Melatonin promotes proliferation of Inner Mongolia cashmere goat hair follicle papilla cells through Wnt10b.

The study demonstrated that melatonin (MT) can induce the development of secondary hair follicles in Inner Mongolian cashmere goats through the Wnt10b gene, leading to secondary dehairing. However, the mechanisms underlying the expression and molecular function of Wnt10b in dermal papilla cells (DPC) remain unknown. This research aimed to investigate the impact of MT on DPC and the regulation of Wnt10b expression, function, and molecular mechanisms in DPC. The findings revealed that MT promotes DPC proliferation and enhances DPC activity. Co-culturing DPC with overexpressed Wnt10b and MT showed a significant growth promotion. Subsequent RNA sequencing (RNA-seq) of overexpressed Wnt10b and control groups unveiled the regulatory role of Wnt10b in DPC. Numerous genes and pathways, including developmental pathways such as Wnt and MAPK, as well as processes like hair follicle morphogenesis and hair cycle, were identified. These results suggest that Wnt10b promotes the growth of secondary hair follicles in Inner Mongolian cashmere goats by regulating crucial factors and pathways in DPC proliferation.

Genome-wide association study for cashmere traits in Inner Mongolia cashmere goat population reveals new candidate genes and haplotypes.

BACKGROUND: The cashmere goat industry is one of the main pillars of animal husbandry in Inner Mongolia Autonomous Region, and plays an irreplaceable role in local economic development. With the change in feeding methods and environment, the cashmere produced by Inner Mongolia cashmere goats shows a tendency of coarser, and the cashmere yield can not meet the consumption demand of people. However, the genetic basis behind these changes is not fully understood. We measured cashmere traits, including cashmere yield (CY), cashmere diameter (CD), cashmere thickness (CT), and fleece length (FL) traits for four consecutive years, and utilized Genome-wide association study of four cashmere traits in Inner Mongolia cashmere goats was carried out using new genomics tools to infer genomic regions and functional loci associated with cashmere traits and to construct haplotypes that significantly affect cashmere traits. RESULTS: We estimated the genetic parameters of cashmere traits in Inner Mongolia cashmere goats. The heritability of cashmere yield, cashmere diameter, and fleece length traits of Inner Mongolia cashmere goats were 0.229, 0.359, and 0.250, which belonged to the medium heritability traits (0.2 ~ 0.4). The cashmere thickness trait has a low heritability of 0.053. We detected 151 genome-wide significantly associated SNPs with four cashmere traits on different chromosomes, which were very close to the chromosomes of 392 genes (located within the gene or within ± 500 kb). Notch3, BMPR1B, and CCNA2 have direct functional associations with fibroblasts and follicle stem cells, which play important roles in hair follicle growth and development. Based on GO functional annotation and KEGG enrichment analysis, potential candidate genes were associated with pathways of hair follicle genesis and development (Notch, P13K-Akt, TGF-beta, Cell cycle, Wnt, MAPK). We calculated the effective allele number of the Inner Mongolia cashmere goat population to be 1.109-1.998, the dominant genotypes of most SNPs were wild-type, the polymorphic information content of 57 SNPs were low polymorphism (0 < PIC < 0.25), and the polymorphic information content of 79 SNPs were moderate polymorphism (0.25 < PIC < 0.50). We analyzed the association of SNPs with phenotypes and found that the homozygous mutant type of SNP1 and SNP3 was associated with the highest cashmere yield, the heterozygous mutant type of SNP30 was associated with the lowest cashmere thickness, the wild type of SNP76, SNP77, SNP78, SNP80, and SNP81 was associated with the highest cashmere thickness, and the wild type type of SNP137 was associated with the highest fleece length. 21 haplotype blocks and 68 haplotype combinations were constructed. Haplotypes A2A2, B2B2, C2C2, and D4D4 were associated with increased cashmere yield, haplotypes E2E2, F1F1, G5G5, and G1G5 were associated with decreased cashmere fineness, haplotypes H2H2 was associated with increased cashmere thickness, haplotypes I1I1, I1I2, J1J4, L5L3, N3N2, N3N3, O2O1, P2P2, and Q3Q3 were associated with increased cashmere length. We verified the polymorphism of 8 SNPs by KASP, and found that chr7_g.102631194A > G, chr10_g.82715068 T > C, chr1_g.124483769C > T, chr24_g.12811352C > T, chr6_g.114111249A > G, and chr6_g.115606026 T > C were significantly genotyped in verified populations (P < 0.05). CONCLUSIONS: In conclusion, the genetic effect of single SNP on phenotypes is small, and SNPs are more inclined to be inherited as a whole. By constructing haplotypes from SNPs that are significantly associated with cashmere traits, it will help to reveal the complex and potential causal variations in cashmere traits of Inner Mongolia cashmere goats. This will be a valuable resource for genomics and breeding of the cashmere goat.

