POLB Regulates Proliferation and Apoptosis of Bovine Primary Myocytes.

DNA polymerase ß (DNA polymerase beta (POLB)) belongs to a member of the DNA polymerase X family, mainly involved in various biological metabolic processes, such as eukaryotic DNA replication, DNA damage repair, gene recombination, and cell cycle regulation. In this study, the muscle development-related gene POLB was screened by selection signature and RNA-seq analysis and then validated for the proliferation and apoptosis of bovine primary myocytes. It was also found that overexpression of the POLB gene had a pro-apoptosis effect, but interfering with the expression of the gene had no significant effect on cells. Then, the analysis of related apoptotic genes revealed that POLB overexpression affected CASP9 gene expression.

Identification and Functional Analysis of Transcriptome Profiles, Long Non-Coding RNAs, Single-Nucleotide Polymorphisms, and Alternative Splicing from the Oocyte to the Preimplantation Stage of Sheep by Single-Cell RNA Sequencing.

Numerous dynamic and complicated processes characterize development from the oocyte to the embryo. However, given the importance of functional transcriptome profiles, long non-coding RNAs, single-nucleotide polymorphisms, and alternative splicing during embryonic development, the effect that these features have on the blastomeres of 2-, 4-, 8-, 16-cell, and morula stages of development has not been studied. Here, we carried out experiments to identify and functionally analyze the transcriptome profiles, long non-coding RNAs, single-nucleotide polymorphisms (SNPs), and alternative splicing (AS) of cells from sheep from the oocyte to the blastocyst developmental stages. We found between the oocyte and zygote groups significantly down-regulated genes and the second-largest change in gene expression occurred between the 8- and 16-cell stages. We used various methods to construct a profile to characterize cellular and molecular features and systematically analyze the related GO and KEGG profile of cells of all stages from the oocyte to the blastocyst. This large-scale, single-cell atlas provides key cellular information and will likely assist clinical studies in improving preimplantation genetic diagnosis.

Copy number variation of the ZNF679 gene in cattle and its association analysis with growth traits.

Copy number variation (CNV) is an important member of genetic structural variation that exists widely in animal genomes and is between 50 bp and several Mb in length and widely used in research's of animal genetics and breeding. ZNF679 is an important transcription factor, which has been found association with diseases in the human genome many times. This gene has also been found to be associated with cattle growth traits in previous re-sequencing studies. We tested the CNVs of the ZNF679 gene in 809 individuals from 7 Chinese cattle breeds and tested the association between the CNVs and growth traits in 552 individuals from 5 breeds. The results demonstrated the correlation the correlation between the CNVs of the ZNF679 gene and some Chinese cattle (QC cattle and XN cattle) growth traits. To sum up, this study indicated that ZNF679-CNVs can be used as a candidate gene for molecular genetic marker-assisted selection breeding for cattle growth traits to contribute to the development of genetic improvement of Chinese cattle.

CNV analysis of VAMP7 gene reveals variation associated with growth traits in Chinese cattle.

Copy number variant (CNV), a common genetic polymorphism, is closely related to the phenotypic variation traits of organisms. Vesicle-associated membrane protein 7 gene (VAMP7) codes a protein, which is a member of the SNARE proteins family and plays an important role in the process of intracellular vesicle transport. In this study, a total of four cattle breeds (Yunling cattle, Xianan cattle, Pinan cattle, Jiaxian red cattle) were used to investigate the copy numbers, and we found an association relationship between CNV of VAMP7 gene and growth traits of cattle by SPSS 20.0 software. The results showed that the CNV type of VAMP7 gene in four cattle breeds had the same distribution, Duplication type occupies a dominant position among the four varieties. In Yunling cattle, the Duplication type of VAMP7 is significantly related to the height at the hip cross (p < 0.05), Individuals with Duplication type commonly have less performance on growth and development, which indicates that the Duplication type of the VAMP7 gene may have a negative effect on cattle growth. Individuals with the other two CNV types may become the breeding direction of the VAMP7 gene. This study provided a new perspective and basic material for the molecular genetics of the CNV of the VAMP7 gene, and also promoted the breeding progress of Chinese local cattle.

