Copy number variation is a part of genomic structural variation and has caused widespread concern. According to the results of high-throughput screening of the MLLT10 gene, we found that the copy number variation region of the MLLT10 gene was correlated with bovine growth traits. We aimed to detect the MLLT10 gene copy number variation and provide materials for the Chinese yellow cattle breed. In this study, the SPSS software was used to analyze the correlation among the copy number type of six different cattle breeds (i.e., Qinchuan, Xianan, Jiaxian, Yanbian, Sinan, Yunling) and the corresponding growth traits. The results showed the following: In Qinchuan cattle, the copy number duplication type was greater than the deletion and normal types; in Xianan cattle, the copy number duplication and normal types were less as compared with the deletion type; and in Yunling cattle, the frequency of the duplication type was dominant among the three types of copy number variants. The correlation analysis result showed that there is a significant correlation between the copy number variation (CNV) of the MLLT10 gene and the growth traits of three cattle breeds. Furthermore, correlation analysis showed that MLLT10 CNV had positive effects on growth traits such as hip width, rump length, hucklebone width, and cannon bone circumference (p < 0.05). This study provides a basis for the molecular-assisted marker breeding of cattle and contributes to the breeding of cattle.

There is growing evidence that non-coding RNAs are emerging as critical regulators of skeletal muscle development. In order to reveal their functional roles and regulatory mechanisms, we constructed a lncRNA-miRNA-mRNA network according to the ceRNA (competitive endogenous RNA) theory, using our high-throughput sequencing data. Subsequently, the network analysis, GO (Gene Ontology) analysis, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were performed for functional annotation and exploration of lncRNA ceRNAs. The results uncovered a scale-free characteristics network which exhibited high functional specificity for bovine skeletal muscle development: co-expression lncRNAs were significantly enriched in muscle development related biological processes and the Wnt signaling pathway. Furthermore, GSEA (Gene Set Enrichment Analysis) indicated that the risk score has a tendency to associate with myogenesis, and differentially expressed RNAs were validated by qPCR, further confirming the credibility of our network. In summary, this study provides insights into lncRNA-mediated ceRNA function and mechanisms in bovine skeletal muscle development and will expand our understanding of lncRNA biology in mammals.

With the inexorable rise of global temperature, heat stress deserves more and more attention in livestock agriculture. Previous studies have shown that the mechanistic target of rapamycin (MTOR) (NC_037343.1:c.2062G>C) gene contributes to the repair of DNA damage repair and is associated with the adaptation of camels in dry and hot environments. However, it is unknown whether this mutation is related to the heat tolerance of Chinese cattle. In this study, PCR and sequencing were used to type the mutation locus in 1030 individuals of 37 cattle breeds. The analysis results showed that the frequency of G allele of the locus gradually diminished from the northern group to the southern group of native Chinese cattle, whereas the frequency of the C allele showed an opposite pattern, displaying a significant geographical difference across native Chinese cattle breeds. Additionally, an analysis of the locus in Chinese indigenous cattle revealed that this SNP was significantly associated with mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI) (p < 0.01), suggesting that cattle with C allele was distributed in regions with higher T, RH and THI. In conclusion, this study proved that the mutation of MTOR gene in Chinese cattle could be associated with the heat tolerance.

SLC45A2 is involved in the synthesis of melanin transporters. We investigated the association between single nucleotide polymorphisms (SNPs) of the SLC45A2 gene and humidity and hot conditions in indigenous cattle habitat. According to the Bovine Genome Variation Database and Selective Signatures (BGVD), we explored the frequency distribution of a missense mutation (NC_037347.1: c.1543A > G, p.ser515gly) in the SLC45A2 gene in Chinese indigenous cattle. This variation from serine to glycine caused a significant change in the protein modeling structure. PCR and partial DNA sequencing were used to genotype 541 individuals, including 28 Chinese indigenous cattle breeds as well as Angus and zebu. From our results, the mutant allele frequency of this SNP in Chinese native cattle increases gradually from north to south, which is consistent with the distribution of climatic conditions in China. In addition, according to association analysis of a missense mutation (NC_037347.1: c.1543A > G) (rs525805167) in Chinese cattle, it is closely related to the annual average temperature (T), relative humidity (RH), temperature and humidity index (THI) and solar radiation time (P < 0.01). Based on the statistical analysis of the data, we assumed that rs525805167 was associated with heat tolerance traits. Simple Summary: The characteristics of Chinese indigenous cattle are closely related to their climatic environment. In China, Bos taurus is mainly distributed in the northern regions; Bos indicus is mainly distributed in southern China. In addition, the average temperature is higher in the south than in the north, and there are many mixed ancestry breeds of B. taurus and B. indicus in the middle area. The SLC45A2 gene is related to melanin synthesis, which may be closely related to heat tolerance in cattle. The purpose of our study was to investigate whether the SLC45A2 gene is related to heat tolerance in Chinese indigenous cattle.

