CFAP58 is involved in the sperm head shaping and flagellogenesis of cattle and mice.

CFAP58 is a testis-enriched gene that plays an important role in the sperm flagellogenesis of humans and mice. However, the effect of CFAP58 on bull semen quality and the underlying molecular mechanisms involved in spermatogenesis remain unknown. Here, we identified two single-nucleotide polymorphisms (rs110610797, A>G and rs133760846, G>T) and one indel (g.-1811_ g.-1810 ins147bp) in the promoter of CFAP58 that were significantly associated with semen quality of bulls, including sperm deformity rate and ejaculate volume. Moreover, by generating gene knockout mice, we found for the first time that the loss of Cfap58 not only causes severe defects in the sperm tail, but also affects the manchette structure, resulting in abnormal sperm head shaping. Cfap58 deficiency causes an increase in spermatozoa apoptosis. Further experiments confirmed that CFAP58 interacts with IFT88 and CCDC42. Moreover, it may be a transported cargo protein that plays a role in stabilizing other cargo proteins, such as CCDC42, in the intra-manchette transport/intra-flagellar transport pathway. Collectively, our findings reveal that CFAP58 is required for spermatogenesis and provide genetic markers for evaluating semen quality in cattle.

Introgression, admixture, and selection facilitate genetic adaptation to high-altitude environments in cattle.

Domestication and subsequent selection of cattle to form breeds and biological types that can adapt to different environments partitioned ancestral genetic diversity into distinct modern lineages. Genome-wide selection particularly for adaptation to extreme environments left detectable signatures genome-wide. We used high-density genotype data for 42 cattle breeds and identified the influence of Bos grunniens and Bos javanicus on the formation of Chinese indicine breeds that led to their divergence from India-origin zebu. We also found evidence for introgression, admixture, and migration in most of the Chinese breeds. Selection signature analyses between high-altitude (≥1800 m) and low-altitude adapted breeds (<1500 m) revealed candidate genes (ACSS2, ALDOC, EPAS1, EGLN1, NUCB2) and pathways that are putatively involved in hypoxia adaptation. Immunohistochemical, real-time PCR and CRISPR/cas9 ACSS2-knockout analyses suggest that the up-regulation of ACSS2 expression in the liver promotes the metabolic adaptation of cells to hypoxia via the hypoxia-inducible factor pathway. High altitude adaptation involved the introgression of alleles from high-altitude adapted yaks into Chinese Bos taurus taurus prior to their formation into recognized breeds and followed by selection. In addition to selection, adaptation to high altitude environments has been facilitated by admixture and introgression with locally adapted cattle populations.

Genome-wide methylation and transcriptome of blood neutrophils reveal the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-infected mastitis cows.

BACKGROUND: Neutrophils are the first effectors of inflammatory response triggered by mastitis infection, and are important defense cells against pathogenic Escherichia coli (E. coli). DNA methylation, as a critical epigenetic mechanism for regulating gene function, is involved in bovine mastitis. RESULTS: In this study, we sequenced the blood neutrophils of healthy and E. coli-infected mastitic half-sib cows for the overall DNA methylation levels using transcriptome sequencing and reduced representation bisulfite sequencing. The methylation levels in the mastitis cows (MCs) were decreased compared with healthy cows (HCs). A total of 494 differentially methylated regions were identified, among which 61 were up-methylated and 433 were down-methylated (MCs vs. HCs). The expression levels of 1094 differentially expressed genes were up-regulated, and 245 genes were down-regulated. Twenty-nine genes were found in methylation and transcription data, among which seven genes' promoter methylation levels were negatively correlated with expression levels, and 11 genes were differentially methylated in the exon regions. The bisulfite sequencing PCR and quantitative real-time PCR validation results demonstrated that the promoter methylation of CITED2 and SLC40A1 genes affected differential expression. The methylation of LGR4 exon 5 regulated its own alternative splicing. The promoter methylation of bta-miR-15a has an indirect effect on the expression of its target gene CD163. The CITED2, SLC40A1, and LGR4 genes can be used as candidates for E. coli-induced mastitis resistance. CONCLUSIONS: This study explored the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-induced mastitis, thereby helping explain the function of DNA methylation in the pathogenesis of mastitis and provided new target genes and epigenetic markers for mastitis resistance breeding in dairy cattle.

