The molecular regulated mechanism of METTL3 and FTO in lipid metabolism of Hu sheep.

BACKGROUND: Balanced lipid metabolism can improve the growth performance and meat quality of livestock. The m6A methylation-related genes METTL3 and FTO play important roles in animal lipid metabolism; however, the mechanism through which they regulate lipid metabolism in sheep is unclear. RESULTS: We established lipid deposition models of hepatocytes and preadipocytes in Hu sheep. In the hepatocyte lipid deposition model, the genes expression levels of FABP4, Accα, ATGL and METTL3, METTL14, and FTO-were significantly up-regulated after lipid deposition (P < 0.05). Transcriptomic and metabolomic analyses showed that lipid deposition had a significant effect on MAPK, steroid biosynthesis, and glycerophospholipid metabolism pathway in hepatocytes. The m6A methylation level decreased but the difference was not significant after METTL3 interference, and the expression levels of FABP4 and ATGL increased significantly (P < 0.05); the m6A methylation level significantly increased following METTL3 overexpression, and LPL and ATGL expression levels significantly decreased (P < 0.05), indicating that overexpression of METTL3 inhibited the expression of lipid deposition-related genes in a m6A-dependent manner. The m6A methylation level was significantly increased, ATGL expression was significantly decreased (P < 0.05), and LPL, FABP4, and Accα expression was not significantly changed following FTO interference (P > 0.05); the m6A methylation level was significantly decreased after FTO overexpression, and LPL, FABP4, and ATGL expression was significantly increased (P < 0.05), indicating that FTO overexpression increased the expression of lipid deposition-related genes in a m6A-dependent manner. Transcriptomic and metabolomic analyses showed that m6A methylation modification mainly regulated lipid metabolism through triglyceride metabolism, adipocytokine signaling, MAPK signaling, and fat digestion and absorption in hepatocytes. In the lipid deposition model of preadipocytes, the regulation of gene expression is the same as that in hepatocytes. CONCLUSIONS: METTL3 significantly inhibited the expression of lipid deposition-related genes, whereas FTO overexpression promoted lipid deposition. Our study provides a theoretical basis and reference for accurately regulating animal lipid deposition by mastering METTL3 and FTO genes to promote high-quality animal husbandry.

Key genes and metabolites that regulate wool fibre diameter identified by combined transcriptome and metabolome analysis.

BACKGROUND: Fibre diameter is an important economic trait of wool fibre. As the fibre diameter decreases, the economic value of wool increases. Therefore, understanding the mechanism of wool fibre diameter regulation is important in improving the value of wool. RESULTS: In this study, we used non-targeted metabolome and reference transcriptome data to detect differences in metabolites and genes in groups of Alpine Merino sheep with different wool fibre diameter gradients, and integrated metabolome and transcriptome data to identify key genes and metabolites that regulate wool fibre diameter. We found 464 differentially abundant metabolites (DAMs) and 901 differentially expressed genes (DEGs) in four comparisons of groups with different wool fibre diameters. Approximately 25% of the differentially abundant metabolites were lipid and lipid-like molecules. These molecules were predicted to be associated with skin development and keratin filament by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. Key genes, including COL5A2, COL5A3, CREB3L4, COL1A1, and SFRP4, were identified by gene set enrichment analysis. CONCLUSIONS: Key genes regulating wool fibre diameter were identified, the effects of lipid molecules on wool performance were investigated, and potential synergies between genes and metabolites were postulated, providing a theoretical framework for fine wool sheep breeding.

Genome-wide detection of structural variation in some sheep breeds using whole-genome long-read sequencing data.