The heat shock factor 1 (HSF1) gene is a regulator of the heat stress response, maximizing HSP protein expression survival. In this research, we explored the frequency distribution of a missense mutation (NC_037341.1 g.616087A > G, rs135258919) in the HSF1 gene in Chinese cattle with amino acid substitution, valine to alanine. This mutation could be related to the heat tolerance in Bos indicus. A total of 941 individuals representing 35 Chinese native cattle breeds, combining pure taurine (Angus) and indicine cattle, were used to determine the genotypes of the mutation through PCR and partial DNA sequencing. The results showed significant differences in allele frequencies and their genotypes amongst Chinese cattle from different regions. Allele G or indicine-specific allele frequency diminished from south to north China, while allele A (genotype AA) or the taurine-specific allele had a contrary pattern, which agreed with the distribution of taurine and indicine cattle. According to the association analysis, the NC_037341.1 g.616087A > G (rs135258919) of the bovine HSF1 gene, annual temperature (T), relative humidity (RH), and the temperature humidity index (THI) (p < 0.01) were interrelated closely, which indicated that the NC_037341.1 g.616087A > G of the HSF1 gene is associated with heat tolerance in indicine cattle.

Muscle development is an important factor affecting meat yield and quality and is coordinated by a variety of the myogenic genes and signaling pathways. Recent studies reported that miRNA, a class of highly conserved small noncoding RNA, is actively involved in regulating muscle development, but many miRNAs still need to be further explored. Here, we identified that the miR-183/96/182 cluster exhibited higher expression in bovine embryonic muscle; meanwhile, it widely existed in other organizations. Functionally, the results of the RT-qPCR, EdU, CCK8 and immunofluorescence assays demonstrated that the miR-183/96/182 cluster promoted proliferation and differentiation of bovine myoblast. Next, we found that the miR-183/96/182 cluster targeted FoxO1 and restrained its expression. Meanwhile, the expression of FoxO1 had a negative correlation with the expression of the miR-183/96/182 cluster during myoblast differentiation. In a word, our findings indicated that the miR-183/96/182 cluster serves as a positive regulator in the proliferation and differentiation of bovine myoblasts through suppressing the expression of FoxO1.

Dianzhong cattle is a classic Chinese indigenous cattle breed with historical records dating back to 200 BC. But with its genomic differences having not been clearly elucidated, the quest for genomic characterization will be an essential step towards understanding the genomic basis of productivity and adaptation to survival under Chinese farming systems. Here we compared 10 Dianzhong cattle (four newly sequenced and six downloaded) with 29 published genomes of three underlying ancestral populations (Chinese zebu, Indian zebu, and Yanbian cattle) to characterize the genomic variations of Dianzhong cattle. Dianzhong cattle has a high nucleotide diversity (0.0034), second only to Chinese zebu. Together with analyses of linkage disequilibrium decay and runs of homozygosity, Dianzhong cattle displayed higher genomic diversity and weaker artificial selection compared with Yanbian cattle. From a selective sweep analysis by four methods (Fst, π-ratio, XP-CLR, and XP-EHH), the positive selective signals were mainly manifested in candidate genes and pathways related to heat resistance, growth and development, fat deposition, and male reproduction. Missense mutations were detected in candidate genes, SDS (c.944C > A and p.Ala315Glu), PDGFD (c.473A > G and p.Lys158Arg), and DDX4 (rs460251486, rs722912933, and rs517668236), which related to heat resistance, fat deposition, and spermatogenesis, respectively. Our findings unravel, at the genome-wide level, the unique diversity of Dianzhong cattle while emphasizing the opportunities for improvement of livestock productivity in further breeding programs.

