Liangzhi Zhang

Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine)

DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development.

Detection of copy number variations and their effects in Chinese bulls

BackgroundCopy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.ResultsWe identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.ConclusionsThe results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle’s evolution and breeding researches.

Effect of an exon 1 mutation in the myostatin gene on the growth traits of the Bian chicken.

Myostatin (MSTN), or growth and differentiation factor 8 (GDF8), is a member of the transforming growth factor (TGF)-β superfamily. This family functions as a negative regulator of skeletal muscle development and growth in mammals. Single-nucleotide polymorphisms in exon 1 of the Bian chicken myostatin gene were detected by polymerase chain reaction-restriction fragment length polymorphism. A mutation (c.234G>A) in exon 1 was found. Female Bian chickens of genotypes AA and GA had significantly higher body weights than those of genotype GG (P < 0.05 or P < 0.01) from 6 to 18 weeks of age. These results suggested that the mutation c.234G>A in exon 1 could be used as a genetic marker for Bian chicken growth traits.

Association of Polymorphisms of Leptin Gene with Body Weight and Body Sizes Indexes in Chinese Indigenous Cattle

PCR-SSCP was used to analyze the polymorphism of leptin gene in 539 samples of six cattle breeds, namely Nanyang (NY), Qinchuan (QC), Jiaxianred (JXR), Xizhen (XZ), Luxi (LX), and Holstein cow (HOL) breeds. PCR products with a 330 bp were amplified and sequenced. The results showed that the frequencies of alleles A/B of NY, QC, JXR, XZ, LX, and HOL breeds were 0.558/0.442, 0.492/0.508, 0.571/0.429, 0.658/0.342, 0.591/0.409, and 0.615/0.385, respectively. The association of variations of leptin gene with growth traits in NY, QC, JXR breeds was analyzed. Some indexes of the individuals with genotype BB were higher than that with genotype AA and AB in NY breed, such as the indexes of body length, heart length, body weight, hucklebone width, body height, and average day gain. The height at hip cross of the individuals with genotype BB was higher than that of those with genotype AA and AB in QC breed (P < 0.05). So leptin gene may be one of the candidate genes for growth traits with height at hip cross, but not for body weight, heart length, and body length trait. However, the height at hip cross and hucklebone width of the individuals with genotype AB and BB were higher than that of those with genotype AA in JXR breed (P < 0.05), but the difference was not statistically significant in body weight and body sizes (body height, body length, and heart length). And the polymorphisms in leptin gene were caused by G --> T transversion at the 66th bp position, A --> C transversion at the 67th bp position and G --> T transversion at the 299th bp position. These results may be applied to marker-assisted selection of Chinese cattle breeds.

Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

A novel c.-274C>G polymorphism in bovine SIRT1 gene contributes to diminished promoter activity and is associated with increased body size

SIRT1, a mammalian homologue for yeast silent information regulator 2 (SIR2), is a NAD(+) -dependent deacetylase that belongs to the class III histone deacetylases. It plays an important role in diverse cellular processes, including stress resistance, mitochondrial function, suppression of inflammation and DNA repair. In this study, we screened and identified a novel polymorphism (c.-274C>G) in the SIRT1 promoter region. In silico prediction reveals that this SNP is in the core of cell cycle-dependent element (CDE)-binding motif. Interestingly, the G allele abolished a CDE-binding site, which suggested its functional significance. In the luciferase assay system, we found that the G allele-containing construct displayed a strikingly lower promoter activity compared with the C allele, which may downregulate SIRT1 expression levels. Additionally, we observed a significant association between the c.-274C>G polymorphism and growth traits in Nanyang cattle, suggesting that anomalous transcription factor-based repression of SIRT1 may increase bovine fat mass and body size.

Novel 35-bp insertion in Visfatin Gene in Chinese cattle

Visfatin is a protein that is preferentially produced in visceral adipose tissue. Both its tissue expression and secreted plasma levels increase in parallel with obesity. Therefore, the present study is focused on detecting insertion in bovine visfatin gene and analyzing its effect on growth traits in four Chinese cattle breeds (Nangyang, Qinchuan, Jiaxian, and Chinese Holstein) using PCR-SSCP, DNA sequencing and agarose electrophoresis methods. For the first time, a 35-bp insertion was described in the gene and two alleles were revealed: W and I. The x2-test analysis demonstrated that all breeds were in agreement with Hardy-Weinberg equilibrium (P > 0.05). The associations of the novel 35 bp-insertion of visfatin gene with growth traits of Nanyang cattle at 6, 12, 18, and 24 months old were analyzed: 6 month-old cows with genotype WW had greater birth weight than cows with genotype II and WI (P < 0.01 or P < 0.05). These results suggest that the presence of 35 bpinsertion in visfatin gene may influence the birth weight and bodyweight in 6 month-old cows.

