Cross-Species Comparative DNA Methylation Reveals Novel Insights into Complex Trait Genetics among Cattle, Sheep, and Goats.

The cross-species characterization of evolutionary changes in the functional genome can facilitate the translation of genetic findings across species and the interpretation of the evolutionary basis underlying complex phenotypes. Yet, this has not been fully explored between cattle, sheep, goats, and other mammals. Here, we systematically characterized the evolutionary dynamics of DNA methylation and gene expression in 3 somatic tissues (i.e. brain, liver, and skeletal muscle) and sperm across 7 mammalian species, including 3 ruminant livestock species (cattle, sheep, and goats), humans, pigs, mice, and dogs, by generating and integrating 160 DNA methylation and transcriptomic data sets. We demonstrate dynamic changes of DNA hypomethylated regions and hypermethylated regions in tissue-type manner across cattle, sheep, and goats. Specifically, based on the phylo-epigenetic model of DNA methylome, we identified a total of 25,074 hypomethylated region extension events specific to cattle, which participated in rewiring tissue-specific regulatory network. Furthermore, by integrating genome-wide association studies of 50 cattle traits, we provided novel insights into the genetic and evolutionary basis of complex phenotypes in cattle. Overall, our study provides a valuable resource for exploring the evolutionary dynamics of the functional genome and highlights the importance of cross-species characterization of multiomics data sets for the evolutionary interpretation of complex phenotypes in cattle livestock.

Genome-wide association study reveals that the IBSP locus affects ear size in cattle.

Ear size is a classical model for hot climate adaptation following the evolution, but the genetic basis of the traits associated with ear size remains to be elucidated. Here, we performed a genome-wide association study on 158 cattle to explain the genetic mechanism of ear size. One region on BTA6 between 36.79 and 38.80 Mb included 50 suggestive SNPs and 4 significant SNPs that were significantly associated with ear size. The most significant locus (P = 1.30 × 10-8) was a missense mutation (T250I) on the seventh exon of integrin-binding sialoprotein (IBSP), which had an allele substitution effect of 23.46 cm2 for ear size. Furthermore, this mutation will cause changes in the three-dimensional structure of the protein. To further identify genes underlying this typical feature, we performed a genome scan among nine cattle breeds with different ear sizes by using SweeD. Results suggested that IBSP was under positive selection among four breeds with relatively large ear sizes. The expression levels of IBSP in ear tissues of large- and small-ear cattle were significantly different. A haplotype diversity survey of this missense mutation in worldwide cattle breeds strongly implied that the origin of this missense mutation event was Bos taurus. These findings have important theoretical importance for the exploration of major genes associated with ear size and provide important molecular markers for the identification of cattle germplasm resources.

Genes related to heat tolerance in cattle-a review.

Heat stress is described as the cumulative detrimental effect caused by an imbalance between heat production within the body and heat dissipation. When cattle are exposed to heat stress with skin surface temperatures exceeding 35 °C, gene networks within and across cells respond to environmental heat loads with both intra and extracellular signals that coordinate cellular and whole-animal metabolism changes to store heat and rapidly increase evaporative heat loss. In this study, we examined evidence from genes known to be associated with heat tolerance (Hsp70, HSF1, HspB8, SOD1, PRLH, ATP1A1, MTOR, and EIF2AK4). This information could serve as valuable resource material for breeding programs aimed at increasing the thermotolerance of cattle.

TRPM4 gene variation associated with climatic conditions in Chinese cattle.

The transient receptor potential (TRP) superfamily has been reported to play an important role in heat tolerance pathways. Based on the Bovine Genome Variation Database and Selective Signatures, a missense mutation (NC_037345.1: c.2237A > G: p. His746Arg) (rs209689836) was identified in the transient receptor potential cation channel subfamily M member 4 (TRPM4) gene, a member of the TRP family, corresponding to heat tolerance. Here, we explored the prevalence of this variant in 19 native Chinese cattle (comprised of 404 individuals) to determine its possible association with heat tolerance in Chinese cattle by using PCR and DNA sequencing. The distribution of alleles of NC_037345.1: c.2237A > G: p. His746Arg displays significant geographical differences across native Chinese cattle breeds, consistent with the distribution of indicine and taurine cattle in China. Additionally, the association analysis indicated that the G allele was significantly associated with mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI) (p < .05), suggesting that cattle carrying allele G were distributed in regions with higher T, RH, and THI. In conclusion, our results suggested that the mutation of the TRPM4 gene in Chinese cattle might be a candidate locus associated with heat tolerance.

Assessing genomic diversity and selective pressures in Bashan cattle by whole-genome sequencing data.