In silico genome-wide miRNA-QTL-SNPs analyses identify a functional SNP associated with mastitis in Holsteins.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) and their target binding sites affect miRNA function and are involved in biological processes and diseases, including bovine mastitis, a frequent inflammatory disease. Our previous study has shown that bta-miR-2899 is significantly upregulated in the mammary gland tissue of mastitis-infected cow than that of healthy cows. RESULTS: In the present study, we used a customized miRNAQTLsnp software and identified 5252 SNPs in 691 bovine pre-miRNAs, which are also located within the quantitative trait loci (QTLs) that are associated with mastitis and udder conformation-related traits. Using luciferase assay in the bovine mammary epithelial cells, we confirmed a candidate SNP (rs109462250, g. 42,198,087 G > A) in the seed region of bta-miR-2899 located in the somatic cell score (SCS)-related QTL (Chr.18: 33.9-43.9 Mbp), which affected the interaction of bta-miR-2899 and its putative target Spi-1 proto-oncogene (SPI1), a pivotal regulator in the innate and adaptive immune systems. Quantitative real-time polymerase chain reaction results showed that the relative expression of SPI1 in the mammary gland of AA genotype cows was significantly higher than that of GG genotype cows. The SNP genotypes were associated with SCS in Holstein cows. CONCLUSIONS: Altogether, miRNA-related SNPs, which influence the susceptibility to mastitis, are one of the plausible mechanisms underlying mastitis via modulating the interaction of miRNAs and immune-related genes. These miRNA-QTL-SNPs, such as the SNP (rs109462250) of bta-miR-2899 may have implication for the mastitis resistance breeding program in Holstein cattle.

Identification of bta-miR-15a∼16a cluster expression, localization and regulated target in Holsteins.

Bovine mastitis is an inflammation response of the mammary gland tissues caused mainly by pathogenic bacteria in cows. Previous studies showed that bta-miR-15a and bta-miR-16a modulate immunity and inflammation responses. In this study, we investigated the expression pattern and tissue localization of bta-miR-15a and bta-miR-16a. The expression levels of bta-miR-15a and bta-miR-16a were significantly upregulated in mammary tissues and blood neutrophils of mastitis-infected cows, compared with those of healthy cows (P < 0.05). Through in situ hybridization, we examined the tissue localization of bta-miR-15a and bta-miR-16a and found that they were expressed in the ductal and acinar cells of mammary gland tissues, where they had a stronger expression signal in the mammary tissues of cows with mastitis than that in healthy cows' tissues. Moreover, we identified CD163 as the target gene of bta-miR-15a and bta-miR-16a. Luciferase assay indicated that bta-miR-15a, bta-miR-16a, and bta-miR-15a∼16a cluster led to the significant reduction in the luciferase activity of CD163 3'UTR vector (P < 0.05). Meanwhile, the luciferase activity had a significantly low value compared with that of single bta-miR-15a or bta-miR-16a plasmid (P < 0.05) in the presence of bta-miR-15a∼16a cluster. The bta-miR-15a∼16a cluster may function more effectively in inhibiting luciferase reporter gene activity of target gene CD163 than single miRNA. Our study provides an insight into the relationship between bovine mastitis and gene expression of bta-miR-15a/16a, which suggested that bta-miR-15a∼16a cluster may play a role against mastitis by binding to target CD163 gene in Holstein dairy cattle.

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis.

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows' mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down-regulation and location of bta-miR-26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real-time PCR, in situ hybridization and dual-luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta-miR-26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.

Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls.

RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (P<0.05). In addition, initial sperm motility was significantly higher in individuals with the GG or GA genotype than in individuals with the AA genotype (P<0.05). The findings of the present study suggest that the difference in semen quality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

Characterization of complete mitochondrial genome of Dezhou donkey (Equus asinus) and evolutionary analysis.