Allelic Polymorphism Detected in the Bovine FTO Gene

Studies in humans have independently identified single nucleotide polymorphisms (SNPs) in the fat mass and obesity associated (FTO) gene associated with obesity in multiple populations. It was shown that FTO participated in the regulation of energy homeostasis and associated with increased lipolytic activity in adipocytes. To ascertain whether there were mutations in the bovine FTO gene, this study investigated the variation of the FTO gene through PCR-SSCP and sequencing. Five synonymous mutations, two missense mutations, and three intronic SNPs were identified in 614 cattle from five independent populations. Haplotype frequencies and linkage disequilibrium (LD) coefficients of these SNPs in three Chinese indigenous cattle breeds were analyzed. Two LD blocks were found in the Qinchuan and Nanyang cattle breeds and three LD blocks were found in the Jiaxian cattle breed, suggesting the possibility of a recombination hotspot between exon 5 and intron 5 of the bovine FTO gene. The variations detected here might have an impact on the FTO gene activity and function.

Three novel single-nucleotide polymorphisms of the bovine LHX3 gene

The LHX3 gene encodes LIM homeodomain class transcription factors that have important roles to play in pituitary and nervous system development. On the one hand, mutations of LHX3 are associated with deficiencies of growth hormone (GH), prolactin (PRL), luteotrophic hormone (LH), follicle-stimulating hormone (FSH) and thyroidstimulating hormone (TSH); on the other hand, mutations of LHX3 are also associated with combined pituitary hormone deficiency (CPHD) diseases in human and animal models. To date, few polymorphisms of the bovine LHX3 gene have been reported. In this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods were employed to screen the genetic variations within the bovine LHX3 gene in 802 Chinese indigenous cattle. The results revealed three novel single-nucleotide polymorphisms (SNPs): AY923832: g.7553G>A, 7631C>T and 7668C>G. Among them, a synonymous mutation of exon II was identified: GAG (Glu) >GAA (Glu) at position 72 aa (AY923832:g.7553G>A) of LHX3 (403aa) in the four Chinese bovine breeds. Significant statistical differences in genotypic frequencies for exon II and its flanking region of the LHX3 gene implied that the polymorphic locus was significantly associated with cattle breeds by the χ2-test (χ2 = 68.975, df = 6, P<0.001). Hence, the three novel SNPs not only extend the spectrum of genetic variations of the bovine LHX3 gene, but could also possibly contribute to conducting association analysis and evaluating these as genetic markers in bovine breeding and genetics, and CPHD detection.

Impact of parental Bos taurus and Bos indicus origins on copy number variation in traditional Chinese cattle breeds

Copy number variation (CNV) is an important component of genomic structural variation and plays a role not only in evolutionary diversification but also in domestication. Chinese cattle were derived from Bos taurus and Bos indicus, and several breeds presumably are of hybrid origin, but the evolution of CNV regions (CNVRs) has not yet been examined in this context. Here, we of CNVRs, mtDNA D-loop sequence variation, and Y-chromosomal single nucleotide polymorphisms to assess the impact of maternal and paternal B. taurus and B. indicus origins on the distribution of CNVRs in 24 Chinese domesticated bulls. We discovered 470 genome-wide CNVRs, only 72 of which were shared by all three Y-lineages (B. taurus: Y1, Y2; B. indicus: Y3), whereas 265 were shared by inferred taurine or indicine paternal lineages, and 228 when considering their maternal taurine or indicine origins. Phylogenetic analysis uncovered eight taurine/indicine hybrids, and principal component analysis on CNVs corroborated genomic exchange during hybridization. The distribution patterns of CNVRs tended to be lineage-specific, and correlation analysis revealed significant positive or negative co-occurrences of CNVRs across lineages. Our study suggests that CNVs in Chinese cattle partly result from selective breeding during domestication, but also from hybridization and introgression.