Genomic structural variants (SVs) constitute a significant proportion of genetic variation in the genome. The rapid development of long-reads sequencing has facilitated the detection of long-fragment SVs. There is no published study to detect SVs using long-read data from sheep. We applied a long-read mapping approach to detect SVs and characterized a total of 30,771 insertions, deletions, inversions and translocations. We identified 716, 916, 842 and 303 specific SVs in Southdown sheep, Alpine merino sheep, Qilian White Tibetan sheep and Oula sheep, respectively. We annotated these SVs and found that these SV-related genes were primarily enriched in the well-established pathways involved in the regulation of the immune system, growth and development and environmental adaptability. We detected and annotated SVs based on NGS resequencing data to validate the accuracy based on third-generation detection. Moreover, five candidate SVs were verified using the PCR method in 50 sheep. Our study is the first to use a long-reads sequencing approach to construct a novel structural variation map in sheep. We have completed a preliminary exploration of the potential effects of SVs on sheep.

Proteome Analysis of Alpine Merino Sheep Skin Reveals New Insights into the Mechanisms Involved in Regulating Wool Fiber Diameter.

Wool fiber is a textile material that is highly valued based on its diameter, which is crucial in determining its economic value. To analyze the molecular mechanisms regulating wool fiber diameter, we used a Data-independent acquisition-based quantitative proteomics approach to analyze the skin proteome of Alpine Merino sheep with four fiber diameter ranges. From three contrasts of defined groups, we identified 275, 229, and 190 differentially expressed proteins (DEPs). Further analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that pathways associated with cyclic adenosine monophosphate and peroxisome proliferator-activated receptor signaling are relevant to wool fiber diameter. Using the K-means method, we investigated the DEP expression patterns across wool diameter ranges. Using weighted gene co-expression network analysis, we identified seven key proteins (CIDEA, CRYM, MLX, TPST2, GPD1, GOPC, and CAMK2G) that may be involved in regulating wool fiber diameter. Our findings provide a theoretical foundation for identifying DEPs and pathways associated with wool fiber diameter in Alpine Merino sheep to enable a better understanding of the molecular mechanisms underlying the genetic regulation of wool fiber quality.

Molecular Mechanism of m6A Methylation Modification Genes METTL3 and FTO in Regulating Heat Stress in Sheep.

Heat stress is an important environmental factor affecting livestock production worldwide. Primary hepatocytes and preadipocytes derived from Hu sheep were used to establish a heat stress model. Quantitative reverse transcriptase-PCR (qRT-PCR) analysis showed that heat induction significantly increased the expression levels of heat stress protein (HSP) genes and the N6-methyladenosine (m6A) methylation modification genes: methyltransferase-like protein 3 (METTL3), methyltransferase-like protein 14 (METTL14), and fat mass and obesity associated protein (FTO). Heat stress simultaneously promoted cell apoptosis. Transcriptome sequencing identified 3980 upregulated genes and 2420 downregulated genes related to heat stress. A pathway enrichment analysis of these genes revealed significant enrichment in fatty acid biosynthesis, degradation, and the PI3K-Akt and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Overexpression of METTL3 in primary hepatocytes led to significant downregulation of HSP60, HSP70, and HSP110, and significantly increased mRNA m6A methylation; FTO interference generated the opposite results. Primary adipocytes showed similar results. Transcriptome analysis of cells under METTL3 (or FTO) inference and overexpression revealed differentially expressed genes enriched in the mitogen-activated protein kinase (MAPK) signaling pathways, as well as the PI3K-Akt and Ras signaling pathways. We speculate that METTL3 may increase the level of m6A methylation to inhibit fat deposition and/or inhibit the expression of HSP genes to enhance the body's resistance to heat stress, while the FTO gene generated the opposite molecular mechanism. This study provides a scientific basis and theoretical support for sheep feeding and management practices during heat stress.

Development of an Improved Method for the Isolation and Culture of Newborn Sheep Primary Hepatocytes.

