Copy number variation (CNV) is a type of genomic variation with an important effect on animal phenotype. We found that the PIGY gene contains a 3600 bp copy number variation (CNV) region located in chromosome 6 of sheep (Oar_v4.0 36,121,601-36,125,200 bp). This region overlaps with multiple quantitative trait loci related to phenotypes like muscle density and carcass weight. Therefore, in this study, the copy number variation of the PIGY gene was screened in three Chinese sheep breeds, namely, Chaka sheep (CKS, May of 2018, Wulan County, Qinghai Province, China), Hu sheep (HS, May of 2015, Mengjin County, Henan Province, China), and small-tailed Han sheep (STHS, May of 2016, Yongjing, Gansu Province, China). Association analyses were performed on the presence of CNV and sheep body size traits. We used real-time quantitative PCR (qPCR) to detect the CNV for association analysis. According to the results, the loss-type CNV was more common than other types in the three breeds (global average: loss = 61.5%, normal = 17.5%, and gain = 21.0%). The association analysis also showed significant effects of the PIGY gene CNV on body weight, chest circumference, and circumference of the cannon bone of sheep. Sheep with gain-type CNV had better growth traits than those with other types. The results indicate a clear relationship between the PIGY gene CNV and growth traits of sheep, suggesting the use of CNV as a new molecular breeding marker.

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.

The potassium inwardly rectifying channel, subfamily J, member 12 (KCNJ12) gene is a promising candidate for economic traits because of its crucial roles in myoblast development. Here, a missense mutation (Cys > Arg) was first detected to be located in exon 3 of KCNJ12 from three Chinese cattle breeds by DNA-pool sequencing. Then, we performed an association analysis of this single-nucleotide polymorphism (SNP) with stature in three Chinese cattle populations (n = 820). A significantly positive correlation was revealed by a reduced animal general linear model and the CC genotype was the most favorable in three breeds. Further, we measured the expression profile of the KCNJ12 gene in various cattle tissues and primary bovine skeletal muscle cells. Ubiquitous expression with high abundance in muscle was observed. Further, in primary bovine skeletal muscle cells, the KCNJ12 mRNA expression was gradually up-regulated in differentiation medium (DM) compared with that in growth medium (GM), suggesting that the KCNJ12 gene is involved in bovine myocyte differentiation. Conclusively, the KCNJ12 gene is a functional candidate gene which can be used as a molecular marker for cattle breeding.

Eukaryotic translation initiation factor 2-alpha kinase 4 (EIF2AK4, also known as GCN2), which pertains to the family of serine-threonine kinase, is involved in oxidative stress and DNA damage repair. A missense single-nucleotide polymorphism (SNP) (NC_037337.1 g.35615224 T > G) in exon 6 of the EIF2AK4 gene which encodes a p.Ile205Ser substitution was observed in the Bovine Genome Variation Database and Selective Signatures (BGVD). The purpose of the current study is to determine the allelic frequency distribution of the locus and analyze its association with thermal tolerance in Chinese indigenous cattle. In our study, the allelic frequency distribution of the missense mutation (NC_037337.1 g.35615224 T > G) in Chinese cattle was analyzed by sequencing 1105 individuals of 37 breeds including 35 Chinese indigenous cattle breeds and two exotic breeds. In particular, association analysis was carried out between the genotypes and three environmental parameters including annual mean temperature (T), relative humidity (RH), and temperature-humidity index (THI). The frequency of the mutant allele G (NC_037337.1 g.35615224 T > G) gradually decreased from the southern cattle groups to the northern cattle groups, whereas the frequency of the wild-type allele T showed an opposite pattern, consistent with the distribution of indicine and taurine cattle in China. In accordance with the association analysis, genotypes were significantly associated with T (P < 0.01), RH (P < 0.01), and THI (P < 0.01), suggesting that the cattle with genotype GG were found in regions with higher T, RH, and THI. Thus, our results suggest that the mutation (NC_037337.1 g.35615224 T > G) of the EIF2AK4 gene is associated with thermal tolerance traits in Chinese cattle.