Mitochondrial DNA (mtDNA) has been widely used in species identification and genetic diversification. Comparisons among mtDNAs of closely related species are valuable for phylogenetic studies. However, only the partial mtDNA Cytb gene and the D-loop sequences were used to analysis the phylogenetic relationship between donkey breeds due to lack of complete mitochondrial genome. Dezhou donkey, as a bigger somatotype ass, is one of Chinese domestic donkey breeds, and used by many places as breeding stock. To further investigate the phylogenetic relationship of Dezhou donkey with other breeds, the complete mtDNA was firstly sequenced and de novo assembled using next generation sequence data from total genomic DNA. The genome was 16,813 bp in length (NCBI submission number: KT182635) and contained 13 protein coding genes, 2 ribosomal RNA genes, 25 transfer RNA genes, and 1 control region. Based on the novel complete mtDNA sequence, the sequences of 13 protein coding genes and 2 ribosomal RNA genes were amplifying in other 2 Dezhou donkeys and 3 Yunnan donkeys, respectively. The pattern of genetic variation in horse, wild ass and domestic donkeys among these 15 genes indicated the sequence polymorphisms. The more accurate phylogenetic relationships of donkey species (Dezhou donkey, Yunnan donkey and previously published donkeys) were first obtained using the combined sequences of 12S rRNA+16S rRNA+13 protein-coding genes. Molecular-based phylogeny supported the hypothesis that Chinese domestic donkey breeds may have originated from Somali wild ass, not from Asian wild ass by analyzing mitochondrial genomes.

A g.-1256 A>C in the promoter region of CAPN1 is associated with semen quality traits in Chinese Holstein bulls.

The micromolar calcium-activated neutral protease gene (CAPN1) is a physiological candidate gene for sperm motility. However, the molecular mechanisms involved in regulating the expression of the CAPN1 gene in bulls remain unknown. In this study, we investigated the expression pattern of CAPN1 in testis, epididymis, and sperm at the RNA and protein levels by qRT-PCR, western blot, immunohistochemistry, and immunofluorescence assay. Results revealed that the expression of CAPN1 levels was higher in the sperm head compared with that in other tissues. Moreover, we identified a novel single-nucleotide polymorphism (g.-1256 A>C, ss 1917715340) in the noncanonical core promoter of the CAPN1 gene between base g.-1306 and g.-1012. Additionally, we observed greater sperm motility in bulls with the genotype CC than in those with the genotype AA (P<0.01), indicating that different genotypes were associated with the bovine semen trait. Furthermore, a higher fluorescence intensity of the C allele than that of the A allele at g. -1256 A>C was revealed by transient transfection in MLTC-1 cells and luciferase report assay. Finally, CAPN1 was highly expressed in the spermatozoa with the CC genotype compared with that with the AA genotype by qRT-PCR. This study is the first report on genetic variant g.-1256 A>C in the promoter region of CAPN1 gene association with the semen quality of Chinese Holstein bulls by influencing its expression. g.-1256 A>C can be a functional molecular marker in cattle breeding.

PCK1 is negatively regulated by bta-miR-26a, and a single-nucleotide polymorphism in the 3' untranslated region is involved in semen quality and longevity of Holstein bulls.

Phosphoenolpyruvate carboxykinase 1 (PCK1) is a multi-functional enzyme that plays important roles in physiological processes, including reproduction. We previously reported that the PCK1 transcript has five splice variants; PCK1-AS4, which lacks exon 5, is enriched in the testis of Holstein bulls. In the present study, we profiled select PCK1 transcript variants in the testis, epididymus, and semen of high- and low-performance bulls, and examined the possibility that microRNAs may be involved in single nucleotide polymorphism (SNP)-mediated modulation of PCK1 expression. PCK1-AS4 abundance is not significantly different between high- and low-performance bulls. Luciferase reporter assays, however, showed that bovine PCK1 expression is repressed by bta-miR-26a in HepG2 hepatocyte cells. One SNP (c. + 2183 G > T) at the miRNA-binding site of PCK1 does not influence PCK1 expression, but is associated with elevated ejaculation volume, fresh sperm motility, and genomic estimated breeding value of longevity, as well as with reduced values of composite index and calving ease. Collectively, the identified 3'-untranslated-region SNP variant highlights the importance of PCK1 in the fecundity of Holstein bulls, and implicates a role for bta-miR-26a in regulating PCK1 abundance. Further study is needed to assess the effects of other genetic variants in 5'-flanking region and exons of PCK1 on enzyme levels in the testis and sperm. Mol. Reprod. Dev. 83: 217-225, 2016. © 2016 Wiley Periodicals, Inc.