The liver plays a crucial role in metabolism, synthesis, biotransformation, secretion, and excretion. Hepatocytes are the main cells of the liver and can be used as a cell model to study liver function. The classic method of collagenase perfusion to isolate hepatocytes is a two-step technique that is time-consuming, labor-intensive, and has high technical requirements. Therefore, in this study, we compared different methods for isolating and culturing primary hepatocytes. We found that the 0.25% trypsin and 0.1 mg/mL type IV collagenase mixture at a 1:1 ratio showed the most efficient cell digestion, and William's Medium E complete medium showed the best growth and proliferation. The isolated cells showed the typical irregular polygonal morphology of hepatocytes. Periodic acid−Schiff staining and immunofluorescence confirmed that the isolated cells were positive for glycogen and hepatocyte-specific markers cytokeratin 18, AFP, and albumin. On subculturing, stable cell lines were obtained. Therefore, we optimized the isolation and in vitro culture method to obtain highly pure (>95%) sheep primary hepatocytes from newborn sheep liver tissue.

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity.

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics ChromiumTM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (Apale and Adark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, Apale and Adark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.

Genome-wide association studies detects candidate genes for wool traits by re-sequencing in Chinese fine-wool sheep.

BACKGROUND: The quality and yield of wool determine the economic value of the fine-wool sheep. Therefore, discovering markers or genes relevant to wool traits is the cornerstone for the breeding of fine-wool sheep. In this study, we used the Illumina HiSeq X Ten platform to re-sequence 460 sheep belonging to four different fine-wool sheep breeds, namely, Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Aohan fine-wool sheep (AHS) and Qinghai fine-wool sheep (QHS). Eight wool traits, including fiber diameter (FD), fiber diameter coefficient of variance (FDCV), fiber diameter standard deviation (FDSD), staple length (SL), greasy fleece weight (GFW), clean wool rate (CWR), staple strength (SS) and staple elongation (SE) were examined. A genome-wide association study (GWAS) was performed to detect the candidate genes for the eight wool traits. RESULTS: A total of 8.222 Tb of raw data was generated, with an average of approximately 8.59X sequencing depth. After quality control, 12,561,225 SNPs were available for analysis. And a total of 57 genome-wide significant SNPs and 30 candidate genes were detected for the desired wool traits. Among them, 7 SNPs and 6 genes are related to wool fineness indicators (FD, FDCV and FDSD), 10 SNPs and 7 genes are related to staple length, 13 SNPs and 7 genes are related to wool production indicators (GFW and CWR), 27 SNPs and 10 genes associated with staple elongation. Among these candidate genes, UBE2E3 and RHPN2 associated with fiber diameter, were found to play an important role in keratinocyte differentiation and cell proliferation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results, revealed that multitude significant pathways are related to keratin and cell proliferation and differentiation, such as positive regulation of canonical Wnt signaling pathway (GO:0090263). CONCLUSION: This is the first GWAS on the wool traits by using re-sequencing data in Chinese fine-wool sheep. The newly detected significant SNPs in this study can be used in genome-selective breeding for the fine-wool sheep. And the new candidate genes would provide a good theoretical basis for the fine-wool sheep breeding.

A global analysis of CNVs in Chinese indigenous fine-wool sheep populations using whole-genome resequencing.

BACKGROUND: Copy number variation (CNV) is an important source of genetic variation that has a significant influence on phenotypic diversity, economically important traits and the evolution of livestock species. In this study, the genome-wide CNV distribution characteristics of 32 fine-wool sheep from three breeds were analyzed using resequencing. RESULTS: A total of 1,747,604 CNVs were detected in this study, and 7228 CNV regions (CNVR) were obtained after merging overlapping CNVs; these regions accounted for 2.17% of the sheep reference genome. The average length of the CNVRs was 4307.17 bp. "Deletion" events took place more frequently than "duplication" or "both" events. The CNVRs obtained overlapped with previously reported sheep CNVRs to variable extents (4.39-55.46%). Functional enrichment analysis showed that the CNVR-harboring genes were mainly involved in sensory perception systems, nutrient metabolism processes, and growth and development processes. Furthermore, 1855 of the CNVRs were associated with 166 quantitative trait loci (QTL), including milk QTLs, carcass QTLs, and health-related QTLs, among others. In addition, the 32 fine-wool sheep were divided into horned and polled groups to analyze for the selective sweep of CNVRs, and it was found that the relaxin family peptide receptor 2 (RXFP2) gene was strongly influenced by selection. CONCLUSIONS: In summary, we constructed a genomic CNV map for Chinese indigenous fine-wool sheep using resequencing, thereby providing a valuable genetic variation resource for sheep genome research, which will contribute to the study of complex traits in sheep.