Specific ecological environments and domestication have continuously influenced the physiological characteristics of Chinese indigenous cattle. Among them, Bashan cattle belongs to one of the indigenous breeds. However, the genomic diversity of Bashan cattle is still unknown. Published whole-genome sequencing (WGS) data of 13 Bashan cattle and 48 worldwide cattle were used to investigate the genetic composition and selection characteristics of Bashan cattle. The population structure analysis revealed that Bashan cattle harbored ancestries with East Asian taurine and Chinese indicine. Genetic diversity analysis implied the relatively high genomic diversity in Bashan cattle. Through the identification of containing >5 nsSNPs or frameshift mutations genes in Bashan cattle, a large number of pathways related to sensory perception were discovered. CLR, θπ ratio, FST, and XP-EHH methods were used to detect the candidate signatures of positive selection in Bashan cattle. Among the identified genes, most of the enriched signal pathways were related to environmental information processing, biological systems, and metabolism. We mainly reported genes related to the nervous system (HCN1, KATNA1, FSTL1, GRIK2, and CPLX2), immune (CD244, SLAMF1, LY9, and CD48), and reproduction (AKR1C1, AKR1C3, AKR1C4, and TUSC3). Our findings will be significant in understanding the molecular basis underlying phenotypic variation of breed-related traits and improving productivity in Bashan cattle.

A missense mutation (rs209302038) of KRT9 gene associated with heat stress in Chinese cattle.

Type I keratin 9 encoded by the KRT9 gene serves an important special function either in the mature palmar and plantar skin tissue. The changes in skin conditions and thickening of the outer layer of the skin may be affected by environmental variables. A missense mutation rs209302038 (NC_037346.1: g.41782870 G > A) was detected in KRT9, which changing the isoleucine into valine. This study aimed to identify the frequency of allele in this locus in Chinese indigenous cattle, and analyze the connection with heat stress. Our results indicated that the frequency of allele A gradually decreases from south to north, while the frequency of G allele showed the opposite pattern. Further analysis of the association of the different genotypes with three climate factors, which showed that the genotypes (GG, GA, AA) were significantly related to climatic conditions (p < 0.01). Therefore, we speculated that the mutation of the rs209302038 in Chinese indigenous cattle might be a genetic marker to detect heat stress.

Muscle transcriptome analysis reveal candidate genes and pathways related to fat and lipid metabolism in Yunling cattle.

Yunling cattle (YL) is a recently developed beef breed harboring a quarter of Yunnan ancestral cattle genome, spanning over past 30 years. Compared with Diqing cattle (DQ), a Yunnan native cattle breed, YL presents various advantages, including rapid growth and exquisite meat quality. However, the molecular mechanisms underlying these phenotypic differences are not clearly understood. To further identify the candidate genes responsible for the quality of the meat in the muscle, longissimus dorsi (LD) muscle was used for RNA-Seq analysis. A total of 508 differentially expressed genes (DEGs) were identified in YL (adjusted p-value <0.01 and log2FoldChange >1), of which 243 were up-regulated and 265 were down-regulated. Functional association analysis showed that the identified DEGs mainly enriched the lipid and fat metabolism pathways. Moreover, it was also observed that several fat-related genes were differentially expressed in both cattle breeds, including three up-regulated genes (MOGAT1, ACSM3, PLPP2) and two down-regulated genes (ADIG, GPAT3). In addition, alternative splice analysis was also performed revealing an important 9-11 exon skipping variation of GPAM gene (crucial for beef marbling) in YL, which is three times higher than that in DQ, suggesting that this variation might have played the central role in the 'snow beef' effect in YL. We believe that our results will help in understanding the mechanism of muscle development and promote the further breeding programs in YL cattle.

A novel SNP of TECPR2 gene associated with heat tolerance in Chinese cattle.

Heat stress affects the animal production and causes serious economic losses to the husbandry. Tectonin beta-propeller repeat containing 2 (TECPR2) gene plays an important role in autophagy which may affect the temperature sensation in animals. A missense mutation (XM_024981840.1:c.3989 G > A p.Arg1330His) of the transcripts X4 in the bovine TECPR2 gene was identified. In this study, the c.3989 G > A variant in TECPR2 gene was genotyped in a total of 25 cattle breeds (520 individuals). Our results indicated that the frequency of A allele showed a decreasing pattern from southern cattle to northern cattle, while the frequency of G allele showed the opposite pattern, which was consistent with the climate distribution of China. Compared with the GG genotype, southern cattle carried more the AA and AG genotypes. Furthermore, the association results carried out that the frequencies of genotypes (GG, AG, AA) and the value of climate parameters (mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI) were significantly correlated (p < 0.01). Hence, we speculated that the c.3989 G > A variant of TECPR2 gene was associated with the heat tolerance trait in Chinese cattle and the locus may be considered as a molecular marker for Chinese cattle breeding.