Myogenesis is controlled by a well-established transcriptional hierarchy that coordinates the activities of a set of muscle genes. Recently, roles in myogenesis have been described for non-coding RNAs, including a role of circular RNA (circRNA) to regulate muscle gene expression. However, the functions of circRNA and the underlying mechanism by which circRNAs affect myogenesis remain poorly understood. In this study, we analyzed circRNA high-throughput sequencing results of bovine skeletal muscle samples and constructed a circRNA-miRNA-mRNA network according to the competitive endogenous RNA (ceRNA) theory. The putative circHUWE1-miR-29b-AKT3 network was analyzed and its involvement in myogenesis was confirmed through a series of assays. To assess the potential function of this regulation, bovine myoblasts were infected with overexpression plasmids and small interfering RNAs (siRNAs) that target circHUWE1. Next, cell proliferation, apoptosis, and differentiation were analyzed using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, western blotting, and qRT-PCR assays. The results suggest that circHUWE1 facilitates bovine myoblast proliferation and inhibits cell apoptosis and differentiation. Next, bioinformatics, dual-luciferase reporter assay, and AGO2 RNA immunoprecipitation (RIP) approaches were used to verify the interaction between circHUWE1, miR-29b, and AKT3. Subsequently, we identified that circHUWE1 could directly interfere with the ability of miR-29b to relieve AKT3 suppression, which ultimately activates the AKT signaling pathway. These findings suggested a new regulatory pathway for bovine skeletal muscle development, and they also expand our understanding of circRNA functions in mammals.

We report genome characterization of three native Chinese cattle breeds discovering ~34.3 M SNPs and ~3.8 M InDels using whole genome resequencing. On average, 10.4 M SNPs were shared amongst the three cattle breeds, whereas, 3.0 M, 4.9 M and 5.8 M were specific to LQ, WN and WS breeds, respectively. Gene ontology (GO)analysis revealed four immune response-related GO terms were over represented in all samples, while two immune signaling pathways were significantly over-represented in WS cattle. Altogether, we found immune related genes (PGLYRP2, ROMO1, FYB2, CD46, TSC1) in the three cattle breeds. Our study provides insights into the genetic basis of Chinese indicine adaptation to the tropic and subtropical environment, and provides a valuable resource for further investigations of genetic characteristics of the three breeds.

The rearrangement of the transfection (RET) gene, which mediates the functions of the ganglion in the gastrointestinal tract, plays an important role in the development of the gastrointestinal nervous system. Therefore, the RET gene is a potential factor influencing animal body measurement. The aim of this study was to reveal the significant genetic variations in the bovine RET gene and investigate the relationship between genotypes and body measurement in two Chinese cattle breeds (Qinchuan and Nanyang cattle). In this study, two SNPs (c.1407A>G and c.1425C>G) were detected in the exon 7 of RET gene by sequencing. For the SNP1 and SNP2, the GG genotype was significantly associated with body height, hip height, and chest circumference in Qinchuan cattle (p < 0.05). Individuals with an AG-CC genotype showed the lowest value of all body measurement in both breeds. Our results demonstrate that the polymorphisms in the bovine RET gene were significantly associated with body measurement, which could be used as DNA marker on the marker-assisted selection (MAS) and improve the performance of beef cattle.

