Bone morphogenetic proteins (BMPs) are peptide growth factors belonging to the transforming growth factor-beta (TGF-β) superfamily, and some members of the BMP family support white adipocyte differentiation. In this study, we focused on the BMP7 which singularly promotes the differentiation of brown preadipocytes. Haplotypes involving 5 single nucleotide polymorphism (SNP) sites in the bovine BMP7 gene were identified and their effect on body weight was analyzed. 16 haplotypes and 18 combined haplotypes were revealed and the linkage disequilibrium was assessed in the cattle population with 602 individuals representing three main cattle breeds from China. The results showed that haplotypes 3, 10 and 14 were predominant and accounted for 75.64%, 69.85%, and 83.36% in Nanyang, Qinchuan and Jiaxian cattle breeds, respectively. The statistical analyses indicated that the SNP 1, 4, and 5 are associated with the body weight, body length, and heart girth at 12 and 24 months in Nanyang cattle population (P<0.05), whereas there is no significant association between their 16 haplotypes and 18 combined haplotypes. Our results provide evidence that some SNPs and haplotypes in BMP7 are associated with growth traits, and may be utilized as a genetic marker in marker-assisted selection for beef cattle breeding programs.

The Mongolian cattle are one of the most widespread breeds with strictly Bos taurus morphological features in northern China. In our current study, we presented a diversity of mitochondrial DNA (mtDNA) D-loop region and Y chromosome SNP markers in 25 male and 8 female samples of Mongolian cattle from the Xinjiang Uygur autonomous region in Western China, and detected 21 B. taurus and four Bos indicus (zebu) mtDNA haplotypes. Among four B. indicus mtDNA haplotypes, two haplotypes belonged to I1 haplogroup and the remaining two haplotypes belonged to I2 haplogroup. In contrast, all 25 male Mongolian cattle samples revealed B. taurus Y chromosome haplotype and no B. indicus haplotypes were found. Historical and archeological records indicate that B. taurus was introduced to Xinjiang during the second millennium BC and B. indicus appeared in this region by the second century AD. The two types of cattle coexisted for many centuries in Xinjiang, as depicted in clay and wooden figurines unearthed in the Astana cemetery in Turfan (3rd-8th century AD). Multiple lines of evidence suggest that the earliest B. indicus introgression in the Mongolian cattle may have occurred during the 2nd-7th centuries AD through the Silk Road around the Xinjiang region. This conclusion differs from the previous hypothesis that zebu introgression to Mongolian cattle happened during the Mongol Empire era in the 13th century.

LIM homeobox transcription factor 4 (LHX4) is a LIM homeodomain transcription factor involved in pituitary gland and nervous system development. The aim of this study was to examine the association of the LHX4 polymorphisms with growth traits in beef cattle breed. A total of 7 single nucleotide polymorphyisms (SNPs) have been identified in the coding region and noncoding region of the bovine LHX4 by sequencing pooled DNA samples (Pool-Seq) and PCR-single strand conformation polymorphism (PCR-SSCP) methods. The linkage disequilibrium was assessed in 871 individuals representing four main cattle breeds from China. The SNPs 2-5 and 7-8 were found to be in complete linkage disequilibrium, respectively. The result of haplotype analysis of 13 SNPs showed that 31 haplotypes were found in four Chinese cattle breeds, and 20 genotypes were only found in Nanyang cattle. The statistical analyses indicated that the SNP1-5, and 6 are associated with the body weight at 18, and 6 months of age in Nanyang cattle population (P<0.05), but no significant associations between their twenty combined genotypes. Our results provide evidence that some polymorphisms in LHX4 are associated with growth traits at certain ages, and may be used as candidates for marker-assisted selection and management in cattle.