Copy number variation (CNV) caused by gene rearrangement is an important part of genomic structural variation. We found that the copy number variation region of the Src Homology 2 Domain Containing E (SHE) gene correlates with a quantitative trait locus of sheep related to milk fat percentage and bone density. The aim of our study was to detect the copy number variation of the SHE gene in four sheep breeds and to conduct a correlation analysis with economic traits, hoping to provide some reference for sheep breeding. In this study, we examined 750 sheep from four Chinese breeds: Chaka sheep (CKS), Hu sheep (HS), Large Tail Han sheep (LTHS) and Small Tail Han sheep (STHS). We used qPCR to evaluate the copy number of the SHE gene, and then used general linear models to analyze the associations between CNV and economic traits. The results showed that there were more individuals with SHE copy number loss in CKS and HS than in STHS and LTHS individuals. Association analyses showed that gain and normal copy number types were correlated to body length, circumference of cannon bone, heart girth, chest width and high at the cross in CKS, HS and STHS (p < 0.05), but this association was not observed for LTHS. Chi-square values (χ2) found prominent differences in CNV distribution among the studied breeds. Overall, the CNV of the SHE gene may be an important consideration for sheep molecular breeding.

Sperm-associated antigen 17 (SPAG17) gene encodes a multifunctional cytoplasmic protein, which influences not only reproduction but also skeletal development related body measurement traits, especially body height. Thus, this study aimed to identify crucial insertion-deletion (indel) variations, which influence the body measurement traits of goats in large goat populations (n = 1725). As a result, two intronic indels (14 bp and 17 bp indel) were identified by sequencing. For the two indel loci, the distributions of genotypes and alleles were significantly different between the Shaanbei white cashmere goat (SBWC) and the Hainan black goat (HNBG). In SBWC goats, the different genotypes of the 14 bp indel were markedly associated with goat body height, chest width, body length and chest depth. The genotypes of the 17 bp indel were significantly related to body height and chest width. At the two loci, for all seven analyzed traits of SBWC goat, the growth data of DD homozygotes were the worst, which means that the 14 bp insertion and the 17 bp deletion were beneficial and detrimental variations, respectively. Moreover, the combined genotypes were significantly related to body height and chest width of SBWC goats and ten traits of HNBG. These results suggested that the 14 and 17 bp indels within SPAG17 can be used in goat growth related traits marker-assisted selection breeding, especially body height.

The SRY-type HMG box 9 (Sox9) gene plays an important role in chondrocyte development as well as changes in hypertrophic chondrocytes, indicating that Sox9 can regulate growth in animals. However, no studies to date have examined the correlation between variations in Sox9 and growth traits in goats. Here, we found a 4 bp indel in the 3'UTR of Sox9 and verified its association with growth traits in Shaanbei white cashmere goats (n = 1109). The frequencies of two genotypes (ID and II) were 0.397 and 0.603, respectively, and polymorphic information content (PIC) values showed that the indel had a medium PIC (PIC > 0.25). The 4 bp indel was significantly correlated with body length (p = 0.006), heart girth (p = 0.001), and hip width (p = 4.37 × 10 -4). Notably, individuals with the ID genotype had significantly superior phenotypic traits compared with individuals bearing the II genotype. Hence, we speculated that the 4 bp indel is an important mutation affecting growth traits in goat, and may serve as an effective DNA molecular marker for marker-assisted selection in goat breeding programs.

Association studies have indicated profound effects of copy number variations (CNVs) on various phenotypes in different species. In this study, we identified the CNV distributions and expression levels of guanylate-binding protein 6 (GBP6) associated with the growth traits of Chinese cattle. The results showed that the phenotypic values of body size and weight of Xianan (XN) cattle were higher than those of Nanyang (NY) cattle. The medium CNV types were mostly identified in the XN and NY breeds, but their CNV distributions were significantly different (adjusted p < 0.05). The association analysis revealed that the body weight, cannon circumference and chest circumference of XN cattle had significantly different values in different CNV types (p < 0.05), with CNV gain types (Log22-ΔΔCt > 0.5) displaying superior phenotypic values. We also found that transcription levels varied in different tissues (p < 0.001) and the CNV gain types showed the highest relative gene expression levels in the muscle tissue, consistent with the highest phenotypic values of body weight and cannon circumference among the three CNV types. Consequently, our results suggested that CNV gain types of GBP6 could be used as the candidate markers in the cattle-breeding program for growth traits.