TNP1 Functional SNPs in bta-miR-532 and bta-miR-204 Target Sites Are Associated with Semen Quality Traits in Chinese Holstein Bulls.

Transition nuclear proteins (TNPs), major proteins found in the chromatin of condensing spermatids, have been implicated in spermatogenesis and male fertility. In this study, DNA samples were collected from 404 Chinese Holstein bulls and sequenced to identify genetic variants in the 3'-untranslated region (UTR) of TNP1 and to investigate genetic variations in the TNP1 gene and their common haplotypes. This study was also conducted to determine whether these variations affect bovine semen quality traits and expression levels by PCR-restriction fragment length polymorphism, bioinformatics analyses, quantitative real-time PCR (qPCR), and fluorescence assay. Results showed that one new single-nucleotide polymorphism (SNP; g. 528 G>A, ss1388116558) and one reported SNP (g. 442 A>G, rs110469441) were found in the 3'-UTR of the TNP1 gene. Bioinformatics analysis results revealed that both loci were located in bta-miR-532-binding and bta-miR-204-binding regions, respectively. Association studies revealed that bulls with H1H1 (AGAG) and H1H3 (AGGG) haplotype combinations exhibited a lower deformity rate than those with other haplotype combinations (P < 0.05). The qPCR results showed that the relative mRNA expression of TNP1 in bulls with H1H1 haplotype combination was significantly higher than that in bulls with H4H4 haplotype combination (P < 0.05). MicroRNA qPCR results suggested that bta-miR-532 expression was downregulated by 5-fold in adult bull testicular tissues compared with that in fetal bull testicular tissues; by contrast, bta-miR-204 expression was downregulated by 1.6-fold. Luciferase assay results also indicated that TNP1 expression was directly targeted by bta-miR-532 and bta-miR-204 in murine Leydig tumor cell lines. These results provide the first indication of g. 442 A>G-mediated and g. 528 G>A-mediated translational suppression in which SNPs altered the binding of bta-miR-204 and bta-miR-532 to the 3'-UTR of TNP1; the mediated translational suppression could be involved in the regulation of TNP1 expression and may influence the morphological characteristics of Chinese Holstein bull sperm. We propose that SNPs on the TNP1 3'-UTR may help select semen quality trait in Chinese Holstein bulls in the dairy industry.

A SNP in intron 8 of CD46 causes a novel transcript associated with mastitis in Holsteins.

BACKGROUND: The membrane protein CD46, a ubiquitous cell surface pathogen receptor, can bind Streptococcus to trigger cell autophagy, which is a critical step in the control of infection. RESULTS: In this study, we found a new splice variant designated CD46 transcript variant (CD46-TV). The splice variant is characterized by the retention of a 48 bp sequence from intron 8 of the bovine CD46 gene, which encodes a putative protein enlarged by 16 amino acids. CD46-TV mRNA was found to be over expressed in mastitis-infected mammary gland tissues relative to healthy tissues. A single nucleotide polymorphism (c. 1033 + 2184 C > T) in the exonic splicing enhancer (ESE) motif region was shown to result in the CD46-TV aberrant splice variant through constructing alternative alleles using the pSPL3 exon capturing vector and transfecting these into 293 T cells. Allelic frequency in 56,682 individuals belonging to 112 Bos taurus, Bos indicus, Bos javanicus, Bos grunniens and Bos mutus, etc. suggests that the C allele (80.09%) is the ancestral allele. Association analysis found that the mean genomic estimated breeding values (gEBV) for milk somatic cell score and the occurrence of clinical mastitis, as well as the milk somatic cell score of Chinese Holsteins with the CT genotype was lower than those of individuals with either the CC or TT genotypes. The mean gEBV for udder health synthesis for the TT genotype was greater than those for the CC or CT genotypes. CONCLUSIONS: Our findings suggest that the CD46 gene likely plays a critical role in the risk of mastitis caused by Streptococcus in dairy cows via an alternative splicing mechanism caused by a functional mutation in intron 8. Our data also underline the importance of variation within ESEs in regulating transcript processing.

iTRAQ-proteomics and bioinformatics analyses of mammary tissue from cows with clinical mastitis due to natural infection with Staphylococci aureus.

