008

175.25 2.47 27.50 0.82

169.42 0.74 25.36 0.25

0.026 0.014

1

The P-value had been adjusted by Bonferroni method.

loci was analysed (Table S4). The analysis revealed that they were not in strong linkage disequilibrium (r2 < 0.33). The association of the combined genotypes of the two loci were also analysed in Nanyang cattle (Table S3). There was no significant association between the traits and the combined genotypes.

Discussion In the present study, g.9232069_9232135dup in the ADIPOQ promoter region of Chinese cattle was detected with PCR-SSCP and confirmed with agarose gel electrophoresis and sequencing. In the mouse and human ADIPOQ promoter, no variable duplication has been reported. It has been demonstrated that short-sequence motifs can affect transcriptional activity, mRNA stability and translational efficacy (Nakamura et al. 1998). Herein, a 67-bp tandem repeat element in the ADIPOQ gene promoter correlated with transcriptional activity and mRNA expression. These results are consistent with previous reports. For example, a 21-bp repeat polymorphism in the 5′-untranslated region (5′-UTR) of the XRCC5 gene could affect promoter activity and gene expression (Wang et al. 2008); a 28-bp repeat polymorphism in TYMS 5′-UTR could affect gene expression (Yawata et al. 2005). To elucidate the influence of the variation, the duplication region was analysed. Comparing the two repeat elements, one different nucleotide was found in the duplication sequence (Fig. 3b). Similarly, one or two different nucleotides were also found in the tandem repeats of the promoter region of XRCC5 gene (Wang et al. 2008). Through the MATINSPECTOR program (http://www.genoma trix.de), one carbohydrate response element (ChRE) was found in the repeat element (Fig. 3b). To date, ChRE has been mapped within the promoter regions of the pyruvate kinase liver and RBC (PKLR), thyroid hormone responsive (THRSP), fatty acid synthase (FASN), acetyl-CoA carboxylase alpha (ACACA) and thioredoxin-interacting protein genes (TXNIP) (Minn et al. 2005; Towle 2005). However, there was no direct evidence of their effects on the ADIPOQ promoter. Moreover, the transcriptional activity reduction may be induced by the binding of repressors to the duplication sequence.

In our study, we selected two tissues to validate the effects of g.9232069_9232135dup on mRNA expression level of the ADIPOQ gene. One was adipose tissue where adiponectin was mainly produced; the other was muscle tissue which, like other tissues, can produce a small amount of adiponectin. Our results indicated that the individuals with the 2D allele had lower mRNA expression levels in both tissues, which was consistent with the promoter activity analysis of the haplotypes. The promoter analysis also revealed that the activity of the reported gene was much higher in the 3T3-L1 cells than in the C2C12 cells, which was consistent with the characteristics of ADIPOQ expression (Scherer et al. 1995). Although previous results have excluded the association between the variable duplication and marbling score in Angus (Morsci et al. 2006), other traits have not been tested. So we selected growth traits (body measurements and body weight) for the association analysis in Chinese cattle. Our association analysis indicated that NW_ 003103812.1:g.9232069_9232135dup was associated with body measurements, especially heart girth and hucklebone width. The differences between the genotypes may be due to ADIPOQ expression. The individuals with the 2D allele had lower ADIPOQ expression, which resulted in more fat in the body. In humans, in addition to the negative correlation with body mass index and waist circumference (Fox et al. 2007; Schober et al. 2007), ADIPOQ is related to bone mass (Biver et al. 2011). The g.9232069_ 9232135dup associated with larger cattle body measurements also was consistent with previous studies on other species (Kotani et al. 2004; Kearns et al. 2006; Gordon et al. 2007). The combined association analysis of g.9232974_ 9232978del revealed no significant relationship between the genotypes and traits, but there was no linkage disequilibrium between the two loci. Thus, g.9232069_ 9232135dup can be used as a candidate marker for body measurements selection in Chinese cattle. In conclusion, a variable duplication (NW_003103812.1: g.9232069_9232135dup) within the ADIPOQ gene promoter region was observed and confirmed in seven breeds of Chinese cattle. Furthermore, we demonstrated that g.9232069_9232135dup inhibited the activation of the promoter and suppressed the expression of ADIPOQ mRNA.


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Zhang et al. These findings provide background for more extensive characterisation of the bovine ADIPOQ gene. Finally, the association analysis indicated that the variation was associated with body measurements.

Acknowledgements This study was supported by the National 863 Program of China (Grant No. 2013AA102505), the National Natural Science Foundation of China (grant no. 30972080), the Agricultural Science and Technology Innovation Projects of Shaanxi Province (no. 2012NKC01-13) and Program of the National Beef Cattle and Yak Industrial Technology System (CARS-38).

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Supporting information Additional supporting information may be found in the online version of this article. Table S1 The association analysed between the genotypes of the g.9232067_9232133dup locus in the ADIPOQ gene and the growth traits in Jiaxian cattle. Table S2 The association analysed between the genotypes of the g.9232067_9232133dup locus in the ADIPOQ gene and the growth traits in Qinchuan cattle. Table S3 The association analysed among the genotypes of g.9232067_9232133dup, g.9232974_9232978del and combined genotypes of the two loci in the ADIPOQ gene and the growth traits in Nanyang cattle. Table S4 The estimated values of linkage disequilibrium analysis between both sites (g.9232974_9232978del and g.9232067_9232133dup) in the bovine ADIPOQ gene and the Hardy–Weinberg equilibrium analysis of the populations.


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Effects of genetic variants in the promoter region of the bovine adiponectin (ADIPOQ) gene on marbling of Hanwoo beef cattle.

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