Genomic analysis of worldwide sheep breeds reveals PDGFD as a major target of fat-tail selection in sheep.

BACKGROUND: Fat tail is a unique trait in sheep acquired during domestication. Several genomic analyses have been conducted in sheep breeds from limited geographic origins to identify the genetic factors underlying this trait. Nevertheless, these studies obtained different candidates. The results of these regional studies were easily biased by the breed structures. RESULTS: To minimize the bias and distinguish the true candidates, we used an extended data set of 968 sheep representing 18 fat-tailed breeds and 14 thin-tailed breeds from around the world, and integrated two statistical tests to detect selection signatures, including Genetic Fixation Index (FST) and difference of derived allele frequency (ΔDAF). The results showed that platelet derived growth factor D (PDGFD) exhibited the highest genetic differentiation between fat- and thin-tailed sheep breeds. Analysis of sequence variation identified that a 6.8-kb region within the first intron of PDGFD is likely the target of positive selection and contains regulatory mutation(s) in fat-tailed sheep. Histological and gene expression analyses demonstrated that PDGFD expression is associated with maturation and hemostasis of adipocytes. Further retrospective analysis of public transcriptomic datasets revealed that PDGFD expression is down-regulated during adipogenesis in both human and mouse, and is higher in fat tissues of obese individuals than that in lean individuals. CONCLUSIONS: These results reveal that PDGFD is the predominant factor for the fat tail phenotype in sheep by contributing to adiopogenesis and maintaining the hemostasis of mature adipocytes. This study provides insights into the selection of fat-tailed sheep and has important application to animal breeding, as well as obesity-related human diseases.

Genome-wide characterization of drought-responsive long non-coding RNAs in sorghum (Sorghum bicolor).

Drought stress strongly affects crop yield. Although knowledge of long non-coding RNAs (lncRNAs) has been updated continuously and rapidly, information about lncRNAs in drought resistance regulation is extremely limited in sorghum. Here, lncRNA-sequencing was performed with seedlings of a sorghum cultivar (Jinza29) under three water control treatments to investigate the mechanism of lncRNAs responsible for drought resistance in sorghum. A total of 377 differentially expressed lncRNAs (DElncRNAs) were identified. We also predicted 4322 and 2827 transcripts as potential cis-target and trans-target genes for drought-responsive lncRNAs, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that those target genes exhibited marked enrichment into "oxidoreductase activity", "signal transducer activity", "DNA repair", "photosynthesis", "glutathione metabolism", and "phenylpropanoid biosynthesis" and other terms associated with abiotic stress resistance. Moreover, several lncRNAs were estimated to modulate the expression of other genes related to stress response and photosynthetic carbon metabolism. Additionally, we found 107 DElncRNAs that might be candidate target mimics for 56 miRNAs. LncRNAs play important roles in drought adaptation of sorghum through interacting with protein-encoding genes. The obtained results provided novel insights into the biological characteristics of lncRNAs and offered potential regulatory factors for genetically enhancing drought resistance in sorghum.

Hybridization promotes growth performance by altering rumen microbiota and metabolites in sheep.