BACKGROUND: Proteomics and bioinformatics may help us better understand the biological adaptations occurring during bovine mastitis. This systems approach also could help identify biomarkers for monitoring clinical and subclinical mastitis. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to screen potential proteins associated with mastitis at late infectious stage. RESULTS: Healthy and mastitic cows' mammary gland tissues were analyzed using iTRAQ combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Bioinformatics analyses of differentially expressed proteins were performed by means of Gene Ontology, metabolic pathways, transcriptional regulation networks using Blast2GO software, the Dynamic Impact Approach and Ingenuity Pathway Analysis. At a false discovery rate of 5%, a total of 768 proteins were identified from 6,499 peptides, which were matched with 15,879 spectra. Compared with healthy mammary gland tissue, 36 proteins were significantly up-regulated (>1.5-fold) while 19 were significantly down-regulated (<0.67-fold) in response to mastitis due to natural infections with Staphylococci aureus. Up-regulation of collagen, type I, alpha 1 (COL1A1) and inter-alpha (Globulin) inhibitor H4 (ITIH4) in the mastitis-infected tissue was confirmed by Western blotting and Immunohistochemistry. CONCLUSION: This paper is the first to show the protein expression in the late response to a mastitic pathogen, thus, revealing mechanisms associated with host tissue damage. The bioinformatics analyses highlighted the effects of mastitis on proteins such as collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan 2. Our findings provide additional clues for further studies of candidate genes for mastitis susceptibility. The up-regulated expression of COL1A1 and ITIH4 in the mastitic mammary gland may be associated with tissue damage and repair during late stages of infection.

Study on genetic variations of PPARα gene and its effects on thermal tolerance in Chinese Holstein.

Peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. In this research, polymerase chain reaction (PCR) technique was used to amplify 766 and 589 bp fragments of intron 3 and 7 of PPARα gene in Chinese Holstein (n = 771). Sequencing results showed that three novel single nucleotide polymorphisms (SNPs) were identified at position 44087 (G/A), 65550 (G/A), and 65676(G/A) in the PPARα gene. PCR-restriction fragment length polymorphism technology was used to genotype the three SNPs. Association analysis showed that cows with H1H8 (P < 0.05), H2H8 (P < 0.01), H5H7 (P < 0.05), H5H8 (P < 0.05), and H8H8 (P < 0.05) haplotype combinations had lower potassium content in erythrocytes than those with H2H6 haplotype combination. Cows with H1H8, and H8H8 haplotype combinations had lower decrease rate of milk yield than those with H2H6 and H6H8 haplotype combinations (P < 0.05). Cows with H2H8 and H8H8 haplotype combinations had lower rectal temperature than those with H5H8 and H7H7 haplotype combinations (P < 0.05). In conclusion, H8H8 haplotype combination may be advantageous for heat resistance traits in Chinese Holstein cattle.

Genetic variations of HSBP1 gene and its effect on thermal performance traits in Chinese Holstein cattle.