Genetic diversity analysis of Inner Mongolia cashmere goats (Erlangshan subtype) based on whole genome re-sequencing.

BACKGROUND: Inner Mongolia cashmere goat (IMCG), renowned for its superior cashmere quality, is a Chinese indigenous goat breed that has been developed through natural and artificial selection over a long period. However, recently, the genetic resources of IMCGs have been significantly threatened by the introduction of cosmopolitan goat breeds and the absence of adequate breed protection systems. RESULTS: In order to assess the conservation effectiveness of IMCGs and efficiently preserve and utilize the purebred germplasm resources, this study analyzed the genetic diversity, kinship, family structure, and inbreeding of IMCGs utilizing resequencing data from 225 randomly selected individuals analyzed using the Plink (v.1.90), GCTA (v.1.94.1), and R (v.4.2.1) software. A total of 12,700,178 high-quality SNPs were selected through quality control from 34,248,064 SNP sites obtained from 225 individuals. The average minor allele frequency (MAF), polymorphic information content (PIC), and Shannon information index (SHI) were 0.253, 0.284, and 0.530, respectively. The average observed heterozygosity (Ho) and the average expected heterozygosity (He) were 0.355 and 0.351, respectively. The analysis of the identity by state distance matrix and genomic relationship matrix has shown that most individuals' genetic distance and genetic relationship are far away, and the inbreeding coefficient is low. The family structure analysis identified 10 families among the 23 rams. A total of 14,109 runs of homozygosity (ROH) were identified in the 225 individuals, with an average ROH length of 1014.547 kb. The average inbreeding coefficient, calculated from ROH, was 0.026 for the overall population and 0.027 specifically among the 23 rams, indicating a low level of inbreeding within the conserved population. CONCLUSIONS: The IMCGs exhibited moderate polymorphism and a low level of kinship with inbreeding occurring among a limited number of individuals. Simultaneously, it is necessary to prevent the loss of bloodline to guarantee the perpetuation of the IMCGs' germplasm resources.

Accuracy of Genomic prediction for fleece traits in Inner Mongolia Cashmere goats.

The fleece traits are important economic traits of goats. With the reduction of sequencing and genotyping cost and the improvement of related technologies, genomic selection for goats has become possible. The research collect pedigree, phenotype and genotype information of 2299 Inner Mongolia Cashmere goats (IMCGs) individuals. We estimate fixed effects, and compare the estimates of variance components, heritability and genomic predictive ability of fleece traits in IMCGs when using the pedigree based Best Linear Unbiased Prediction (ABLUP), Genomic BLUP (GBLUP) or single-step GBLUP (ssGBLUP). The fleece traits considered are cashmere production (CP), cashmere diameter (CD), cashmere length (CL) and fiber length (FL). It was found that year of production, sex, herd and individual ages had highly significant effects on the four fleece traits (P < 0.01). All of these factors should be considered when the genetic parameters of fleece traits in IMCGs are evaluated. The heritabilities of FL, CL, CP and CD with ABLUP, GBLUP and ssGBLUP methods were 0.26 ~ 0.31, 0.05 ~ 0.08, 0.15 ~ 0.20 and 0.22 ~ 0.28, respectively. Therefore, it can be inferred that the genetic progress of CL is relatively slow. The predictive ability of fleece traits in IMCGs with GBLUP (56.18% to 69.06%) and ssGBLUP methods (66.82% to 73.70%) was significantly higher than that of ABLUP (36.73% to 41.25%). For the ssGBLUP method is significantly (29% ~ 33%) higher than that with ABLUP, and which is slightly (4% ~ 14%) higher than that of GBLUP. The ssGBLUP will be as an superiors method for using genomic selection of fleece traits in Inner Mongolia Cashmere goats.

Knockdown of miR-361-5p promotes the induced activation of SHF-stem cells through FOXM1 mediated Wnt/ß-catenin pathway in cashmere goats.

The inducing activation event of secondary hair follicle (SHF)-stem cells is considered a key biological process in the SHF regeneration, and the morphogenesis of cashmere fiber in cashmere goats. The miR-361-5p was essentially implicated in the induced activation of SHF-stem cells of cashmere goats, but its functional mechanisms are unclear. Here, we confirmed miR-361-5p was significantly downregulated in anagen SHF bugle of cashmere goats compared with that at telogen, and miR-361-5p expression was significantly lower in SHF-stem cells after activation than its counterpart before activation. Further, we found that miR-361-5p could negatively regulate the induced activation event of SHF-stem cells in cashmere goats. Mechanistically, through dual-luciferase reporter assays, miR-361-5p specifically bound with FOXM1 mRNA in SHF-stem cells of cashmere goats and negatively regulated the expression of FOXM1 gene. Also, through overexpression/knockdown analysis of FOXM1 gene, our results indicated that FOXM1 upregulated the expression of Wnt/ß-catenin pathway related genes in SHF-stem cells. Moreover, based on TOP/FOP-flash Wnt report assays, the knockdown of miR-361-5p promotes the Wnt/ß-catenin pathway activation through upregulating the FOXM1 expression in SHF-stem cells. Finally, we demonstrated that miR-361-5p negatively regulated the induced activation of SHF-stem cells through FOXM1 mediated Wnt/ß-catenin pathway in cashmere goats.