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.

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.

In the beef industry, fat tissue is closely related to meat quality. In this study, high-throughput RNA sequencing was utilized for adipose tissue transcriptome analysis between cattle-yak, Qaidamford cattle, and Angus cattle. The screening and identification of differentially expressed genes (DEGs) between different breeds of cattle would facilitate cattle breeding. Compared to Angus cattle adipose tissue, a total of 4167 DEGs were identified in cattle-yak adipose tissue and 3269 DEGs were identified in Qaidamford cattle adipose tissue. Considering cattle-yak as a control group, 154 DEGs were identified in Qaidamford cattle adipose tissue. GO analysis indicatedthe significant enrichment of some DEGs related to lipid metabolism. The KEGG pathway database was also used to map DEGs and revealed that most annotated genes were involved in ECM-receptor interaction and the PI3K-Akt signal pathway, which are closely related to cell metabolism. Eight selected DEGs related to adipose tissue development or metabolism were verified by RT-qPCR, indicating the reliability of the RNA-seq data. The results of this comparative transcriptome analysis of adipose tissue and screening DEGs suggest several candidates for further investigations of meat quality in different cattle breeds.

The casein alpha s1 (CSN1S1) gene encodes α-s1 casein, one of the proteins constituting milk, which affects milk performance, as well as improving the absorption of calcium and bone development in mammals. A previous study found that an 11-bp insertion/deletion (indel) of this gene strongly affected litter size in goats. However, to our knowledge, the relationships between this polymorphism and the milk performance and body measurement traits of goats have not been reported. In this paper, the previously identified indel has been recognized in three Chinese goat breeds, namely the Guanzhong dairy goat (GZDG; n = 235), Shaanbei white cashmere goat (SBWC; n = 1092), and Hainan black goat (HNBG; n = 278), and the following three genotypes have been studied for all of the breeds: insertion/insertion (II), deletion/deletion (DD), and insertion/deletion (ID). The allele frequencies analyzed signified that the frequencies of the "D" allele were higher (47.8%-65.5%), similar to the previous report, which indicates that this polymorphism is genetically stable in different goat breeds. Further analysis showed that this indel was markedly associated with milk fat content, total solids content, solids-not-fat content, freezing point depression, and acidity in GZDG (p < 0.05), and also affected different body measurement traits in all three breeds (p < 0.05). The goats with II genotypes had superior milk performance, compared with the others; however, goats with DD genotypes had better body measurement sizes. Hence, it may be necessary to select goats with an II or DD genotype, based on the desired traits, while breeding. Our study provides information on the potential impact of the 11-bp indel polymorphism of the CSN1S1 gene for improving the milk performance and body measurement traits in goats.

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.

Chinese indigenous cattle breeds have abundant genetic resources, which are valuable for the molecular breeding of cattle around the world. Thus, identifying important candidate genes and their genetic markers is very important for cattle molecular breeding. A previous study found that a nonsense mutation (rs378652941, c.483C > A, p.Cys161X) in the bovine transmembrane protein 95 gene (TMEM95) seriously reduced the reproductive performance in bulls, but few studies have detected this mutation in Chinese indigenous cattle breeds. Since the mutation c.483C > A may serve as a potential genetic marker for selecting higher fertility bulls, in the present study, using tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR), forced PCR restriction fragment length polymorphism (forced PCR-RFLP), and DNA sequencing methods, the mutation c.483C > A was detected in 765 individuals from 13 Chinese cattle breeds. However, the results showed that this mutation did not exist at this locus in our analyzed breeds. Interestingly, we identified a newly frameshift insertion/deletion (indel) mutation (NC_037346.1: g.27056998_27057000delCT) in the bovine TMEM95 gene in 11 cattle breeds, which changed the location of the termination codon and changed the 16 amino acids in the C-terminal to 21 amino acids. Combined with previous studies, our study provides evidence that in Chinese cattle breeds the mutation c.483C > A cannot be used as a genetic marker in molecular breeding.

