Transcriptomics analysis of cashmere fineness functional genes.

Liaoning cashmere goat (LCG) is a famous cashmere goat breed in China. Cashmere fineness, as an important index to evaluate cashmere quality, is also one of the problems to be improved for Liaoning cashmere goats. Transcriptome studies all mRNA transcribed by a specific tissue or cell in a certain period. It is a key link in the study of gene expression regulation. It plays an important role in the analysis of biological growth and disease. Transcriptome is spatio-temporal specific, that is, gene expression varies in different tissues or at different times. Three coarser and three fine LCG skin samples were sequenced by RNA-seq technology, and a total of 427 differentially expressed genes were obtained, including 291 up-regulated genes and 136 down-regulated genes. In the experiment, we screened out 16 genes that had significant differences in the expression of coarse and fine cashmere of Liaoning cashmere goats, so it was inferred that these 16 genes might have regulatory effects on cashmere fineness. Moreover, GO gene set enrichment analysis revealed that differential genes mainly consist of immune response, MHC protein complex, Heme binding and other pathways. KEGG analysis showed that transplant-versus-host disease and allograft rejection were the main pathways of differential genes.

High-throughput analysis of lncRNA in cows with naturally infected Staphylococcus aureus mammary gland.

LncRNA (long non-coding RNA) is an RNA molecule with a length between 200 and 100,000 nt. It does not encode proteins and is involved in a variety of intracellular processes, becoming a research hotspot of genetics. To identify key lncRNAs associated with dairy mastitis, we collected mammary epithelial tissue samples of Normal disease-free Holstein cows (HCN) and unhealthy Holstein cows with Staphylococcus aureus (HCU) and performed RNA sequencing (RNA-seq) on the samples. A total of 270 differentially expressed lncRNAs and 500 differentially expressed mRNAs were identified by high-throughput sequencing and bioinformatics analysis. Furthermore, Hydrolase activity is the most enriched in GO, and ErbB signaling pathway is significantly enriched in KEGG. In addition, through qPCR validation of 5 candidate lncRNAs in HCN and HCU, four differentially expressed lncRNAs MSTRG.498, MSTRG57.1, MSTRG.41.1 and MSTRG 124.1 were confirmed to have significant differentially expressed in cow mastitis. Also, lncRNA MSTRG.498 and its target gene, SMC4, might directly or indirectly play a role in cow mastitis. The regulatory network of lncRNA-miRNA-mRNA has been inferred from a bioinformatics perspective, which may assist understand the underlying molecular mechanism of lncRNAs involved in regulating mastitis in cows. Our findings will provide meaningful resources for further research on the regulatory function of lncRNAs in cow mastitis.

CeRNA regulates network and expression and SNP effect on NFKBIA of cashmere fineness.

In this study, a total of 1140 Liaoning Cashmere Goats (LCG) were genotyped for single nucleotide polymorphism (SNP) of NFKBIA gene. There are 15 SNPs and 7 genotypes have been found, and G1547A (GG) genotype has been associated with cashmere fineness and cashmere yield. An integrated ceRNA regulatory network of NFKBIA gene was made. To prove NFKBIA and these non-coding RNAs (ncRNAs) may be related to cashmere fineness, we performed qPCR on these ncRNA in LCG coarse type skin (CT-LCG) and LCG fine type skin (FT-LCG). The result of qPCR showed lncRNA XLOC_011060 and ciRNA452 are at high expression level in CT-LCG, all miRNAs appear high expressed in FT-LCG, and mir-93 was the most significant difference between CT-LCG and FT-LCG. In addition, five miRNAs were selected for qPCR in different genotypes. The qPCR results showed that mir-93 might negatively regulate cashmere fineness and mir-17-5p may play a positive role in regulating cashmere fineness of individuals with G1355A (AG) genotype. These results demonstrated that NFKBIA gene is associated with cashmere fineness of LCG and G1547A (GG) genotype is the preferred marker genotype for cashmere fineness.

Analysis of m6A methylation in skin tissues of different sex Liaoning cashmere goats.

N6-methyladenosine (m6A) is the most frequent internal modification of mRNA and lncRNA in eukaryotes. We used two high-throughput sequencing method, m6A-seq and RNA-seq to identify pivotal m6A-modified genes in cashmere fineness and fiber growth. 8062 m6A peaks were detected by m6A-seq, including 2157 upregulated and 6445 downregulated. Furthermore, by comparing m6A-modified genes of the male Liaoning Cashmere Goat (M-LCG) and female Liaoning Cashmere Goat (F-LCG) skin tissues, we get 862 differentially expressed m6A-modified genes. To identify differently expressed m6A genes associated with cashmere fineness, 11 genes were selected for validation using real time fluorescent quantitative PCR in M-LCG and F-LCG. This study provides an acadamic basis on the molecular regulation mechanism of m6A modification in cashmere growth process.