Hybridization can substantially improve growth performance. This study used metagenomics and metabolome sequencing to examine whether the rumen microbiota and its metabolites contributed to this phenomenon. We selected 48 approximately 3 month-old male ♂Hu × â™€Hu (HH, n = 16), ♂Poll Dorset × â™€Hu (DH, n = 16), and ♂Southdown × â™€Hu (SH, n = 16) lambs having similar body weight. The sheep were fed individually under the same nutritional and management conditions for 95 days. After completion of the trial, seven sheep close to the average weight per group were slaughtered to collect rumen tissue and content samples to measure rumen epithelial parameters, fermentation patterns, microbiota, and metabolite profiles. The final body weight (FBW), average daily gain (ADG), and dry matter intake (DMI) values in the DH and SH groups were significantly higher and the feed-to-gain ratio (F/G) significantly lower than the value in the HH group; additionally, the papilla height in the DH group was higher than that in the HH group. Acetate, propionate, and total volatile fatty acid (VFA) concentrations in the DH group were higher than those in the HH group, whereas NH3-N concentration decreased in the DH and SH groups. Metagenomic analysis revealed that several Prevotella and Fibrobacter species were significantly more abundant in the DH group, contributing to an increased ability to degrade dietary cellulose and enrich their functions in enzymes involved in carbohydrate breakdown. Bacteroidaceae bacterium was higher in the SH group, indicating a greater ability to digest dietary fiber. Metabolomic analysis revealed that the concentrations of rumen metabolites (mainly lysophosphatidylethanolamines [LPEs]) were higher in the DH group, and microbiome-related metabolite analysis indicated that Treponema bryantii and Fibrobacter succinogenes were positively correlated with the LPEs. Moreover, we found methionine sulfoxide and N-methyl-4-aminobutyric acid were characteristic metabolites in the DH and SH groups, respectively, and are related to oxidative stress, indicating that the environmental adaptability of crossbred sheep needs to be further improved. These findings substantially deepen the general understanding of how hybridization promotes growth performance from the perspective of rumen microbiota, this is vital for the cultivation of new species and the formulation of precision nutrition strategies for sheep.

[Characterization of sequences, expression profiling, and natural allelic variation analysis of the MYC gene family in sorghum (Sorghum bicolor)].

Sorghum aphid (Melanaphis sacchari) and head smut fungi (Sporisorium reilianum) infesting sorghum cause delayed growth and development, and reduce yield and quality. This study use bioinformatics and molecular biological approaches to profile the gene expression pattern during sorghum development and under pest infestation, and analyzed the natural allelic DNA variation of sorghum MYC gene family. The findings provide insights for potential application in breeding the stress resistant and high productivity sorghum varieties. The results indicated that there are 28 MYC genes identified in sorghum genome, distributed on 10 chromosomes. The bHLH_MYC_N and HLH domains are the conserved domains of the MYC gene in sorghum. Gene expression analysis showed that SbbHLH35.7g exhibited high expression levels in leaves, SbAbaIn showed strong expression in early grains, and SbMYC2.1g showed high expression levels in mature pollen. In anti-aphid strains at the 5-leaf stage, SbAbaIn, SbLHW.4g and SbLHW.2g were significantly induced in leaves, while SbbHLH35.7g displayed the highest expression level in panicle tissue, which was significantly induced by the infection of head smut. Promoter cis-element analysis identified methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA) and MYB-binding sites related to drought-stress inducibility. Furthermore, genomic resequencing data analysis revealed natural allelic DNA variations such as single nucleotide polymorphism (SNP) and insertion-deletion (INDEL) for the key SbMYCs. Protein interaction network analysis using STRING indicated that SbAbaIn interacts with TIFYdomain protein, and SbbHLH35.7g interacts with MDR and imporin. SbMYCs exhibited temporal and spatial expression patterns and played vital roles during the sorghum development. Infestation by sugarcane aphids and head smut fungi induced the expression of SbAbaIn and SbbHLH35.7g, respectively. SbAbaIn modulated the jasmonic acid (JA) pathway to regulate the expression of defensive genes, conferring resistance to insects. On the other hand, SbbHLH35.7g participated in detoxification reactions to defend against pathogens.

Analysis of Geographic and Pairwise Distances among Chinese Cashmere Goat Populations.