TAS2R16 introgression from banteng into indigenous Chinese cattle.

Historical hybridization between southern indigenous Chinese cattle and banteng has been well-documented and has resulted in gene introgression. Bitter taste receptors were reported in indigenous cattle as a result of introgression from banteng. To determine the level of introgression of the taste 2 receptor member 16 (TAS2R16) gene from banteng into Chinese cattle, two missense mutations in the bovine TAS2R16 gene were examined. Here, we explored the prevalence of the two variants in 28 indigenous Chinese cattle and banteng breeds (comprising 750 individuals) to determine the influence of banteng introgressions on Chinese cattle based on PCR and DNA sequencing. In our study, the two mutant alleles had a higher frequency distribution in southern China with strong geographic distribution, especially in the south-central and southeast areas. In conclusion, this study examines the impact of introgression on the frequency distributions of mutations in variable regions and the subsequent adaptation of Chinese indigenous cattle to different environmental conditions.

Copy number variation of GAL3ST1 gene is associated with growth traits of Chinese cattle.

Copy number variation (CNV) is a type of genomic structural variation, and the research on it has flourished in recent years. According to the high-throughput sequencing data, we found that the copy number variation region of the GAL3ST1 gene was correlated with the growth traits of bovine. It is significant that we study the CNV of GAL3ST1 gene and process the association analysis between results of Q-PCR and growth traits of Chinese cattle. In this research, SPSS software was used to detect the distribution of GAL3ST1 gene copy number in four cattle breeds and the correlation of growth traits was analyzed. Correlation analysis showed that GAL3ST1 CNV had positive effects on some growth traits of bovine (p < 0.05). In addition, the study detects the expression of GAL3ST1 gene in different tissues of Xia'nan cattles on mRNA level. The result showed that GAL3ST1 gene has different expression conditions in different tissues, results showed that the expression level was high in intestine and low in liver tissue. In a word, we speculated that the GAL3ST1 gene can be used as a molecular marker and this study confirmed that the CNV of it can provide theoretical basis for molecular breeding of cattle in China.

Deletions in GSN gene associated with growth traits of four Chinese cattle breeds.

The aim of this study was to assess the potential of 21 bp mutation in the second intron of the GSN gene as a molecular marker-assisted by exploring the effect of 21 bp mutation on growth traits in four beef cattle breeds. Gelsolin (GSN), a member of the superfamily of gel proteins, is involved in the regulation of a variety of cellular activities in the organism and plays an important function in cell motility, apoptosis, signal transduction and inflammatory responses. Gelatin can not only negatively regulate the pro-apoptotic effect of P53 protein, but also promote apoptosis by blocking the interaction between actin and deoxyribonuclease, so, the GSN gene was selected as a candidate gene in this study. In this study, a 21 bp mutation on the second intron to the GSN gene was verified in 573 individuals of Yunling (YL, n = 220), Jiaxian (JX, n = 140), Xianan (XN, n = 114) and Qinchuan (QC, n = 97) cattle breeds using Once PCR and agarose gel electrophoresis. The association analysis of polymorphisms in the GSN gene with growth traits in four breeds was revealed: in YL cattle, the heart girth and forehead size of heterozygous ID genotype were significantly higher than II genotype (P < 0.05). In JX cattle, the withers height, body length and heart girth of II and ID genotype were significantly highest than DD genotype (P < 0.01); the height at hip cross and height at sacrum of II genotype was significantly highest than DD genotype (P < 0.01), but ID genotype was significantly higher than DD genotype. In XN cattle, the abdominal girth and circumference of the cannon bone of II genotype were significantly higher than ID genotype (P < 0.05). In QC cattle, the hucklebone width of ID genotype was significantly the highest than II genotype (P < 0.01). The results suggest that GSN may be an important candidate gene and that a 21 bp mutation on the second intron to the GSN gene can be used for molecular marker-assisted selection of four beef cattle breeds.

The distribution of four missense mutations in SPTBN5 gene across native Chinese breeds.