Association analysis between reproduction genes INHA, PGR, RARG with lamb and other traits of Liaoning cashmere goats.

Reproductive traits have a high economic value in goat breeding, and increasing the number of lambs produced by ewes is of great importance to improve the production efficiency of goat farming. Lambing traits in goats are low heritability traits, but their genetic basis is ultimately determined by genes. This study aimed to investigate the relationship between INHA, RARG, and PGR gene polymorphisms and production performance, such as lambing, cashmere production, milk production, and body size in Liaoning cashmere goats. A total of six single nucleotide polymorphisms (SNPs) loci were identified in these three genes, G144A and T504C on the INHA gene, A56G, G144A, G490C on the RARG gene, and G109519T on the PGR gene. For lambing and cashmere production traits, the AA genotype of G144A on the INHA gene, TT on the T504C genotype, GG genotype of G144A on the INHA gene, A56G, G144A, and T504C on RARG and G109519T on PGR gene are dominant genotypes. AATT is a dominant haplotype combination. Allele G can be used as a molecular marker for lambing, cashmere, and milk production traits in Liaoning cashmere goats. Marker-assisted selection can be used for early selection to achieve improvement of genetic traits in Liaoning cashmere goats.

Association analysis for SNPs of BAAT and COL1A1 genes with cashmere production performance and other production traits in Liaoning cashmere goats.

This study aimed to investigate the relationship between the polymorphism of bile acid-CoA: amino acid N-acyltransferase (BAAT) and collagen type I alpha 1 chain (COL1A1) genes and the production performance of Liaoning Cashmere goat (LCG). The potential single nucleotide polymorphisms (SNPs) of LCG were detected by sequence comparison of BAAT and COL1A1 genes and PCR-Seq polymorphism, and the effect of SNPs on production performance was analyzed by SPSS software. The results showed that three SNPs loci were detected in BAAT gene: G7900A, T7967C, C7998T, and one SNP locus T6716C was detected in COL1AL gene. At G7900A locus, the dominant genotype for cashmere performance was GG, and the dominant genotype for body measurement traits and milk production traits was AG. At T7967C locus, the dominant genotype for cashmere performance was TT, and the dominant genotype for body measurement traits and milk production traits was CC. At C7998T locus, TT was the dominant genotype for cashmere performance, body measurement traits, and milk production traits. At the T6716C locus, TT was the dominant genotype for cashmere performance, body measurement traits, and milk production traits. H1H1: AACC is the dominant haplotype combination. Therefore, this study will provide a reliable reference for future research on cashmere production performance, body measurement traits, and milk production traits of LCG.

Association analysis for SNPs of KRT26 and TCHH genes with cashmere production performance, body measurement traits and milk production traits in Liaoning cashmere goats.

Cashmere fineness is getting thicker, which is one of the key problems in cashmere breeding, however, there have been no systematic studies on the molecular regulation of cashmere fineness. The aim of this study was to investigate the relationship between KRT26 and TCHH gene polymorphism and production performance in Liaoning cashmere goats (LCG). The potential single nucleotide polymorphisms (SNPs) of LCG were detected by sequence alignment and PCR-Seq polymorphism of KRT26 and TCHH genes and analyzed the effect of SNPs on production performance by SPSS software. Two SNPs sites (A559T and A6839G) of two genes were detected. The AA genotype of KRT26 A559T locus was the dominant genotype. AG and GG at TCHH A6839G locus were the dominant genotypes. AAAA was the dominant haplotype combination. The results showed that KRT26 and TCHH genes were associated with cashmere fineness of LCG, and A559T (AA) and A6839G (GG) genotypes were the preferred marker genotypes for cashmere fineness, which provided more theoretical basis for further research on cashmere fineness.

Association analysis for SNPs of MSTN and IGFBP-3 genes with body size and other production traits in Liaoning Cashmere Goats.