With the rapid development of economy, the demand for beef, with regard to quantity and quality, by consumers has been increasing in China. Chinese native cattle are characterized by their abundant genetic resources, unique origins, large breeding stocks, and robust environmental adaptability. Thus, to explore the genetic mechanisms on regulating meat quality in Chinese native cattle is of great importance to satisfy increased requirements for beef production. In this study, we investigated three breeds of cattle, namely Yunling, Wenshan, and Simmental, at the age of 12 months. Animals were classified into three groups (n = 5/breed). Growth traits including body weight and body size and plasma hormone levels were measured. Body weight of Wenshan cattle was significantly lower than that of Yunling and Simmental cattle (P < 0.05). Again, body size indexes, such as withers height, body slanting length, chest circumference, and hip and rump length, were significantly lower in Wenshan cattle than those in Yunling and Simmental cattle (P < 0.05). However, there were no significant differences in those indexes between Yunling and Simmental cattle (P > 0.05). Cattle were slaughtered at the age of 18 months and then meat color, pH, pressing losses, muscle tenderness, and cooking losses were measured at 0, 1, 2, 3, 5, and 7 days. Data revealed differences in meat quality among the three breeds analyzed. Based on transcriptomic sequencing and bioinformatic analysis, we observed 3,198 differentially expressed genes related to meat quality, of which 1,750 genes were upregulated. Moreover, we found two important signaling pathways closely linked to meat quality, namely adipocytokine signaling pathway [e.g., Leptin receptor (LEPR)] and protein processing in the endoplasmic reticulum [e.g., signal transducer and activator of transcription 3 (STAT3), heat shock protein (HSPA12A), and calpain 1 (CAPN1)]. The results of transcripts were further verified by qRT-PCR. Using correlation analysis between gene expression levels and shear force, we also identified two functional genes (e.g., HSPA12A and CAPN1) associated with meat quality. Overall, this study provides new sights into novel targets and underlying mechanisms to modulate meat quality in Chinese native cattle.

The KRT75 gene (Keratin 75) is a member of the type II epithelial α-keratin gene family which plays a key role in hair and nail formation. And the coat conformation affects heat tolarence in mammals. Therefore, the aim of this study was to identify novel single nucleotide polymorphisms (SNPs) of the KRT75 gene and further evaluate its relation to heat stress in Chinese cattle. A missense mutation (NC_037332.1: g.1052 T > C) of the bovine KRT75 was identified using the Bovine Genome Variation Database (BGVD). The g.1052 T > C variant was then genotyped in 519 individuals of 22 cattle breeds. Further analyses showed that the frequency of T allele in Chinese indigenous cattle breeds gradually diminished from northern groups to southern groups, whereas the frequency of C allele displayed a contrary patternl. Simultaneously, the frequency of the CC and CT genotype for southern groups was much greater than that of the TT genotype. Additionally, association analysis showed the genotypes were remarkably associated with mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI) (P < 0.01). Our results demonstrated that the KRT75 gene might be a candidate gene associated with the heat stress in Chinese cattle.

Pleomorphic adenoma gene 1 (PLAG1) belongs to the PLAG family of zinc finger transcription factors. In cattle, a 19-bp insertion/deletion (indel) was identified in intron 1 of the PLAG1 gene (GenBank Accession No. AC_000171.1). Researches showed that the indel is polymorphic in Chinese cattle breeds such as Qinchuan cattle, Pinan cattle, Xianan cattle, and Jiaxian red cattle, and correlation analysis showed that the polymorphism is related to the height of these cattle breeds. Chinese cattle breeds show a difference in height related to geographical distribution. We investigated the distribution of the 19-bp indel polymorphism in 37 cattle breeds, including 1354 individuals. The results showed that there were three genotypes and two alleles (W, 366 bp; D, 347 bp). From northern cattle to southern cattle, the frequency of W allele gradually decreased, while the frequency of D allele showed an opposite trend, which was consistent with the distribution of cattle breeds of different height in China. Therefore, the polymorphism of this indel may be related to the regional distribution of cattle breeds in China. In addition, we chose Yunling cattle with a mixed genetic background to study the genetic effects of the 19-bp indel on body size traits. Statistical analysis showed that PLAG1 was significantly associated with the body height, cross height, and chest circumference of Yunling cattle (p < 0.05). This study provides new evidence that the 19-bp indel of the PLAG1 gene is a highly effective trait marker that can be used as a candidate molecular marker for cattle breeding.