Forkhead box A2 (Foxa2) has been recognized as one of the most potent transcriptional activators that is implicated in the control of feeding behavior and energy homeostasis. However, similar researches about the effects of genetic variations of Foxa2 gene on growth traits are lacking. Therefore, this study detected Foxa2 gene polymorphisms by DNA pool sequencing, PCR-RFLP and PCR-ACRS methods in 822 individuals from three Chinese cattle breeds. The results showed that four sequence variants (SVs) were screened, including two mutations (SV1, g. 7005 C>T and SV2, g. 7044 C>G) in intron 4, one mutation (SV3, g. 8449 A>G) in exon 5 and one mutation (SV4, g. 8537 T>C) in the 3'UTR. Notably, association analysis of the single mutations with growth traits in total individuals (at 24months) revealed that significant statistical difference was found in four SVs, and SV4 locus was highly significantly associated with growth traits throughout all three breeds (P<0.05 or P<0.01). Meanwhile, haplotype combination CCCCAGTC also indicated remarkably associated to better chest girth and body weight in Jiaxian Red cattle (P<0.05). We herein described a comprehensive study on the variability of bovine Foxa2 gene that was predictive of molecular markers in cattle breeding for the first time.

Copy number variations (CNVs) recently have been recognized as another important genetic variability followed single nucleotide polymorphisms (SNPs). The guanylate binding protein 2 (GBP2) gene plays an important role in cell proliferation. This study was performed to determine the presence of GBP2 CNV (relative to Angus cattle) in 466 individuals representing six main cattle breeds from China, identify its relationship with growth, and explore the biological effects of gene expression. There were two CNV regions in the GBP2 gene, for three types, CNV1 loss type (relative to Angus cattle) was more frequent in XN than other breeds, and CNV2 loss type (relative to Angus cattle) was more frequent in XN and CDM than other breeds. Though the GBP2 gene copy number presented no correlation with the transcriptional expression of JX (P > .05), but the transcriptional expression in heart is higher than other tissues, and the copy number in muscles and fat of JX is higher than others breeds. Statistical analysis revealed that the GBP2 gene CNV1 and CNV2 were significantly associated with growth traits (P < .05). In conclusion, this research established the correlations between CNVs of GBP2 gene and growth traits in different cattle breeds, and our results suggested that the CNVs in GBP2 gene may be considered markers for the molecular breeding of Chinese beef cattle.

Previous, studies have shown that the dynein transporter compound has a role in diseases such as intellectual disability and cerebral malformations. However, the study of CNV in DYNC1I2 gene has not been reported. Q-PCR and data association analysis were used for DYNC1I2 gene copy in this study. In this study, blood samples were collected from five breeds of Chinese cattle (Qingchuan cattle, Xianan cattle, Yunling cattle, Pinan cattle and Guyuan cattle) for DYNC1I2 gene CNV type detection. SPSS 20.0 software and method of ANOVA were used to analyzed the association between types of CNV and growth traits. Results reveal that the distribution of different copy number types in different cattle breeds is different. Association analysis indicate that CNV of DYNC1I2 gene showed a positive effect in cattle growth: in XN cattle, individuals with deletion types showed better performance on height at hip cross (P < 0.05); individuals with duplication types have better performance on body length (P < 0.05) in PN cattle; individuals with deletion types was significantly correlated with chest width and Hucklebone width (P < 0.05) in QC cattle; individuals with duplication types in Yunling cattle were better than the normal types, and there was a significant correlation between copy number variant and chest depth (P < 0.05). The results showed that CNV markers closely related to cattle production traits were detected at DNA level, which could be used as an important candidate molecular marker for marker-assisted selection of growth traits in Chinese cattle, and provided a new research basis for genetics and breeding of Chinese beef cattle.

Deleted in breast cancer 1 (DBC1, KIAA1967, p30 DBC) is a novel protein that has been recently shown to bind and regulate SIRT1. Loss of function of DBC1 increased SIRT1 deacetylase activity, which promotes "browning" of WAT by deacetylating peroxisome proliferator-activated receptor (PPARγ) on Lys268 and Lys293. In the present study, we have cloned and characterized the bovine DBC1 gene. Two transcript variants of bovine DBC1 were identified, designated DBC1-A and DBC1-B, respectively, which were both located in nucleus. Protein sequence analysis showed that DBC1-A was well conserved across species. Expression analysis of DBC1 in seven different tissues of calves and bulls by RT-PCR indicated that the two transcripts were ubiquitously expressed. However, the relatively level of DBC1-A was higher when compared to DBC1-B in all examined tissues. Surprisingly, the expression of DBC1-A was extraordinary high in calves adipose tissue, which implicated its potential key role in regulating calve adipocyte development. These findings provide new insight into our understanding of the biochemical characteristics and physiological role of bovine DBC1.