Bta-miR-200b promotes endometrial epithelial cell apoptosis by targeting MYB in cattle.

Embryonic mortality is considered to be one of the main reasons for reduced conception rates in the cattle industry. Insufficient endometrial receptivity is a major factor resulting in embryo implantation failure and losses. Apoptosis of endometrial epithelial cells is an important process during establishment of uterine receptivity and embryo implantation. The aim of this study was to explore the role of bta-miR-200b on endometrial epithelial cell apoptosis in cattle. Overexpression of bta-miR-200b upregulated the expression of proapoptotic gene BCL2 associated X, apoptosis regulator (BAX) and endometrial receptivity marker gene osteopontin (OPN) at mRNA and protein level in bovine endometrial epithelial cells. Moreover, overexpression of bta-miR-200b was able to inhibit proliferation and promote apoptosis of bovine endometrial epithelial cells by arresting the cell cycle at the G0/G1 phase. MYB Proto-Oncogene (MYB) was verified to be a target of bta-miR-200b in bovine endometrial epithelial cells using dual-luciferase reporter assay. Transfection of bta-miR-200b mimics decreased the mRNA and protein expression of MYB. Overexpression of MYB decreased the effect of bta-miR-200b on apoptosis of bovine endometrial epithelial cells. Our findings suggest that bta-miR-200b can affect the apoptosis of endometrial epithelial cells in cattle by targeting the MYB gene.

Copy number variation of EIF4A2 loci related to phenotypic traits in Chinese cattle.

BACKGROUND: Generally, copy number variation (CNV) is a large-scale structural variation between 50 bp and 1 kb of the genome. It can affect gene expression and is an important reason for genetic diversity and phenotypic trait diversity. Studies have shown that the eukaryotic translation initiation factor 4A2 (EIF4A2) gene plays an essential role in muscle development in both humans and pigs. However, the influence of bovine EIF4A2's copy number change on phenotypic traits has not been reported. OBJECTIVES: To detect the tissue expression profile of the EIF4A2 gene in adult cattle and individuals' CNV type of variation. Then, we explored the correlation between EIF4A2-CNV and growth traits in Chinese cattle breeds. METHODS: Real-time fluorescent quantitative reverse transcription PCR (qRT-qPCR) was used to determine the expression profile of the EIF4A2 gene. Real-time fluorescent quantitative PCR (qPCR) was used to detect the CNV type of bovine populations. Then, SPSS 26.0 was used for association analysis. RESULTS: In this study, a total of 513 individuals in four cattle breeds (Qinchuan cattle [QC], Yunling cattle [YL], Pinan cattle [PN] and Jiaxian cattle [JX]) were detected for EIF4A2 gene's CNV. The results showed that EIF4A2-CNV has an essential impact on hip width (HW) and rump length (RL) in QC, heart girth (HG), chest depth (CD) and RL in YL and HW in PN. However, it had no significant effect on JX. CONCLUSIONS: The above results suggest that EIF4A2 gene's CNV can be used as a molecular marker for cattle breeding, which is helpful to accelerate the breeding of superior beef cattle breeds.

Assessing genomic diversity and signatures of selection in Pinan cattle using whole-genome sequencing data.