Considerable evidence has demonstrated that the μ -calpain (CAPN1) gene and its inhibitor calpastatin (CAST) gene are major factors affecting meat quality. Marker-assisted selection (MAS) has been widely used to improve beef quality traits. Therefore, the objective of the present study was to investigate the single nucleotide polymorphisms (SNPs) of bovine CAPN1 and CAST genes using 367 animals representing the four main Chinese cattle breeds and to explore the effects of these SNPs on meat quality traits. Two SNPs within CAPN1 and one SNP in CAST were successfully identified in cattle. Genetic diversity analyses suggested that most SNPs in the four breeds exhibited a moderate genetic diversity. Moreover, associations between individual markers and meat quality traits were analyzed in Chinese Simmental cattle. The CAPN1 4558 A > G locus was found to be significantly associated with shear force value (SFV) and marbling score (BMS), and CAPN1 4684 C > T exerted a significant effect on SFV, while the CAST genotype was not significantly associated with any of the measured traits. SFV, commonly used to measure meat tenderness, represents an important quality trait as it contributes to the flavor of cooked meat. This work confirms the effect of CAPN1 on beef tenderness and lays an important foundation for future cattle breeding.

Marigold (Tagetes erecta L) accounts for over half of the world's loose flower production, and marigold crop residue (MCR) are abundantly available and should be used as a forage. In this study, MCR from the last commercial flower pickings was ensilaged with lactic acid bacteria (LAB) and the shift in their volatile organic compounds (VOCs) profiles was monitored. Samples were collected at 6 different times during ensilage (3, 6, 9, 12, 15, 30 days) to determine and quantify the VOCs changes using a solid-phase microextraction (SPME) technique and gas chromatography - mass spectrometry (GC-MS).

With increasing acreage of cash crops, the use of their by-products as supplements for livestock feed becomes an important factor. Marigold (Tagetes erecta L.) account for more than half of the world's loose flower production. However, there is no precedent for the abundantly available marigold crop residue (MCR) being used as feed in agricultural production, probably because of its strong pungent taste. This study aimed to evaluate the biotransformation of the volatile organic compounds (VOCs) of MCR by mixed ensilage and assess its palatability by cattle. Caryophyllene, the most prevalent VOC in MCR, decreased by 29.11% (P < 0.05), 38.85% (P < 0.05), 37.15% (P < 0.05), and 28.36% (P < 0.05) ensilage with corn meal (CM), bran (BR), crop corn (CC), and straw (ST), respectively. The acetic acid content increased by 686.05% (P < 0.05), 1337.21% (P < 0.05), 1244.19% (P < 0.05), and 1795.34% (P < 0.05) after mixed ensilage with CM, BR, CC, and ST, respectively. The total amount of alcoholic VOCs followed an overall increasing trend during mixed storage and 10 new alcohols were obtained. Over seven days, feed intake of mixed ensilage MCR by cattle differed significantly (P < 0.05) among treatments compared with MCR and was highest in MCRCM. Combined with palatability trials, the best MCR feed intake was achieved with MCRCM. The findings shed light on how feed odor can be improved and how degradation of terpenes can be enhanced in practical applications by mixed ensilage.

Bovine leukocyte antigen, class II, DO beta (BoLA-DOB) is related to antigen presentation, which can triggered by multicul factors. And the condition of immune function determines how much cattle load to heat stress. To evaluate the relationship between heat-resistance and single nucleotide polymorphisms (SNPs) in BoLA-DOB gene, our study has taken further analysis in Chinese indigenous cattle for the first time. A missense single nucleotide polymorphism (rs464874590) was detected in BoLA-DOB gene. We directly sequenced rs464874590 (NM_001013600.1 g.7122762 A > G) in BoLA-DOB gene of 522 individuals of 26 cattle breeds. The frequency of allele G gradually decreases from south to north with distinct climatic distribution characteristics. Further association analysis was carried out between different genotypes and environmental parameters, including annual mean temperature (T), relative humidity (RH), and temperature-humidity index (THI). The result showed that three genotypes were significantly correlated with T, H, and THI (P < 0.01), indicating that GG genotype was distributed in areas with hot and moist conditions. Therefore, our results suggested that the rs464874590 could be applied as a genetic marker to detect the heat-resistance of Chinese indigenous cattle.