Liaoning cashmere goat (LCG) have tall bones, high cashmere production and outstanding meat production performance. In recent years, good breeding progress has not been made in terms of body size, meat yield, milk yield and other properties in terms of production. The study focused on the correlation between the SNPs of MSTN and IGFBP-3 genes with the body size performance, cashmere production and milk performance. The MSTN and IGFBP-3 gene sequence alignment and PCR-Seq polymorphism were used to detect the potential SNPs, and the correlation with production performance was analyzed by SPSS and SHEsis software. The results showed that the TT genotype at the T1662G locus of the MSTN gene is dominant and has significant advantages in body measurements such as sacrum height, chest width, and waist height. The C allele at the C4021T locus of IGFBP-3 gene shows an advantage in the body measurement performance. Among the haplotype combinations, H2H2:TGTC is preponderant combination for body size performance, H2H2:TGTC and H1H2:TGCC are preponderant combinations for cashmere production performance, H1H3:GGCC is preponderant combination for milk production performance. It may be a molecular marker for future selection and breeding.

Association analysis for SNPs of LIPE and ITGB4 genes with cashmere production performance, body measurement traits and milk production traits in Liaoning cashmere goats.

Liaoning cashmere goat (LCG) is one of the excellent cashmere goat breeds in China. Because of its larger size, better cashmere, and better cashmere production performance, people pay special attention to it. This article mainly studied the relationship between SNP loci of LIPE gene and ITGB4 gene and milk production, cashmere production and body measurement traits of LCGs. We further identified potential SNP loci by PCR-Seq polymorphism detection and gene sequence comparison of LIPE and ITGB4 genes. Further, we use SPSS and SHEsis software to analyze their relationship to production performance. The consequence indicated that CC genotype of LIPE gene T16409C locus was dominant genotype in milk production and cashmere production, while CT genotype of LIPE gene T16409C locus was dominant in body size. The CT genotype of C168T locus of ITGB4 gene is the dominant genotype of body type and cashmere production, while the dominant genotype of milk production is TT genotype. Through joint analysis, in haploid combinations, H1H2:CCCT is the dominant haplotype combination in cashmere fineness. H3H4:TTCT is a dominant haplotype combination of milk production traits and body measurement traits. These dominant genotypes can provide a reliable basis for the study of production performance of LCG.

Proteomic analysis of coarse and fine skin tissues of Liaoning cashmere goat.

Proteomics is the study of all proteins expressed by a cell or even an organism. However, knowledge of proteins that regulate the fineness of cashmere is limited. Liaoning cashmere goat (LCG) is a valuable genetic resource of China. The skin samples of Liaoning cashmere goats during the growing period were collected, performed tandem mass tag (TMT) method, and identified 117 differentially expressed proteins in CT_LCG (course type) and FT_LCG (fine type). To verify proteins differentially expressed in LCG, we performed PRM validation on three candidate proteins (ALB, SDC1, and ITGB4) in CT-LCG and FT-LCG. Furthermore, primary metabolic process and lysosome are most enriched in the GO and KEGG pathways, respectively. In addition, we also derived a protein-protein interaction (PPI) regulatory network from the perspective of bioinformatics. This study sought to elucidate the molecular mechanism of differential proteins regulating cashmere fineness of Liaoning cashmere goats by using TMT quantitative proteomics analysis. Differentially expressed proteins ALB and SDC1 may regulate cashmere fineness; ITGB4 can become a promising protein for further study. They can be used as key proteins to lay a foundation for studying cashmere fineness of Liaoning cashmere goats.

High-throughput analysis of CircRNA in cows with naturally infected Staphylococcus aureus mammary gland.

Circular RNAs (CircRNA) are a special type of non-coding RNA molecule with a closed ring structure and are not affected by RNA exonucases. It has stable expression, is not easy to degrade, and exists in most eukaryotes. However, circRNA regulation of cow mastitis has not been widely recognized. Mammary epithelial tissues were collected from healthy Holstein cows (HCN) and mastitis Holstein cows (HCU). RNA sequencing (RNA SEQ) was performed for the differentially expressed circRNAs, and analysis results showed that 19 differentially expressed circRNAs were identified in HCN and HCU, among which 6 circRNAs were up-regulated and 13 circRNAs were down-regulated. We randomly selected nine circRNAs for Q-PCR verification, and the results showed consistent expression. Three circRNAs: circRNA2860, circRNA5323 and circRNA4027 were confirmed to be significantly differentially expressed circRNAs in cow mastitis. Also, their host genes TRPS1, SLC12A2 and MYH11 might be directly or indirectly play a role in cow mastitis. Furthermore, RNA polymerase transcription factor binding and tight junction are most enriched in GO and KEGG pathways, respectively. In addition, the regulatory network of circRNA-miRNA has been inferred from a bioinformatics perspective, which may help to understand the underlying molecular mechanism of circRNAs involved in regulating mastitis in cows.