BACKGROUND: Crossbreeding is an important way to improve production beef cattle performance. Pinan cattle is a new hybrid cattle obtained from crossing Piedmontese bulls with Nanyang cows. After more than 30 years of cross-breeding, Pinan cattle show a variety of excellent characteristics, including fast growth, early onset of puberty, and good meat quality. In this study, we analyzed the genetic diversity, population structure, and genomic region under the selection of Pinan cattle based on whole-genome sequencing data of 30 Pinan cattle and 169 published cattle genomic data worldwide.  RESULTS: Estimating ancestry composition analysis showed that the composition proportions for our Pinan cattle were mainly Piedmontese and a small amount of Nanyang cattle. The analyses of nucleotide diversity and linkage disequilibrium decay indicated that the genomic diversity of Pinan cattle was higher than that of European cattle and lower than that of Chinese indigenous cattle. De-correlated composite of multiple selection signals, which combines four different statistics including θπ, CLR, FST, and XP-EHH, was computed to detect the signatures of selection in the Pinan cattle genome. A total of 83 genes were identified, affecting many economically important traits. Functional annotation revealed that these selected genes were related to immune (BOLA-DQA2, BOLA-DQB, LSM14A, SEC13, and NAALADL2), growth traits (CYP4A11, RPL26, and MYH10), embryo development (REV3L, NT5E, CDX2, KDM6B, and ADAMTS9), hornless traits (C1H21orf62), and climate adaptation (ANTXR2). CONCLUSION: In this paper, we elucidated the genomic characteristics, ancestry composition, and selective signals related to important economic traits in Pinan cattle. These results will provide the basis for further genetic improvement of Pinan cattle and reference for other hybrid cattle related studies.

Copy number variation of the CCDC39 gene is associated with growth traits in Chinese cattle.

BACKGROUND: Copy number variation (CNV) has become an essential part of genetic structural variation. Coiled-coil domain containing 39 (CCDC39) is a gene that related to the growth and development of organs and tissues. It is identified that it has a CNV region by animal genome resequencing. OBJECTIVE: In this study, we detected the phenotypic traits and different distributions of CCDC39 gene copy numbers in five Chinese cattle breeds (Qinchuan (QC) cattle, Yunling (YL) cattle, Xianan (XN) cattle, Pinan (PN) cattle and Jiaxian (JX) cattle). METHODS: Five hundred and six cattle were randomly selected for CNV distribution detection. Blood samples were taken and genomic DNA was extracted. Different tissues were obtained from adult (n = 3) XN cattle, including heart, liver, kidney, skeletal muscle and lung. The genome qPCR experiment was performed with SYBR Green in triplicate. CDNA qPCR was used to detect the expression level of CCDC39 in different tissues and varieties. Using SPSS v20.0 software, the relationship between CCDC39 CNV and the growth traits of PN, XN, QC, NY and YL cattle breeds was analyzed by one-way analysis of variance (ANOVA). RESULTS: The results showed that the expression of CCDC39 in lung was higher than that in other tissues. The expression in liver and kidney was similar, but the expression in heart and muscle was less. It can be seen that the duplication type of QC cattle CCDC39 CNV is higher than the deletion or normal in the height at hip cross. The normal type of PN cattle in body length and hip width was better than duplication and deletion (p < 0.05). In XN cattle, the deletion type of CNV had superior growth characteristics in heart girth and cannon bone circumference compared with the duplication type and the normal type (p < 0.05). CONCLUSION: The study revealed a significant association between CNV of CCDC39 gene and growth traits in different Chinese cattle breeds.

The relationship between MFN1 copy number variation and growth traits of beef cattle.

Copy number variation, as a kind of genetic submicroscopic structural variation, refers to the deletion or repetition of a large segment of genomic DNA, involving a segment size ranging from 50 bp to several MB. Mitochondrial fusion protein (MFN1) gene regulates the fusion of mitochondrial outer membrane in cells and maintains the dynamic needs of reticular mitochondria in cells. In this study, we conducted to tested the dstribution characteristics of MFN1-CNV in 522 cattles across Xianan cattle (XN), Pinan cattle (PN), Qinchuan cattle (QC), Jiaxian cattle (JX), Yunling cattle (YL), and correlated it with phenotypic traits. Then we observed the expression of MFN1 in various tissues of QC cattle (n = 3), and the expression levels were higher in lung and muscle. The results showed that there was significant correlation between MFN1 gene CNV and hucklebone width of QC cattle, hip width and height at sacrum of JX red cattle, chest width and rump length of YL cattle (P < 0.05). Individuals with duplication type were better than the type of normal or deletion in phenotypic traits. In conclusion, our data showed the correlation between MFN1 gene and growth traits of Chinese cattle. MFN1 gene can be used as a molecular marker for cattle selection and breeding, and accelerate the improvement of Chinese cattle.

miRNA profiling in intrauterine exosomes of pregnant cattle on day 7.