Copy number variations (CNVs) are an important source of genetic variation, which can affect a wide range of economic traits by diverse mechanisms. KMT2D (Lysine methyltransferase 2D) is an important positional candidate for growth traits. Quantitative trait loci (QTLs) with large effects on economically important traits cover the KMT2D gene. The KMT2D gene overlays a CNV within its exons, hence it was chosen as a crucial candidate gene to study the association between CNV and growth traits. Further, KMT2D, a major mammalian histone H3K4 mono-methyltransferase, plays a critical role in regulating development, differentiation, metabolism, and tumor suppression. Therefore, we proposed the hypothesis that KMT2D CNV may have phenotypic effects on sheep growth traits. In our study, KMT2D CNVs in three Chinese sheep breeds were detected by quantitative polymerase chain reaction (qPCR), and the loss copy was found to be the dominant genotype. Association analysis between growth traits and KMT2D CNV was also performed, which revealed that individuals with the median copy showed better performance than those with the loss copy in all three breeds. This research suggested that KMT2D CNV can be used as a promising marker for sheep molecular breeding.

MiRNAs have emerged as key regulators of skeletal muscle development, but the knowledge of miRNAs in the molecular network of muscle development remains poorly understood. In this study, we designed to examine the biological function of bovine-miR-101-1. The bovine miR-101-1 was detected in the skeletal muscle of fetal, calf and adult cattle. Its abundance was significantly higher in the skeletal muscle of calf cattle than that in fetal and adult cattle. In the course of C2C12 myoblast differentiation, the expression of miR-101-1 gradually increased. Transfected the exogenous miR-101-1 into the C2C12 myoblast could decrease myotube formation, and the mRNA expression levels of the myogenic marker genes MyOD, MyOG and MyHC were up-regulated. The protein level of MyOD, MyOG and MyHC were also up-regulated. Through TargetScan to predict the target gene of bovine miR-101-1, and the dual luciferase system was used for target gene verification. The results show that amyloid precursor protein (APP) is the target gene of miR-101-1. Therefore, our results shed light on miR-101-1 in the regulation of the skeletal muscle development.

The aim of this study was to examine the association of cofilin2 (CFL2) gene polymorphisms with growth traits in Chinese Qinchuan cattle. Three single nucleotide polymorphisms (SNPs) were identified in the bovine CFL2 gene using DNA sequencing and (forced) PCR-RFLP methods. These polymorphisms included a missense mutation (NC_007319.5: g. C 2213 G) in exon 4, one synonymous mutation (NC_007319.5: g. T 1694 A) in exon 4, and a mutation (NC_007319.5: g. G 1500 A) in intron 2, respectively. In addition, we evaluated the haplotype frequency and linkage disequilibrium coefficient of three sequence variants in 488 individuals in QC cattle. All the three SNPs in QC cattle belonged to an intermediate level of genetic diversity (0.250.33). Association analysis indicated that SNP G 1500 A, T 1694 A and C 2213 G were significantly associated with growth traits in the QC population. The results of our study suggest that the CFL2 gene may be a strong candidate gene that affects growth traits in the QC cattle breeding program.

Adipose differentiation-related protein (ADFP) is important for regulation of lipid metabolism and insulin secretion in beta-cells. In this study, we investigated polymorphisms within the caprine ADFP gene and determined its relationship with production traits. As there was no sequence information available for the caprine ADFP gene, we generated DNA sequence data and examined the genomic organisation. The caprine ADFP gene is organised into 7 exons and 6 introns that span approximately 8.7 kbp and is transcribed into mRNA containing 1,353 bp of sequence coding for a protein of 450 amino acids. The protein sequences showed substantial similarity (71-99%) to orthologues from cattle, human and mouse. We identified polymorphisms in the sequences using DNA sequencing, PCR-RFLP and forced PCR-RFLP methods. Seven single nucleotide polymorphisms (SNPs) were identified using samples from 4 different goat populations consisting of 1408 healthy and unrelated individuals. Six haplotypes involving the 7 SNPs from the caprine ADFP gene were identified and their effects on production traits were analysed. Haplotype 6 had the highest haplotype frequency and was highly significantly associated with chest circumference and milk yield in the analysed populations. The results of this study suggest that the ADFP gene is a strong candidate gene affecting production traits and may be used for marker-assisted selection and management in Chinese dairy goat breeding programmes.