Modern cattle belong to two subspecies, Bos taurus and Bos indicus. Since divergence, cattle types have accumulated different genetic variations, which have contributed to highly differentiated phenotypes. The mammalian inner ear possesses functional and morphological innovations that contribute to its unique hearing capacities. The spectrin beta, non-erythrocytic 5 (SPTBN5) gene has been shown to play an important function in the inner ear. Four missense mutations: rs522333459 (c.7232G > C:p.Cys2411Ser), rs718838405 (c.6568A > C:p.Met2190Leu), rs516536785 (c.6283C > T:p.Leu2095Phe) and rs480278206 (c.4201T > C:p.Cys1401Arg) were identified in the bovine SPTBN5 gene by whole genome resequencing (http://animal.nwsuaf.edu.cn/code/index.php/BosVar), which might be candidate mutations related with hearing of both taurine and indicine cattle. In our study, PCR and DNA sequencing were used to explore the allele frequencies of four mutations of 971 individuals belonging to 38 native Chinese cattle breeds. We find that four mutant alleles showing strong geographic distribution, consisting with the ancestry distribution of taurine and indicine in China. In addition, we identified four mutations of SPTBN5 were diverged in taurine and indicine cattle showing signatures of adaptive evolution in two subspecies, which might participate in bovine inner ear development.

Distribution of the variant at AKIRIN2: c.*188G > A in Chinese cattle.

Marbling score (MS), is an economically important trait in cattle. Previous results showed that a SNP (c.*188G > A) of akirin 2 (AKIRIN2) gene was associated with MS in Japanese Black cattle and Korean cattle. However, the distribution of the genotypic frequency of the single nucleotide polymorphism (SNP) has not been explored in Chinese cattle. In this study, we used polymerase chain reaction (PCR) and DNA sequencing to detect the variation in 1296 individuals from 39 Chinese cattle breeds, one semi-wild bovine species (Dulong) and three introduced breeds (Angus, Holstein and Brahman). Our study found the frequency of the A allele at this locus roughly diminished from north to south in Chinese cattle, and we detected statistically significant differences between Angus and Brahman (p < 0.05), Dulong and another two breeds (Angus and Holstein; p < 0.01) using Chi-Square Independence Test. Our results reflected the variation of AKIRIN2: c.*188G > A in Chinese cattle, which would help us better understand Chinese cattle genetic resources and provide reference for further research.

Large-scale genomic analysis reveals the genetic cost of chicken domestication.

BACKGROUND: Species domestication is generally characterized by the exploitation of high-impact mutations through processes that involve complex shifting demographics of domesticated species. These include not only inbreeding and artificial selection that may lead to the emergence of evolutionary bottlenecks, but also post-divergence gene flow and introgression. Although domestication potentially affects the occurrence of both desired and undesired mutations, the way wild relatives of domesticated species evolve and how expensive the genetic cost underlying domestication is remain poorly understood. Here, we investigated the demographic history and genetic load of chicken domestication. RESULTS: We analyzed a dataset comprising over 800 whole genomes from both indigenous chickens and wild jungle fowls. We show that despite having a higher genetic diversity than their wild counterparts (average π, 0.00326 vs. 0.00316), the red jungle fowls, the present-day domestic chickens experienced a dramatic population size decline during their early domestication. Our analyses suggest that the concomitant bottleneck induced 2.95% more deleterious mutations across chicken genomes compared with red jungle fowls, supporting the "cost of domestication" hypothesis. Particularly, we find that 62.4% of deleterious SNPs in domestic chickens are maintained in heterozygous states and masked as recessive alleles, challenging the power of modern breeding programs to effectively eliminate these genetic loads. Finally, we suggest that positive selection decreases the incidence but increases the frequency of deleterious SNPs in domestic chicken genomes. CONCLUSION: This study reveals a new landscape of demographic history and genomic changes associated with chicken domestication and provides insight into the evolutionary genomic profiles of domesticated animals managed under modern human selection.

DGAT1 K232A polymorphism is associated with milk production traits in Chinese cattle.

The production traits of cattle, especially milk trait, are of great significance to human life. A quantitative trait loci (QTL) associated with milk fat content was detected in the centromeric region of cattle chromosome 14. This QTL harbors a strong candidate gene called DGAT1 responsible for the milk quality. A non-conservative substitution of lysine by alanine (K232A) was found in DGAT1 gene producing a strong effect on milk composition and yield. The lysine (K allele) is associated with increased milk fat content, while the decreased milk fat content is linked to the alanine (A allele) amino acid. To estimate the frequencies of the DGAT1 K232A polymorphism in Chinese cattle breeds, PCR and DNA sequencing methods were used to investigate the polymorphism of DGAT1 K232A in a total of 682 individuals, including 655 Chinese cattle and 27 Holstein cattle. The results demonstrated that the frequency of K allele gradually elevated from the northern group to the southern group of native Chinese cattle, whereas the frequency of A allele showed a contrary pattern, displaying a significant geographical difference across native Chinese cattle breeds. Our results confirm that the southern cattle group has higher milk fat content than that of the northern group.