Intrauterine exosomes have been identified to be involved in the embryo development and implantation. The aim of this study was to explore the role of miRNAs in intrauterine exosomes in bovine pregnancy. Intrauterine exosomes were collected from uterine flushing fluids of three donor and three recipient Xianan cows 7 days after fertilization. Intrauterine exosomes miRNAs were extracted and the exosomal miRNAs expression levels were analyzed. Sixty miRNAs differed significantly in their amounts between donors and recipients (p-value < 0.05, |log2(FoldChange)| > 1). Twenty-two miRNAs were upregulated and 38 downregulated in the group of donor cows. The bta-miR-184 was the most significant (P Benjamini-Hochberg < 0.001). A total of 9,775 target genes were predicted using the 60 miRNAs. GO and KEGG analysis showed that the target genes were enriched in several biological processes or pathways associated with embryo implantation and endometrial development, such as cell adhesion, cell junction, focal adhesion, and Rap1 signaling pathway. Our findings suggest that, in cattle early pregnancy stage, these differently expressed miRNAs in intrauterine exosomes involved in embryo implantation and endometrial development, which may exert a significant effect and influence the uterine microenvironment for embryo implantation. These results could provide reference for screening and exploring the intrauterine exosomal miRNA affecting embryo implantation.

Copy number variation of the USP16 gene and its association with milk traits in Chinese Holstein cattle.

Copy number variations (CNVs) were similar to single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel), regarded as genetic variations in many species. CNV is defined as the variable change of DNA segment length compared with the reference genome, including gains or losses from 50 bp to several mega bases. The functions of USP16 gene are diverse, such as regulating the cell cycle, DNA damage, histone H2A deubiquitination or mitotic nuclear division. To analyze the relationship between CNV of USP16 gene and milk traits in Chinese Holstein, we used qPCR to detect the individuals of Chinese Holstein (n = 180). The results showed that the effect of USP16 gene CNV on daily milk yield and fat percentage had significant difference (p < 0.05). The gain was the advantage type in daily milk yield and the loss was the advantage type in fat percentage. Therefore, CNV of USP16 gene is an important factor of milk traits in Chinese Holstein. Meanwhile, it may be used as a molecular marker for assisted selection of milk traits in Chinese Holstein, which provides a theoretical basis for the genetic improvement of cow breeds in China.

A molecular marker of milk composition traits in NCAM2 gene of Chinese Holstein.

The aim of this study was to detect the novel copy number variation (CNV) locus of NCAM2 gene in Chinese Holstein, and to analyze the effect of the novel CNV locus in NCAM2 gene on milk composition traits. The novel CNV locus of NCAM2 gene in 310 Chinese Holstein was detected by real-time quantitative fluorescent PCR (qPCR) and association analysis was performed between the novel CNV locus in NCAM2 gene and milk composition traits in Chinese Holstein. There are three CNV types of NCAM2 gene in Chinese Holstein: gain (increased copy number), median (normal copy number) and loss (deleted copy number). Statistical analysis revealed that there was a significant association between CNV types and milk fat rate (p < 0.05). Moreover, we also discovered that the milk production and milk protein rate of gain type is higher than that of loss type, but that of mediate type is lower than that of loss type. However, in terms of somatic cell score, loss type is higher than that of gain type, but that of mediate type is lower than that of gain type. These observations suggested that gain type can be used as a candidate molecular genetic marker of milk fat rate.HighlightsThe CNVs of the NCAM2 gene were detected and validated in Chinese Holstein.The type of CNV was successfully implemented using qPCR.The statistical analysis indicated that the CNV of the NCAM2 gene are significantly associated with milk fat rate.