Polymorphic adenoma gene 1 (PLAG1) is a member of the pleomorphic adenoma gene family. PLAG family of proteins as a nuclear transcription factor mainly play a role in regulating a variety of important genes in the body. The aim of this study was to examine the association of the PLAG1 polymorphism with growth traits in 566 cattle. A novel 19-bp indel mutation was identified in the PLAG1 by sequencing pooled DNA samples (Pool-Seq) and agarose gel electrophoresis methods. The PCR products of PLAG1 exhibited 3 genotypes and 2 alleles: 142 bp (denoted as W) and 123 bp (denoted as D). Genotype WW and allele W were predominant in the studied populations. In addition, the 19-bp indel was significantly associated with the growth traits in cattle breeds, with the hip width and rump length of Pinan cattle (P < 0.05), heart girth and cannon bone circumference of Xianan cattle (P < 0.01 or P < 0.05), as well as the heart girth, hip width, hucklebone width, rump length, height at sacrum and chest depth of the Jiaxian cattle (P < 0.05). Our results indicate that the Indel marker of PLAG1 gene can be used as candidate molecular markers for the breeding in cattle.

As an important epigenetic modification DNA methylation is catalyzed by DNA methylation transferases (DNMTs) and occurs mainly in CpG islands. DNA methylation plays an important role in regulates gene expression, cell differentiation, genetic imprinting and tumor therapy. Retinoic acid-binding proteins (RAC) is vital for the absorption, transport, metabolism and maintenance of homeostasis of retinoic acid, which in turn regulates the differentiation and proliferation of cells by regulating the transcription of many target genes, therefore, these proteins influence differentiation and proliferation of adipocytes and muscle fibroblasts. Thus, cellular retinoic acid binding protein 2 (CRABP2) may be a candidate gene which affects beef quality, yield and fat deposition. The aim of this study was to evaluate the expression and the methylation pattern on the differentially methylated region (DMR) of the promoter of CRABP2. The DNA methylation pattern was tested by bisulfite sequencing polymerase chain reaction (BSP), the quantitative real-time PCR (qPCR) was used to analysis the expression of CRABP2 gene. The results showed that the DNA methylation level was higher in purebred cattle breed than that in hybrid cattle breeds which was negative correlation with the expression of the gen. These results indicate that the methylation status of the CRABP2 DMR can regulate mRNA expression. What's more, there are different methylation and expression patterns in different breeds and tissues which may influence the phenotype, and the results may be a useful parameter to investigate the function of CRABP2 in muscle and fat developmental in Chinese cattle.

Copy number variation (CNV) related to complex traits, such as disease and quantitative phenotype, is considered an important and wealthy source of genetic and phenotypic diversity. It suggests that the copy number variation of function gene maybe leads to the phenotypic changes. Kupple like factor 3 (KLF3) gene is a vital transcription factor associated with the growth and development of muscle and adipose tissue. It has been mapped in a CNV region by animal genome re-sequencing. In this study, we detected the distribution diversity of KLF3 gene copy numbers in six Chinese cattle breeds (QC, NY, XN, PN, QDM and JX) and associated the phenotypic traits with it. Then, we analyzed the KLF3 gene transcription expression level in different tissues of Jiaxian (JX) cattle. Furthermore, we detected mRNA expression level of muscle and fat tissues of Jiaxian cattle (JX), Angus × Jiaxian (AJ). The results showed that the copy number in CNV loss was more frequent in QC than others. And we revealed a positive effect of KLF3 CNV on growth traits, such as body mass and heart girth (P < 0.05). In a word, we ascertained the significance between CNVs of KLF3 gene and growth traits in different cattle breeds, and our data indicates that the CNVs of KLF3 gene may as a marker for the future molecular breeding of Chinese beef cattle.