Metabolomic Analysis and MRM Verification of Coarse and Fine Skin Tissues of Liaoning Cashmere Goat.

One of the critical elements in evaluating the quality of cashmere is its fineness, but we still know little about how it is regulated at the metabolic level. In this paper, we use UHPLC-MS/MS detection and analysis technology to compare the difference in metabolites between coarse cashmere (CT_LCG) and fine cashmere (FT_LCG) skin of Liaoning cashmere goats. According to the data, under positive mode four metabolites were significantly up-regulated and seven were significantly down-regulated. In negative mode, seven metabolites were significantly up-regulated and fourteen metabolites were significantly down-regulated. The two groups' most significant metabolites, Gly-Phe and taurochenodeoxycholate, may be crucial in controlling cashmere's growth, development, and fineness. In addition, we enriched six KEGG pathways, of which cholesterol metabolism, primary bile acid biosynthesis, and bile secretion were enriched in positive and negative modes. These findings offer a new research idea for further study into the critical elements influencing cashmere's fineness.

Association analysis of single-nucleotide polymorphism in prolactin and its receptor with productive and body conformation traits in Liaoning cashmere goats.

The results of this study showed that the single-nucleotide polymorphism (SNP) sites of the PRL and PRLR genes have a certain association with the milk production performance, body size and cashmere performance of Liaoning cashmere goats (LCGs). Through our designed experiment, the potential SNPs of LCG were detected by sequence alignment, and two SNPs were found on two genes. The CC genotype of the PRL gene is the dominant genotype among the three genotypes. The GG genotype of the PRLR gene is the dominant genotype among the two genotypes. At the same time, the two genotypes also have good performance in cashmere production and body size. Through the screening of haplotype combination, the milk fat rate >  7.6 %, the milk protein rate >  5.6 %, the milk somatic cell number <  1500  × â€¯10 3  mL - 1 , the cashmere fineness <  15.75  µ m, the chest girth >  105 cm, the chest depth >  33 cm, and the waist height >  67.5 cm are considered as screening indexes for comprehensive production performance of Liaoning cashmere goats. It is concluded that the GCGC type is the dominant haplotype combination. According to our research data, we found that the biological indicators of Liaoning cashmere goat milk are higher than the national standards, so we think it is very significant to study the milk production performance of our experiment. Further research can be done on goat milk production and body conformation traits around PRL gene and PRLR gene.

Screening of cashmere fineness-related genes and their ceRNA network construction in cashmere goats.

Competitive endogenous RNA (ceRNA) is a transcript that can be mutually regulated at the post-transcriptional level by competing shared miRNAs. The ceRNA network connects the function of protein-encoded mRNA with the function of non-coding RNA, such as microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA). However, compared with the ceRNA, the identification and combined analysis of lncRNAs, mRNAs, miRNAs, and circRNAs in the cashmere fineness have not been completed. Using RNA-seq technology, we first identified the miRNAs presented in Liaoning Cashmere Goat (LCG) skin, and then analyzed the mRNAs, lncRNAs, circRNAs expressed in LCG and Inner Mongolia cashmere goat (MCG) skin. As a result, 464 known and 45 new miRNAs were identified in LCG skin. In LCG and MCG skin, 1222 differentially expressed mRNAs were identified, 170 differentially expressed lncRNAs and 32 differentially expressed circRNAs were obtained. Then, qRT-PCR was used to confirm further the representative lncRNAs, mRNAs, circRNAs and miRNAs. In addition, miRanda predicted the relationships of ceRNA regulatory network among lncRNAs, circRNAs, miRNAs and mRNAs, the potential regulatory effects were investigated by Go and KEGG analysis. Through the screening and analysis of the results, the ceRNA network regulating cashmere fineness was constructed. LncRNA MSTRG14109.1 and circRNA452 were competed with miRNA-2330 to regulated the expression of TCHH, KRT35 and JUNB, which may provide a potential basis for further research on the process of regulating the cashmere fineness.

Selection of Cashmere Fineness Functional Genes by Translatomics.