This study investigated the geographic and pairwise distances of nine Chinese local Cashmere goat populations through the analysis of 20 microsatellite DNA markers. Fluorescence PCR was used to identify the markers, which were selected based on their significance as identified by the Food and Agriculture Organization of the United Nations (FAO) and the International Society for Animal Genetics (ISAG). In total, 206 alleles were detected; the average allele number was 10.30; the polymorphism information content of loci ranged from 0.5213 to 0.7582; the number of effective alleles ranged from 4.0484 to 4.6178; the observed heterozygosity was from 0.5023 to 0.5602 for the practical sample; the expected heterozygosity ranged from 0.5783 to 0.6464; and Allelic richness ranged from 4.7551 to 8.0693. These results indicated that Chinese Cashmere goat populations exhibited rich genetic diversity. Further, the Wright's F-statistics of subpopulation within total (FST) was 0.1184; the genetic differentiation coefficient (GST) was 0.0940; and the average gene flow (Nm) was 2.0415. All pairwise FST values among the populations were highly significant (p<0.01 or p<0.001), suggesting that the populations studied should all be considered to be separate breeds. Finally, the clustering analysis divided the Chinese Cashmere goat populations into at least four clusters, with the Hexi and Yashan goat populations alone in one cluster. These results have provided useful, practical, and important information for the future of Chinese Cashmere goat breeding.

Association of SLIT3 and ZNF280B Gene Polymorphisms with Wool Fiber Diameter.

The SLIT3 gene encodes a secreted protein, and the ZNF280B gene is a member of the transcription factor family. Both genes have multiple biological functions. This study was conducted to investigate the association between SLIT3 and ZNF280B gene polymorphisms and wool fiber diameter and to determine potential molecular marker sites for breeding sheep with fine wool. We used Kompetitive Allele-Specific PCR to type the single nucleotide polymorphism (SNP) loci in the SLIT3 and ZNF280B genes within 1081 Alpine Merino sheep and associated these SNPs with wool fiber diameter. The results revealed one SNP in SLIT3 and ZNF280B, which were each related to sheep fiber diameter. The wool fiber diameters of sheep with the CC genotype in SLIT3 g.478807C>G and AA genotype in ZNF280B g.677G>A were the smallest and differed significantly from the diameters of other genotypes (p < 0.05). These results suggest potential molecular marker sites for fine-wool sheep breeding.

Transcriptomics and metabolomics reveal improved performance of Hu sheep on hybridization with Southdown sheep.

Consumers are increasingly demanding high-quality mutton. Cross breeding can improve meat quality and is widely used in sheep breeding. However, little is known about the molecular mechanism of cross breeding sheep meat quality. In this study, male Southdown and female Hu sheep were hybridized. The slaughter performance and longissimus dorsi quality of the 6-month-old hybrid offspring were measured, and the longissimus dorsi of the hybrid offspring was analyzed by transcriptomics and metabolomics to explore the effect of cross breeding on meat quality. The results showed that the production performance of Southdown × Hu F1 sheep was significantly improved, the carcass fat content was significantly decreased, and the eating quality of Southdown × Hu F1 sheep were better. Compared with the HS group (Hu × Hu), the NH group (Southdown × Hu) had 538 differentially expressed genes and 166 differentially expressed metabolites (P < 0.05), which were significantly enriched in amino acid metabolism and other related pathways. Up-regulated genes METTL21C, PPARGC1A and down-regulated gene WFIKKN2 are related to muscle growth and development. Among them, the METTL21C gene, which is related to muscle development, was highly correlated with carnosine, a metabolite related to meat quality (correlation > 0.6 and P < 0.05). Our results provide further understanding of the molecular mechanism of cross breeding for sheep muscle growth and meat quality optimization.

Global DNA Methylation, miRNA, and mRNA Profiles in Sheep Skeletal Muscle Promoted by Hybridization.