Adipose tissue (AT) is a multi-depot organ in mammals. AT from various depots differs in composition and function. Revealing the composition feature of AT depots will provide valuable information for further research on the development and fat deposition patterns in buffalo. This study explored the cellular morphology and gene expression profiles of brown and beige markers in seven AT depots of fattened buffalo: three subcutaneous depots (back, sternum, and inguinal) and four visceral depots (perirenal, mesenteric, pericardial, and omental). Histological results showed unilocular adipocytes in all seven AT depots. Uncoupling protein 1 (UCP1) mRNA, a brown and beige adipocyte gene, was detected in all depots with the highest level in VAT depots, and a limited number of UCP1-positive unilocular adipocytes were observed in the three VAT depots. The mRNAs of PPARG coactivator 1 alpha (PGC1 α ) and transmembrane protein 26 (TMEM26), brown or beige adipocyte markers, were identified in all seven depots and were mainly expressed in VAT depots. However, the mRNA of zinc finger protein of the cerebellum 1 (ZIC1), a brown adipocyte-specific marker, was almost undetectable. Our results demonstrated that all seven AT depots are white adipose tissue (WAT), with potential function of non-shivering thermogenesis in fattened buffalo. Beige adipocytes are more active in VAT depots than in WAT depots. These results improve our knowledge on the feature of different adipose tissue depots in buffalo, which will be useful for the research of fat deposition.

Abnormal neurotransmitter concentration is one of the factors that affect the health status, behavioral personality, and welfare level of animals, but the genetic basis of the abnormality is still largely unknown. The objective of this study is to identify putative genomic loci associated with neurotransmitter concentration in cattle. We measured serotonin (5HT), dopamine (DA), cortisol, glutamate (Glu), and ACTH concentrations in blood serum using double-antibody sandwich ELISA in 30 Brahman cattle and 127 Yunling cattle. Interestingly, we found that ACTH concentration was positively correlated with body weight, cannon circumference, and hip width (P < 0.05). Genome-wide association study (GWAS) was performed with mixed linear models using autosomal SNPs derived from the whole-genome sequence. We identified five, five, two, three, and five suggestive loci associated with 5HT, DA, cortisol, Glu, and ACTH concentration, respectively. These 20 associated loci implicated 18 candidate genes. For Glu concentration, the most significant association locus was assigned to MCHR1, a G-coupled receptor that could modulate glutamate release. For dopamine concentration, a very strong association locus was located in the intron of SLC18A2, which is a critical mediator of dopamine dynamics. However, for ACTH concentration, a very strong association locus was assigned to HTR1F, a G protein-coupled receptor that can influence the release of ACTH. Other candidate genes of interest identified for neurotransmitter concentration were PRMT6, GADD45A, PCCA, ANGPT1, ACCS, LOC100336971, TNR, GSDMA, CNTN3, CARMIL1, CDKAL1, RBFOX1, PCDH15, and LGALS12. Our findings will provide targets for the genetic improvement of neurotransmitter-related traits in domestic cattle and basic materials for studying the mechanism of neurotransmitter synthesis, release, and transport in human and animals.

Yunling cattle are a composite beef cattle breed, combining Brahman (1/2), Murray Grey (1/4) and Local Yunnan cattle (1/4), that was developed in Yunnan, China in the 1980s. Understanding the genetic information of Yunling cattle is of great significance to the development of reasonable breeding strategies for this breed. In the present study, we assessed the current genetic status of Yunling cattle in Yunnan Province (China) by analyzing the variability of the whole mitochondrial genome of 129 individuals. Altogether, 129 sequences displayed 47 different haplotypes. The haplotype diversity and the average number of nucleotide differences were 0.964 and 128.074, respectively. Phylogenetic analyses classified Yunling cattle into seven haplogroups: T1, T2, T3, T4, T6, I1 and I2. Haplogroup I1 was found to be predominant (41.86%), followed by T3 (28.68%). Furthermore, we also identified a novel haplogroup, T6, and defined the sub-haplogroup I1a in Yunling cattle. According to the formation process of Yunling cattle (local Yunnan cattle as the maternal line), the high genetic diversities in the mitochondria of Yunling cattle could be due to the complex maternal origin of local Yunnan cattle. Further studies about local Yunnan breeds are necessary to determine the exact source of haplogroup T6 in Yunling cattle. Our results will be useful for the evaluation and effective management of Yunling cattle.