Hongbao Wang

Whole-genome sequencing of the endangered bovine species Gayal (Bos frontalis) provides new insights into its genetic features.

Gayal (Bos frontalis) is a semi-wild and endangered bovine species that differs from domestic cattle (Bos taurus and Bos indicus), and its genetic background remains unclear. Here, we performed whole-genome sequencing of one Gayal for the first time, with one Red Angus cattle and one Japanese Black cattle as controls. In total, 97.8 Gb of sequencing reads were generated with an average 11.78-fold depth and >98.44% coverage of the reference sequence (UMD3.1). Numerous different variations were identified, 62.24% of the total single nucleotide polymorphisms (SNPs) detected in Gayal were novel, and 16,901 breed-specific nonsynonymous SNPs (BS-nsSNPs) that might be associated with traits of interest in Gayal were further investigated. Moreover, the demographic history of bovine species was first analyzed, and two population expansions and two population bottlenecks were identified. The obvious differences among their population sizes supported that Gayal was not B. taurus. The phylogenic analysis suggested that Gayal was a hybrid descendant from crossing of male wild gaur and female domestic cattle. These discoveries will provide valuable genomic information regarding potential genomic markers that could predict traits of interest for breeding programs of these cattle breeds and may assist relevant departments with future conservation and utilization of Gayal.

Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

Novel polymorphism of the bovine fat mass and obesity-associated (FTO) gene are related to backfat thickness and longissimus muscle area in five Chinese native cattle breeds

In this study, genetic variation of the fat mass and obesity-associated gene (FTO) was detected by PCRSSCP and DNA sequencing in 618 individuals from five Chinese indigenous cattle breeds, and their genetic effects on meat quality traits were evaluated. The results showed that a novel single nucleotide polymorphism C1071T was detected in exon 5 and the allelic frequencies for the C and T alleles of the five breeds were 0.666/0.334, 0.583/0.417, 0.631/0.369, 0.653/0.347 and 0.689/0.311, respectively. Animals with CT genotype had higher mean values for backfat thickness than those with CC or TT genotypes (P < 0.01). Individuals with CC or CT genotypes had higher longissimus muscle area than those with TT genotype (P < 0.05). The FTO gene may be a candidate gene for identifying differences in meat quality traits and therefore, could be applied to marker-assisted selection of native Chinese cattle breeds.

Polymorphic CA microsatellites in the third exon of the bovine BMP4 gene.

We examined the variation of the BMP4 gene in four Chinese indigenous cattle breeds and investigated the association of this polymorphism with body measurement traits. Using PCR-SSCP and DNA sequencing, a polymorphic microsatellite was detected in the third exon of the bovine BMP4 gene in 459 samples from four Chinese indigenous cattle breeds, Qinchuan, Luxi, Nanyang, and Jiaxian red. The two alleles were named A and B. Allele frequencies of BMP4-A/B in the four breeds were 0.939/0.061, 0.928/0.072, 0.929/0.071, and 0.938/0.062, respectively. Least squares analysis revealed significant effects of genotype on withers height in the four breeds, on hip height in two breeds (Luxi and Nanyang, P < 0.05) and on chest circumference in Qinchuan (P < 0.05), while no significant effects of genotype on body length and rump length were found. These results can be applied to marker-assisted selection of Chinese cattle breeds, but a much larger number of animals will be needed for association analysis.

Polymorphism of somatostatin gene and its association with growth traits in Chinese cattle.

Somatostatins play a crucial role in the regulation of growth and development in vertebrates, especially muscle growth. We assessed the association of somatostatin gene polymorphisms with growth traits by PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and DNA sequencing methods in 694 individuals from six Chinese cattle breeds. A novel single nucleotide polymorphism, G126A, was detected, and significant associations were found with body length, body height, hip width, heart girth, and hucklebone width index. Polymorphism of the somatostatin gene was found to be highly associated with growth traits in the Qinchuan breed at various ages. Gene frequency analysis showed significant differences among the breeds. Individuals with genotype AA had significantly lower body height, body length, hip width, and hucklebone width values compared to AG at 1.5 years old, and had significantly lower hip width, body length and hucklebone width compared to AG at 2 years old. At 2.5 years old, populations with genotype AA had significantly lower body length, hip width and hucklebone width than AG individuals, with the exception of the Luxi breed, in which two genotypes were found. The Luxi and Ximentaer crossbreed had the lowest frequency of the G allele, while the highest G allele frequencies were found in the Luxi breed.

Genetic Architecture and Selection of Chinese Cattle Revealed by Whole Genome Resequencing

&NA; The bovine genetic resources in China are diverse, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Chinese cattle, we analyzed the whole genomes of 46 cattle from six phenotypically and geographically representative Chinese cattle breeds, together with 18 Red Angus cattle genomes, 11 Japanese black cattle genomes and taurine and indicine genomes available from previous studies. Our results showed that Chinese cattle originated from hybridization between Bos taurus and Bos indicus. Moreover, we found that the level of genetic variation in Chinese cattle depends upon the degree of indicine content. We also discovered many potential selective sweep regions associated with domestication related to breed‐specific characteristics, with selective sweep regions including genes associated with coat color (ERCC2, MC1R, ZBTB17, and MAP2K1), dairy traits (NCAPG, MAPK7, FST, ITFG1, SETMAR, PAG1, CSN3, and RPL37A), and meat production/quality traits (such as BBS2, R3HDM1, IGFBP2, IGFBP5, MYH9, MYH4, and MC5R). These findings substantially expand the catalogue of genetic variants in cattle and reveal new insights into the evolutionary history and domestication traits of Chinese cattle.

Association and expression analyses of the Ucp2 and Ucp3 gene polymorphisms with body measurement and meat quality traits in Qinchuan cattle.