The combination between NCSTN gene copy number variation and growth traits in Chinese cattle.

Copy number variation (CNV) has been used as an important source of phenotypic and genetic diversity in recent years. Nicastrin (NCSTN) gene is usually attached to human diseases such as Alzheimer's disease, and Acne inversa. However, there are no essays about the NCSTN gene combining with cattle breeds. In our study, we discovered different distributions of NCSTN gene copy number and associated it with phenotypic traits in four Chinese yellow cattle breeds (XN, PN, QC and YL). The result turned out that the CNV of the NCSTN gene was associated with several growth traits, such as cannon circumference, chest girth and rump length (p < 0.05). In general, we revealed the eminence over CNV of NCSTN gene and economic traits, suggesting that the CNV of the NCSTN gene can be considered to be a promising molecular breeding marker of Chinese beef cattle.

Whole-genome resequencing reveals diversity, global and local ancestry proportions in Yunling cattle.

Yunling cattle, a three-breed cross consisting of 1/2 Brahman cattle, 1/4 Murray Grey cattle and 1/4 Yunnan Yellow cattle, has advantage of rapid growth, good meat quality, enhanced tolerance towards a hot and humid climate, tick resistance and crude feed forbearance. Here, we investigated the genetic diversity, global and local ancestry proportions by sequencing the genomes of 131 Yungling cattle and 31 Brahman cattle (for control). Furthermore, we used 35 published genomes of ancestry breeds (including Angus cattle [ancestry of Murray Grey], Wannan cattle [ancestry of Yunnan Yellow cattle: Wenshan and Dianzhong], Wenshan and Dianzhong cattle) to characterize the formation process of Yunling cattle. The highest nucleotide diversity was found in the Wannan cattle, followed by Wenshan, Dianzhong, Brahman, Yunling and Angus cattle. The results of LD decay in each breed was largely consistent with the results of nucleotide diversity, except for the faster decay in Angus cattle out of a smaller effective population size and a strong bottleneck during the breed formation. The population-structure analysis revealed that the cross-breed Yunling cattle harboured the ancestry with Angus (0.44), Brahman (0.48) and Wannan cattle (0.08). Subsequently, we used RFmix to infer local ancestry in Yunling cattle and then performed chi-squared test to obtain the segments whose proportions of a certain ancestry were excessive compared with the whole-genome level, leading to 391 Angus, 49 Brahman and 2,312 Wannan segments. Gene annotation and KEGG enrichment analysis revealed that the excessive Angus, Brahman and Wannan segments might contribute to the rapid growth, immune resistance and indigenous adaptation, respectively, in Yunling cattle. Our results help understand ancestry components and formation process in Yunling cattle and will provide an opportunity for selective breeding by molecular approaches in future.

Genome-wide association study identifies genomic loci associated with flight reaction in cattle.

Lower flight reaction is closely related to higher production in cattle, but the genetic basis of lower flight reaction is not clearly understood. Here, we sampled a total of 45 Brahman cattles and 166 Yunling cattles with flight distance (FD), and 73 Brahman cattles and 288 Yunling cattles with crush score (CS) and flight speed (FS), whereas there were 45 Brahman cattles and 161 Yunling cattles with all three traits. The FD, CS and FS in Brahman cattle were significantly lower than those in Yunling cattle. The flight reaction traits had negative correlation with conformational traits (e.g., body weight, withers height and body length). Based on SNPs derived from a subset of 162 whole genomes (25 Brahman genomes and 100 Yunling genomes with FD, 30 Brahman and 131 Yunling genomes with CS and FS), genome-wide association study with mixed linear model was performed to test potential associations between flight reaction traits and genomic variants. We identified five, two and two genomic loci suggestively associated with FD, CS and FS, respectively. Five out of five candidate genes for FD (LOC789753, LRP6, CTIF, SLC9A9 and ZEB1) were reported to be related to Alzheimer's disease representing cognitive impairment in human, which was consistent with the finding that cognitive-behavioural intervention decreased the FD of cows to human. In CS, a very strong association locus was assigned to CDH8, a cadherin involved in synaptic adhesion, axon outgrowth and guidance, whose deletion was associated with autism spectrum disorder. In FS, a very strong association locus was assigned to GABRG2, a gamma-aminobutyric acid (a major inhibitory neurotransmitter in brain) receptor, whose polymorphisms were associated with suicidal behaviour in schizophrenia patients. Our findings will provide targets for molecular-marker selection and genetic manipulation of cattle improvement to meeting the growing demand for lower flight reaction to human.