Mutations are heritable changes at the base level of genomic DNA. Furthermore, mutations lead to genetic polymorphisms and may alter animal growth phenotypes. Our previous study found that mutations in the bovine Actin-like protein 8 (ACTL8) gene may be involved in muscle growth and development. This study explored several mutations of the ACTL8 gene and their influence on body size in Chinese beef cattle, as well as tested the tissue expression profile of the ACTL8 gene in Qinchuan cattle at different ages. Five single nucleotide polymorphisms (SNPs) (including one synonymous mutation (c.2135552895G > A)) and two insertion/deletion polymorphisms (indels) were identified in the ACTL8 gene from 1138 cattle by DNA-seq, RFLP and other methods. Then, the expression profile of the ACTL8 gene in Qinchuan cattle showed that it was expressed in heart, spleen, lung, liver, muscle, and fat tissues. Moreover, the expression level of ACTL8 was increased with cattle growth (p < 0.01). The ACTL8 mRNA expression level in kidney and muscle tissues was the highest in the calves, while lowest in the fetal stage. Overall, we showed that the mutations could act as markers in beef molecular breeding and selection of the growth traits of cattle.

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.

As a gene contributing to spermatogenesis, the Boule gene (also called Boll), whose mutations result in azoospermia and sterility of flies and mice, was conserved in reductional maturation divisions. However, in goats, the polymorphisms of Boule, especially with regard to their fundamental roles in female reproduction traits, are still unknown. Therefore, the aims of this study were to detect a potential mutation (rs661484476: g.7254T>C) located in intron 2 of the Boule gene by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) and to explore its potential association with the litter size of Shaanbei White-Cashmere goats (SBWGs). In this study, g.7254T>C was firstly detected. The TT genotype was the dominant genotype in the single-lamb group, and T was also the dominant allele in all tested groups. Additionally, the detected locus displayed moderate polymorphism with polymorphism information content (PIC) values among all studied goats ranging from 0.303 to 0.344. Notably, according to the χ2 test, the distribution differences for the genotypic frequencies between the single- and multi-lamb groups was significant (p = 0.014). Furthermore, the polymorphisms of the goat Boule gene were significantly associated with the goat litter size in SBWGs (p < 0.05), which indicated that g.7254T>C could be a potential marker in the marker-assisted selection process for potential litter size in goats. These results also indicated that the Boule gene might exercise important functions in female goat reproduction, which provided new insight for female goat breeding and could accelerate the process of goat breeding.

Based on the previous GWAS research related to bovine heat tolerance trait, this study aimed to explore the effect of myosin-1a (MYO1A) gene on bovine heat tolerance trait, and find the molecular markers related to the heat tolerance of Chinese cattle. In our study, four novel candidate SNPs highly conserved in B. indicus breeds but barely existed in B. taurus were identified in MYO1A gene according to Bovine Genome Variation Database and Selective Signatures (BGVD). PCR and DNA sequencing were used to genotype 1072 individuals including 34 Chinese indigenous cattle breeds as well as Angus and Indian zebu. Two synonymous mutations (rs208210464 and rs110123931), one missense mutation (rs209999142; Phe172Ser), and one intron mutation (rs135771836) were detected. The frequencies of mutant alleles of the four SNPs gradually increased from northern groups to southern groups of Chinese cattle, which was consistent with the distribution of various climatic conditions of China. Additionally, four SNPs were significantly associated with four climatic conditions including annual mean temperature (T), relative humidity (H), temperature-humidity index (THI), and average annual sunshine hours (100-cloudiness) (SR). Among these, rs209999142 and Hap 1/1 had better performance than others. Our results suggested that rs209999142 was associated with heat-tolerance trait and rs208210464, rs110123931, and rs135771836 showed high phenotypic effect on heat-tolerance trait because of the strong linkage with rs209999142. These SNPs could be used as candidates for marker-assisted selection (MAS) in cattle breeding.