Effect of copy number variation of PLA2G2A gene to growth traits in Chinese cattle.

SIMPLE SUMMARY: As a member of genetic polymorphism, copy number variation has been a commonly used method in the world for investigating effect of genetic polymorphism on gene expression. Effect of genetic polymorphism made on livestock development has been more and more important in beef cattle molecular breeding. The characteristics of Chinese cattle are excellent meat quality, tolerant to rough feeding, good environmental adaptability and so on. But there are some obvious weaknesses still exist in the process of cattle growth and development, such as weak hindquarters and growth slowly. To improve the growth performance and market competitiveness of Chinese cattle, a lot of studies have been made about finding and investigating effective molecular marker. In this study, Q-PCR and data association analysis were used for PLA2G2A gene copy number variation detection and related effect analysis in Chinese cattle. Results showed that PLA2G2A gene has a significant effect on two breeds of Chinese cattle on growth traits, which could be a basic materials and effective information of cattle molecular markers breeding. PLA2G2A, member of secreted phospholipases A2 (sPLA2) in superfamily of phospholipase A2, could catalyze the process of glycerophospholipids hydrolysis from position of sn-2 acyl with the release of free fatty acids and lysophospholipids. Researches about PLA2G2A gene are mostly focus on disease, including tumors and diabetes, the number of study occurred on animal breeding is weak. 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 PLA2G2A 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. In QC, XN and GY cattle, the frequencies of Deletion and Duplication are about 40%; in YL cattle, the frequency of Deletion type exceeds 60%; in PN cattle, the frequency of Duplication is closed to 80%. Association analysis indicate that CNV of PLA2G2A gene showed a positive effect in cattle growth: in QC cattle, Chest depth with Normal type copy number possess a increased trend (P < 0.05); individuals with Deletion type copy number have better performance on Height at sacrum, Heart girth and Body height in GY cattle (P < 0.05). The functional role and molecular mechanism of PLA2G2A gene in animal growth and development are still unclear, and it is necessary for processing a further research. This research aims to provide basic materials for molecular breeding of Chinese cattle.

Assessing genomic diversity and signatures of selection in Jiaxian Red cattle using whole-genome sequencing data.

BACKGROUND: Native cattle breeds are an important source of genetic variation because they might carry alleles that enable them to adapt to local environment and tough feeding conditions. Jiaxian Red, a Chinese native cattle breed, is reported to have originated from crossbreeding between taurine and indicine cattle; their history as a draft and meat animal dates back at least 30 years. Using whole-genome sequencing (WGS) data of 30 animals from the core breeding farm, we investigated the genetic diversity, population structure and genomic regions under selection of Jiaxian Red cattle. Furthermore, we used 131 published genomes of world-wide cattle to characterize the genomic variation of Jiaxian Red cattle. RESULTS: The population structure analysis revealed that Jiaxian Red cattle harboured the ancestry with East Asian taurine (0.493), Chinese indicine (0.379), European taurine (0.095) and Indian indicine (0.033). Three methods (nucleotide diversity, linkage disequilibrium decay and runs of homozygosity) implied the relatively high genomic diversity in Jiaxian Red cattle. We used θπ, CLR, FST and XP-EHH methods to look for the candidate signatures of positive selection in Jiaxian Red cattle. A total number of 171 (θπ and CLR) and 17 (FST and XP-EHH) shared genes were identified using different detection strategies. Functional annotation analysis revealed that these genes are potentially responsible for growth and feed efficiency (CCSER1), meat quality traits (ROCK2, PPP1R12A, CYB5R4, EYA3, PHACTR1), fertility (RFX4, SRD5A2) and immune system response (SLAMF1, CD84 and SLAMF6). CONCLUSION: We provide a comprehensive overview of sequence variations in Jiaxian Red cattle genomes. Selection signatures were detected in genomic regions that are possibly related to economically important traits in Jiaxian Red cattle. We observed a high level of genomic diversity and low inbreeding in Jiaxian Red cattle. These results provide a basis for further resource protection and breeding improvement of this breed.

Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3'UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

Genetic effects of MOGAT1 gene SNP in growth traits of Chinese cattle.

Single nucleotide polymorphism (SNP) has recently become one of the ideal genetic markers. SNP refers to the DNA sequence polymorphism caused by double nucleotide variation in the genome, including the conversion or transversion of segmented bases. The synthesis and metabolism of triglycerides are related to the changes of energy in the body of livestock, which in turn affects their growth and development. Studies have shown that MOGAT1 gene plays a role in the route of triglyceride synthesis. PCR-RFLP and agarose gel electrophoresis technology were used to type the SNP site of MOGAT1 gene at g.25940T > C in this study. Association analysis between typing results and growth trait data was detected by SPSS 20.0 software. Results show that MOGAT1 gene was in a low level of heterozygosity in Xianan, Qinchuan and Pinan cattle population (0 < PIC < 0.25), and in middle level of heterozygosity in YL cattle population(0.25 < PIC < 0.5). And genotype 'AA' was dominant gene in Chinese cattle population. In QC and XN cattle, genotype of GG possess advantage on Body weight (P < 0.05); in YL cattle, individuals with genotype of homozygous mutation decreased significantly on Chest depth (P < 0.05). The purpose of this research is to provide theoretical materials for molecular breeding of yellow cattle and to promote the process of improving the growth traits of Chinese local yellow cattle.

Identification of key bacterial populations affecting early embryonic development in cattle uterus.

Superovulation is an important animal breeding biotechnology, while the quality of embryos obtained from superovulation is unstable in cattle. The relationship between the microorganisms in the cattle uterus and embryo qualities was determined to identify the key bacterial populations affecting early embryonic development. A total of 10 Xia Nan cows underwent superovulation, we collected cervical mucus and flush samples to investigated by 16S rDNA sequencing. Results showed that there were abundant microorganisms in cervical mucus, but no obvious relationship with the quality of embryos. The clustering results of flush samples were consistent with the grouping of embryo quality. Proteobacteria accounted for more than 95% of the total bacterial community in group A with the best embryo quality (qualified embryo ratio above 0.8), and as embryo quality decreased, the Proteobacteria proportion also decreased. In contrast to the proportion of Proteobacteria, the proportions of Firmicutes and Bacteroidetes significantly increased as embryo quality decreased. For group C with the worst embryo quality, the proportions of Firmicutes and Bacteroidetes increased to 4.7 times and 12.3 times of group A, respectively. These results showed that the quantities and proportions of Firmicutes and Bacteroidetes may be related to early embryonic development in cattle.

Association Analysis to Copy Number Variation (CNV) of Opn4 Gene with Growth Traits of Goats.

Extensive research has been carried out regarding the correlation between the growth traits of livestock and genetic polymorphisms, including single nucleotide polymorphisms and copy number variations (CNV). The purpose of this study was to analyze the CNV and its genetic effects of the Opn4 gene in 284 Guizhou goats (Guizhou black goat: n = 186, Guizhou white goat: n = 98). We used qPCR to detect the CNV of the Opn4 gene in Guizhou goats, and the classification results were correlated with the corresponding individual growth traits by SPSS software. The results showed that the Opn4 gene had a superior effect on growth traits with multiple copy variants in Guizhou black goats, and there was a significant correlation between copy number variation sites and body length traits. Contrary to the former conclusion, in Guizhou white goats, individuals with the Normal copy number type showed superior growth traits and copy number variant sites were significantly associated with body weight traits. Therefore, the CNV of the Opn4 gene can be used as a candidate molecular genetic marker to improve goat growth traits, speeding up the breeding process of goat elite varieties.