Molecular chaperones have been understood to be preferentially transcribed to prevent perturbations in response to various stresses. In this study, three single nucleotide polymorphisms (SNPs), g.324G>C, g.589C>T and g.651C>G in Heat shock factor binding protein 1 (HSBP1) gene were found and genotyped in 930 Chinese Holstein cattle. The results indicated that only g.589C>T polymorphism locus met Hardy-Weinberg equilibrium (P > 0.05). Pair linkage disequilibrium analysis and haplotype construction of HSBP1 gene were performed using SHEsis software. Seven haplotypes were constructed and fourteen haplotype combinations were found. Association analysis showed that H2H2 haplotype combination was advantageous for thermo tolerance breeding in Chinese Holstein. The cows with H2H2 haplotype combination have lower decrease rate of milk yield than those with H2H3 haplotype combination (P < 0.05) and lower potassium content in erythrocytes (PCE) than those with H2H5 (P < 0.05), H4H4 (P < 0.05) and H4H5 (P < 0.01) haplotype combination. The association between SNP and thermo tolerance traits showed that PCE of cows with GG genotype was lower than those with CG genotype at g.651C>G locus (P < 0.01). Pair linkage disequilibrium analysis revealed that the three loci were at a strong disequilibrium state. So we presumed that the effect of H2H2 haplotype combination on thermo tolerance traits major due to the SNP of g.651C>G.

Multiple promoters and targeted microRNAs direct the expressions of HMGB3 gene transcripts in dairy cattle.

HMGB3 (high-mobility group box 3) is an X-linked member of a family of sequence-independent chromatin-binding proteins and functions as a universal sentinel for nucleic acid-mediated innate immune responses. The splice variant expression, promoter characterization and targeted microRNAs of the bovine HMGB3 gene were investigated to explore its expression pattern and possible regulatory mechanism. The results revealed that the expression of HMGB3 transcript variants 1 and 2 (HMGB3-TV1 and HMGB3-TV2) mRNA in the mastitis-infected mammary gland tissues was up-regulated by 8.46- and 5.31-fold respectively compared with that in healthy tissues (P < 0.05). HMGB3-TV1 was highly expressed in the mammary gland tissues, whereas HMGB3-TV2 was expressed primarily in liver. Functional analyses indicated that HMGB3 transcription is regulated by three distinct promoters - promoters 1, 2 and 3 (P1, P2 and P3) - resulting in two alternative transcripts with the same 3'-untranslated region. Promoter luciferase activity analysis suggested that the core sequences of P1 and P2 were mapped in the region of g.1535 to ~g.2076 and g.2074 to ~g.2491 respectively. The g.5880C>T SNP in P3 affected its base promoter activity, and different genotypes were associated with the bovine somatic count score. The expression of targets bovine miR-17-5p, miR-20b and miR-93 of the HMGB3 gene was down-regulated 1.56-, 1.72- and 2.94-fold respectively in mammary gland tissues as compared with that in healthy tissues (P < 0.05). The findings suggest that HMGB3 expression is under complex transcriptional and post-transcriptional control by alternate promoter usage, alternative splicing mechanism and microRNAs in dairy cattle.

Signatures of selection in indigenous Chinese cattle genomes reveal adaptive genes and genetic variations to cold climate.

Cold climate shapes the genome of animals and drives them to carry sufficient genetic variations to adapt to changes in temperature. However, limited information is available about the genome-wide pattern of adaptations to cold environments in cattle. In the present study, we used 777K SNP genotyping (15 Chinese cattle breeds, 198 individuals) and whole genome resequencing data (54 cattle breeds of the world, 432 individuals) to disentangle divergent selection signatures, especially between the cold-adapted (annual average temperature of habitat, 6.24 °C to 10.3 °C) and heat-adapted (20.2 °C to 24.73 °C) Chinese native cattle breeds. Genomic analyses revealed a set of candidate genes (e.g., UQCR11, DNAJC18, EGR1, and STING1) were functionally associated with thermogenesis and energy metabolism. We also characterized the adaptive loci of cattle exposed to cold temperatures. Our study finds new candidate genes and provides new insights into adaptations to cold climates in cattle.

Cold climates can affect cattle performance, survival, and health. Local cattle breeds have been adapted to the local environments including extremely cold temperatures after a long period of natural and artificial selection. Selection and local adaptation are shaping populations. However, identifying loci associated with cold adaptation has been a major challenge. We used high-density SNP arrays and resequencing data to comprehensively analyze and compare the genomic selection signatures of Chinese northern and southern cattle, and elucidated several adaptive genes and alleles involved in cold adaptation. The complexity of genetic adaptation mechanism among different low-temperature adapted cattle breeds was also emphasized.