Cashmere fineness is an important index to evaluate cashmere quality. Liaoning Cashmere Goat (LCG) has a large cashmere production and long cashmere fiber, but its fineness is not ideal. Therefore, it is important to find genes involved in cashmere fineness that can be used in future endeavors aiming to improve this phenotype. With the continuous advancement of research, the regulation of cashmere fineness has made new developments through high-throughput sequencing and genome-wide association analysis. It has been found that translatomics can identify genes associated with phenotypic traits. Through translatomic analysis, the skin tissue of LCG sample groups differing in cashmere fineness was sequenced by Ribo-seq. With these data, we identified 529 differentially expressed genes between the sample groups among the 27197 expressed genes. From these, 343 genes were upregulated in the fine LCG group in relation to the coarse LCG group, and 186 were downregulated in the same relationship. Through GO enrichment analysis and KEGG enrichment analysis of differential genes, the biological functions and pathways of differential genes can be found. In the GO enrichment analysis, 491 genes were significantly enriched, and the functional region was mainly in the extracellular region. In the KEGG enrichment analysis, the enrichment of the human papillomavirus infection pathway was seen the most. We found that the COL6A5 gene may affect cashmere fineness.

Single-Cell Sequencing Reveals Differential Cell Types in Skin Tissues of Liaoning Cashmere Goats and Key Genes Related Potentially to the Fineness of Cashmere Fiber.

Cashmere fineness is one of the important factors determining cashmere quality; however, our understanding of the regulation of cashmere fineness at the cellular level is limited. Here, we used single-cell RNA sequencing and computational models to identify 13 skin cell types in Liaoning cashmere goats. We also analyzed the molecular changes in the development process by cell trajectory analysis and revealed the maturation process in the gene expression profile in Liaoning cashmere goats. Weighted gene co-expression network analysis explored hub genes in cell clusters related to cashmere formation. Secondary hair follicle dermal papilla cells (SDPCs) play an important role in the growth and density of cashmere. ACTA2, a marker gene of SDPCs, was selected for immunofluorescence (IF) and Western blot (WB) verification. Our results indicate that ACTA2 is mainly expressed in SDPCs, and WB results show different expression levels. COL1A1 is a highly expressed gene in SDPCs, which was verified by IF and WB. We then selected CXCL8 of SDPCs to verify and prove the differential expression in the coarse and fine types of Liaoning cashmere goats. Therefore, the CXCL8 gene may regulate cashmere fineness. These genes may be involved in regulating the fineness of cashmere in goat SDPCs; our research provides new insights into the mechanism of cashmere growth and fineness regulation by cells.

m6A Methylation Analysis of Differentially Expressed Genes in Skin Tissues of Coarse and Fine Type Liaoning Cashmere Goats.

N6-methyladenosine (m6A) is the most common internal modification in mRNAs of all higher eukaryotes. Here we perform two high-throughput sequencing methods, m6A-modified RNA immunoprecipitation sequence (MeRIP-seq) and RNA sequence (RNA-seq) to identify key genes with m6A modification in cashmere fiber growth. A total of 9,085 m6A sites were differentially RNA m6A methylated as reported from by MeRIP-seq, including 7,170 upregulated and 1,915 downregulated. In addition, by comparing m6A-modified genes between the fine-type Liaoning cashmere goat (FT-LCG) and coarse-type Liaoning Cashmere Goat (CT-LCG) skin samples, we obtain 1,170 differentially expressed genes. In order to identify the differently methylated genes related to cashmere fiber growth, 19 genes were selected to validate by performing qRT-PCR in FT-LCG and CT-LCG. In addition, GO enrichment analysis shows that differently methylated genes are mainly involved in keratin filament and intermediate filament. These findings provide a theoretical basis for future research on the function of m6A modification during the growth of cashmere fiber.

Fabrication of Metal-Substituted Polyoxometalates for Colorimetric Detection of Dopamine and Ractopamine.

A novel colorimetric detection method based on the peroxidase-like activity of metal-substituted polyoxometalates (POMs) of SiW9M3 (M = Co2+, Fe3+, Cu2+, Mn2+) has been established. POMs can catalyze oxidation of dopamine (DA) and ractopamine (RAC) by H2O2 in aqueous solutions. SiW9Co3-based POMs detect DA at concentrations as low as 5.38 × 10−6 mol·L−1 simply by observation of the color change from colorless to orange using the naked eye. RAC is detected by observing the change from colorless to slight red by SiW9Cu3 with a detection limit of 7.94 × 10−5 mol·L−1. This study shows that colorimetric DA and RAC detection using SiW9Co3 and SiW9Cu3 is highly selective and sensitive as well as visually observable.