Copy number variation is a large genome variation which usually happens in the noncoding-region, and it may occur at the locus associated with the functional gene to further influence the phenotype. Potassium inwardly-rectifying channel, subfamily J 12 (KCNJ12) gene expressed widely in cardiomyocytes and neurons, plays an important role in tumor therapy and muscle movement regulation. In this study, we detected the distribution of CNVs for KCNJ12 gene in 404 individuals belonging to four Chinese cattle breeds (NY, JX, JA and GF). We also investigated the KCNJ12 gene expression in different tissues of JX cattle. Additionally, we examined the association of two CNV regions (CNV1: 1,600 bp, intron 1; CNV2: 4,800 bp, intergenic) with growth traits. The statistical analyses indicated that the CNV1 is associated with the body length, rump length and weight in JX cattle population (P < 0.05); and there has a significant association with the body length, chest circumference, and body weight in GF cattle (P < 0.05).The CNV2 had a significant effect on the body length and body weight in JX cattle (P < 0.05); the body length, chest circumference, rump length and body weight in GF cattle (P < 0.01 or P < 0.05). The copy numbers of KCNJ12 gene presented the negative correlations with the transcript level of gene in skeletal muscles (P < 0.05). Our results provide evidence that CNV1 and CNV 2 in KCNJ12 are associated with growth traits in two cattle populations and may be used as candidates for marker-assisted selection and breeding management in cattle.

Qinchuan cattle is one of the five yellow cattle breeds in China with good performance of meat. The proliferation and differentiation level of muscle and fat are closely related to the growth and development of the organism and are the key factors affecting the quality of meat. In order to study the effect of lncRNA on the fat tissues of Qinchuan cattle, six calf and adult bovine adipose tissues were selected for high-throughput sequencing. We obtained 3,716 lncRNA candidates from calves and adult cattle fat samples, among them 789 lncRNA were annotated and 2,927 lncRNA were novel lncRNA. A number of lncRNAs were highly abundant, and 119 lncRNA were differentially expressed between two developmental stages. We further validated several differentially expressed lncRNAs using qPCR, and the results were consistent with the sequencing data. Therefore, we conclude that lncRNA may play an important role in adipose tissue in different age groups of cattle.

Transmembrane protein 95 (TMEM95) is closely related to male reproductive performance in cattle, but does not affect semen quality. Alternative splicing plays an important role in regulating biological function as well as in generating proteomic and functional diversity in metazoan organisms. Thus, the aim of this study was to clone and identify transcripts of the TMEM95 gene in cattle using RT-PCR, characterize them via bioinformatics analysis, and detect their expression patterns using qRT-PCR. Two transcripts of TMEM95 were identified in cattle, including TMEM95-SV1 and TMEM95-SV2. Bioinformatics predicted that TMEM95-SV1 has a leucine-rich repeat C-terminal domain and a Pfam: IZUMO. These regions are closely related to protein interactions and the acrosome reaction, respectively. Interestingly, the two transcripts were exclusively expressed in the testes and brain in male fetus cattle, and TMEM95-SV1 was expressed in the brain at significantly higher levels than in the testis (P<0.05, 4.06-fold) and TMEM95-SV2 in the brain (P<0.05, 4.95-fold). These findings enrich the understanding of the TMEM95 gene function and benefit for enhancing male reproduction in cattle industry.

DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P<0.05 or P<0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P<0.05 or P<0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.

Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to examine the association of the IGF2 polymorphism with growth traits in beef cattle breed. Four single nucleotide polymorphisms (SNPs: 1-4) were identified in the bovine IGF2 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (Forced PCR-RFLP) methods. The result of haplotype analysis of four SNPs showed that eight haplotypes and eighteen combined genotypes were revealed, and the linkage disequilibrium and evolutionary relationship were assessed in 1522 individuals representing four purebred cattle breeds from China. The statistical analyses indicated that the 4 SNPs and 18 combined genotypes or haplotypes are associated with the body weight at 18 and 24 months in Jiaxian cattle population (P<0.05 or P<0.01). Our results provide evidence that polymorphisms in the IGF2 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.

The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26T>C, g.156T>C, g.587A>G, g.598C>T, g.1485C>T, g.2115A>G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A>G and g.598C>T residing in the 5'UTR region were novel SNPs identified by this study. The g.2115A>G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.