With the development of high-throughput sequencing technology, several nongenetic variations, including noncoding RNAs such as miRNAs, and DNA methylation, have been found to play an important role in animal muscle development and fat metabolism. In this study, Southdown and Suffolk were selected as male parents for hybridization with Hu sheep (Southdown × Hu (NH), Suffolk × Hu (SH), and Hu × Hu (HH)). RNA sequencing, bisulfite sequencing, and small-RNA sequencing were used to study the methylation patterns and differences in miRNA and mRNA expression in the F1 sheep longissimus dorsi muscle tissue. We identified 765 differentially expressed genes (DEGs), 10,161 differentially methylated regions (DMRs), and 164 differentially expressed miRNAs, which were significantly enriched in AMPK signaling, fatty acid degradation, metabolism, and other related pathways (P < 0.05). In addition, we constructed a DNA methylation-mRNA and miRNA-mRNA coexpression network. A total of 42 common genes were identified from DMRs and DEGs. Importantly, we predicted that 33 differentially expressed miRNAs directly or indirectly targeted the SLC27A6. The data obtained in this study provide useful information and evidence to support further understanding of the miRNA and DNA methylation of key genes regulating muscle growth and fat metabolism in hybrid sheep populations.

Correlation of 20 Single-Nucleotide Polymorphisms with Weight and Wool Traits in Alpine Merino Sheep.

SNPs associated with important traits of fine-wool sheep that were previously obtained through genome-wide association analysis screening were verified and analyzed. A total of 20 SNPs related to birth weight, bundle strength, cleaning rate, and fiber diameter were screened using whole-genome resequencing, and the SNPshot assay was used to detect and analyze polymorphisms. This study found that, among the 20 SNPs associated with important traits in Alpine Merino sheep, 8 were monomorphic and 12 were polymorphic, of which 6 showed moderate polymorphisms and 6 showed low polymorphisms. The heterozygosity of the 12 polymorphic loci ranged from 0.10 to 0.49, the effective number of alleles ranged from 1.11 to 1.98, and the polymorphic information content ranged from 0.09 to 0.37. The chi-square test showed that only RHPN2:g.42678119T>G and RALYL:g.90030866A>G were in Hardy-Weinberg disequilibrium (p < 0.05); the other loci were in equilibrium (p > 0.05). These SNPs associated with important traits in Alpine Merino sheep provide a theoretical basis for genomic selection and molecular design breeding.

[Identification, expression and DNA variation analysis of high affinity nitrate transporter NRT2/3 gene family in Sorghum bicolor].

Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3, SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.

Effects of Sheep Sires on Muscle Fiber Characteristics, Fatty Acid Composition and Volatile Flavor Compounds in F1 Crossbred Lambs.

Crossbreeding significantly improves meat production performance in sheep; however, whether hybridization changes the meat quality characteristics of lambs is uncertain. We analyzed the effects of three different hybrid sires on muscle fiber characteristics (MFCs), fatty acid composition (FAC), and volatile flavor compounds (VFCs) in lambs under identical feeding conditions. Compared with those of purebred lambs, the muscle fiber diameter and cross-sectional areas of the crossbred lambs were significantly decreased (p < 0.05), and the collagen fiber content was significantly increased (p < 0.05). The numbers and area ratios of the fast and slow muscle fibers did not significantly differ between the purebred and crossbred lambs, but the expressions of four MyHC gene types differed significantly (p < 0.05). Twenty-three fatty acids were identified in both the purebred and crossbred lambs, of which thirteen were differentially expressed (p < 0.05). Saturated fatty acid (SFA) contents in the crossbred lambs were significantly increased (p < 0.05), whereas the monounsaturated fatty acid content was significantly decreased (p < 0.05). Polyunsaturated fatty acid/SFA and n-6/n-3 ratios were significantly lower in the crossbred lambs than in the purebred lambs (p < 0.05). Twenty-five VFCs were identified among the three hybrids, and aldehydes were the main VFCs. Eleven VFCs were differentially expressed in the crossbred lambs (p < 0.05). Hybrid sires affected the MFCs, FAC, and VFCs of the F1 lambs, thus providing a reference for high-quality mutton production.