Coordinated alternation of DNA methylation and alternative splicing of PBRM1 affect bovine sperm structure and motility.

DNA methylation and gene alternative splicing drive spermatogenesis. In screening DNA methylation markers and transcripts related to sperm motility, semen from three pairs of full-sibling Holstein bulls with high and low motility was subjected to reduced representation bisulphite sequencing. A total of 948 DMRs were found in 874 genes (gDMRs). Approximately 89% of gDMR-related genes harboured alternative splicing events, including SMAD2, KIF17, and PBRM1. One DMR in exon 29 of PBRM1 with the highest 5mC ratio was found, and hypermethylation in this region was related to bull sperm motility. Furthermore, alternative splicing events at exon 29 of PBRM1 were found in bull testis, including PBRM1-complete, PBRM1-SV1 (exon 28 deletion), and PBRM1-SV2 (exons 28-29 deletion). PBRM1-SV2 exhibited significantly higher expression in adult bull testes than in newborn bull testes. In addition, PBRM1 was localized to the redundant nuclear membrane of bull sperm, which might be related to sperm motility caused by sperm tail breakage. Therefore, the hypermethylation of exon 29 may be associated with the production of PBRM1-SV2 in spermatogenesis. These findings indicated that DNA methylation alteration at specific loci could regulate gene splicing and expression and synergistically alter sperm structure and motility.

The exon 29 c.3535A>T in the alpha-2-macroglobulin gene causing aberrant splice variants is associated with mastitis in dairy cattle.

Alpha-2-macroglobulin (A2M) binds proteases, thereby acting as defense barriers against pathogens in the plasma and tissues of vertebrates and invertebrates. Quantitative real-time polymerase chain reaction (PCR) and the isobaric tags for relative and absolute quantitation method were used to determine the expression levels of A2M mRNA and proteins in mastitis-infected mammary tissues. A2M mRNA and protein expression were significantly higher in mastitis-infected mammary tissues than those in healthy tissues. We also identified 23 novel A2M splice variants in the bovine mammary tissues using reverse transcription PCR combined with clone sequencing. These splice variants predominantly affected the bait region, the inhibitory region, and the thioester region of the protein, which have the functional key roles in inhibiting the proteases of pathogens. Genomic sequencing analysis revealed a nonsynonymous c.3535A>T single-nucleotide polymorphism (SNP) in exon 29, which is located within a putative exonic splice enhancer and may be the reason why the A2M gene produces the aberrant splice variant A2M-AS4. Our findings suggest that the A2M gene can play its role by alternative splicing mechanism and it may be of significance against mastitis. This study provides clues to better understand the function of the bovine A2M gene and the effects of the exonic SNP on the production of aberrant splice variants.

One SNP in the 3'-UTR of HMGB1 gene affects the binding of target bta-miR-223 and is involved in mastitis in dairy cattle.

High-mobility group box protein 1 (HMGB1) gene has a universal sentinel function for nucleic acid-mediated innate immune responses and acts as a pathogenic mediator in the inflammatory disease. In an effort to identify the functional single-nucleotide polymorphism (SNP) in the 3'-untranslated region (UTR) of the bovine HMGB1 gene that affects the binding to its target microRNA, first, the expression of HMGB1 mRNA in different genotypes and its candidate bta-miR-223 was investigated. Quantitative real-time polymerase chain reaction results showed that the relative expression of HMGB1 mRNA in cows with the genotype GG is significantly higher than those in cows with the genotype AA (P < 0.05). The expression of bta-miR-223 was significantly upregulated by 1.95-fold (P < 0.05) in the bovine mastitis-infected mammary gland tissues compared with that in the healthy tissues. Subsequently, luciferase assay indicated that the HMGB1 expression was directly targeted by bta-miR-223 in human embryo kidney 293 T (HEK 293T) cells. One novel SNP (g. +2776 A > G) in the HMGB1 3'-UTR, altering the binding of HMGB1 and bta-miR-223, was found to be associated with somatic count scores in cows. Taken together, the g. +2776 A > G-GG was an advantageous genotype which can be used as a candidate functional marker for mastitis resistance breeding program.