The uncoupling proteins (UCPs) belong to the mitochondrial inner membrane anion carrier superfamily and play an important role in energy homeostasis. Genetic studies have demonstrated that Ucp2 and Ucp3 gene variants are involved in obesity and metabolic syndrome. The aim of this study was to identify associations between polymorphisms of Ucp2 and Ucp3 genes and economically-important traits in Qinchuan cattle. In the present study, one single-nucleotide polymorphism (SNP) in the 5 ′UTR region (SNP1: g.C-754G) of the Ucp2 gene was identified by direct sequencing of 441 Qinchuan cattle. Two SNPs in exon 3 (SNP2: g.G4877A; SNP3: g.C4902T) of the Ucp3 gene were identified by sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) among 441 Qinchuan cattle. Association analysis showed that SNP1 and SNP2 were associated with the meat quality traits (MQTs) including back fat thickness, loin muscle area and intramuscular fat content. SNP3 was found to be associated with part of the body measurement traits (BMTs) which referred to withers height and chest depth. In addition, QTL pyramiding analysis showed that individuals with diplotype P3P3 (GG–GG–CC) exhibited the best performance in terms of back fat thickness, loin muscle area, intramuscular fat content, rump length, hip width, chest depth and chest circumference. With regard to the G4877A mutation, real time PCR analysis revealed that individuals with AA genotype of the Ucp3 gene expressed higher mRNA levels than those with GG genotype. These results suggest that the diplotype P3P3 (GG–GG–CC) could be used as a molecular marker of the combined genotypes for future selection of body measurement traits and meat quality traits in Qinchuan cattle.

Production of transgenic beef cattle rich in n-3 PUFAs by somatic cell nuclear transfer

ObjectivesOmega-3 polyunsaturated fatty acids (n-3 PUFAs) are beneficial to human health. However, the n-3 PUFAs contents of the livestock meat that we consume daily are relatively low. Utilization of transgenic technology to increase n-3 PUFAs contents in livestock may solve this problem.ResultsThe omega-3 fatty acid desaturase (FAD3), encoded by fat1 gene derived from Caenorhabditis elegans (C. elegans), converts omega-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. In the study, a plasmid containing the codon-optimized C. elegansfat1 gene (mfat1) was constructed and used to produce transgenic beef cattle by somatic cell nuclear transfer. Fourteen transgenic calves were obtained, and the ratio of n-6 to n-3 PUFAs in the transgenic calves decreased from 5.33: 1 to 0.95: 1 compared with negative controls.ConclusionsOur results demonstrated that the codon-optimized C. elegansmfat1 gene can be functionally expressed in the beef cattle and converts n-6 PUFAs to n-3 PUFAs.

Lack of an association between a single nucleotide polymorphism in the bovine myogenic determination 1 (MyoD1) gene and meat quality traits in indigenous Chinese cattle breeds.

The myogenic determination 1 (MyoD1) gene is a member of the MyoD gene family. It encodes for skeletal muscle-specific transcription factors containing highly conserved basic helix-loop-helix regions that perform important roles in the initiation, maintenance, and regulation of phenotypic traits. We investigated a new single nucleotide polymorphism (SNP) in the MyoD1 gene to evaluate whether this polymorphism affects meat quality traits in five Chinese indigenous cattle breeds, namely Qinchuan (QC), Xia-Nan (XN), Nan-yang (NY), Luxi (LX), and Jia-xian red (JXR). A C→G transversion at position 624 was detected in exon 1 of the MyoD1 gene; it causes an amino acid substitution ((624)serine/(624)cysteine). Least squares analysis showed that this SNP is not significantly associated with back fat thickness, eye muscle area, intramuscular fat, or marbling. The A/B allelic frequencies in the five breeds were 0.810/0.189, 0.779/0.220, 0.768/0.231, 0.820/0.180, and 0.801/0.198, respectively. Based on the χ(2) test, the genotype distributions of four cattle breeds (LX, NY, QC, and XN) did not agree with Hardy-Weinberg equilibrium (P < 0.05); one breed (JXR) did not deviate significantly from Hardy-Weinberg equilibrium (P > 0.05). The genotypic frequencies among all five cattle breeds showed moderate diversity (0.25 < polymorphism information content < 0.5). We concluded that the C624G SNP of the MyoD1 gene does not influence meat quality traits in indigenous Chinese cattle breeds; however, this SNP could be included in breed composition and population admixture analyses due to the marked differences in allelic frequencies among these five breeds.

Myocyte enhancer factor 2A promotes proliferation and its inhibition attenuates myogenic differentiation via myozenin 2 in bovine skeletal muscle myoblast

Myocyte enhancer factor 2A (MEF2A) is widely distributed in various tissues or organs and plays crucial roles in multiple biological processes. To examine the potential effects of MEF2A on skeletal muscle myoblast, the functional role of MFE2A in myoblast proliferation and differentiation was investigated. In this study, we found that the mRNA expression level of Mef2a was dramatically increased during the myogenesis of bovine skeletal muscle primary myoblast. Overexpression of MEF2A significantly promoted myoblast proliferation, while knockdown of MEF2A inhibited the proliferation and differentiation of myoblast. RT-PCR and western blot analysis revealed that this positive effect of MEF2A on the proliferation of myoblast was carried out by triggering cell cycle progression by activating CDK2 protein expression. Besides, MEF2A was found to be an important transcription factor that bound to the myozenin 2 (MyoZ2) proximal promoter and performed upstream of MyoZ2 during myoblast differentiation. This study provides the first experimental evidence that MEF2A is a positive regulator in skeletal muscle myoblast proliferation and suggests that MEF2A regulates myoblast differentiation via regulating MyoZ2.