Drivers of plateau adaptability in cashmere goats revealed by genomic and transcriptomic analyses.

BACKGROUND: The adaptive evolution of plateau indigenous animals is a current research focus. However, phenotypic adaptation is complex and may involve the interactions between multiple genes or pathways, many of which remain unclear. As a kind of livestock with important economic value, cashmere goat has a high ability of plateau adaptation, which provides us with good materials for studying the molecular regulation mechanism of animal plateau adaptation. RESULTS: In this study, 32 Jiangnan (J) and 32 Tibetan (T) cashmere goats were sequenced at an average of 10. Phylogenetic, population structure, and linkage disequilibrium analyses showed that natural selection or domestication has resulted in obvious differences in genome structure between the two breeds. Subsequently, 553 J vs. T and 608 T vs. J potential selected genes (PSGs) were screened. These PSGs showed potential relationships with various phenotypes, including myocardial development and activity (LOC106502520, ATP2A2, LOC102181869, LOC106502520, MYL2, ISL1, and LOC102181869 genes), pigmentation (MITF and KITLG genes), hair follicles/hair growth (YAP1, POGLUT1, AAK1, HES1, WNT1, PRKAA1, TNKS, WNT5A, VAX2, RSPO4, CSNK1G1, PHLPP2, CHRM2, PDGFRB, PRKAA1, MAP2K1, IRS1, LPAR1, PTEN, PRLR, IBSP, CCNE2, CHAD, ITGB7, TEK, JAK2, and FGF21 genes), and carcinogenesis (UBE2R2, PIGU, DIABLO, NOL4L, STK3, MAP4, ADGRG1, CDC25A, DSG3, LEPR, PRKAA1, IKBKB, and ABCG2 genes). Phenotypic analysis showed that Tibetan cashmere goats has finer cashmere than Jiangnan cashmere goats, which may allow cashmere goats to better adapt to the cold environment in the Tibetan plateau. Meanwhile, KRTs and KAPs expression in Jiangnan cashmere goat skin was significantly lower than in Tibetan cashmere goat. CONCLUSIONS: The mutations in these PSGs maybe closely related to the plateau adaptation ability of cashmere goats. In addition, the expression differences of KRTs and KAPs may directly determine phenotypic differences in cashmere fineness between the two breeds. In conclusion, this study provide a reference for further studying plateau adaptive mechanism in animals and goat breeding.

CircRNA-1967 participates in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression.

Circular RNAs (circRNAs), a novel class of non-coding RNAs, can interact with miRNAs through a sequence-driven sponge mechanism, thereby regulating the expression of their downstream target genes. CircRNA-1967 was found in secondary hair follicles (SHFs) of cashmere goats, but its functions are not clear. Here, we showed that both circRNA-1967 and its host gene BNC2 had significantly higher expression in SHF bulge at anagen than those at telogen of cashmere goats. Also, circRNA-1967 participates in the differentiation of SHF stem cells (SHF-SCs) into hair follicle lineage in cashmere goats. RNA pull-down assay verified that circRNA-1967 interacts with miR-93-3p. We also indicated that circRNA-1967 promoted LEF1 expression in SHF-SCs of cashmere goats. By dual-luciferase reporter analysis, we found that circRNA-1967 up-regulated LEF1 expression through the miR-93-3p-mediated pathway. The results from this study demonstrated that circRNA-1967 participated in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression. Our founding might constitute a novel pathway for revealing the potential mechanism of the differentiation of SHF-SCs into hair follicle lineage in cashmere goats. Also, these results provided a valuable basis for further enhancing the intrinsic regeneration of cashmere goat SHFs with the formation and growth of cashmere fibers.

MiR-125b regulates the differentiation of hair follicles in Fine-wool Sheep and Cashmere goats by targeting MXD4 and FGFR2.

With the development of miRNAs identification technology, more and more miRNAs have been discovered, and the role of miRNAs in the development of animal hair follicles has become a focus of research on hair-producing animals. In the previous experiment, compare the microRNA (miRNA) trancriptomes of goats and sheep skin using Solexa sequencing and differentially expressed miR-125b was screened. However, the mechanism of miR-125b regulating hair follicle development is not clear. Therefore, in the present study, the expression of miR-125b, MXD4 and FGFR2 in skin tissue of Fine-wool Sheep and Cashmere goats and HEK-293T cells was examined by qPCR and Western blot. Furthermore, the correlation between miR-125b and the predicted target gene (MXD4, FGFR2) was verified using the Dual-luciferase Reporter assay. We demonstrated that the expression of MXD4 and FGFR2 in Cashmere goats was significantly higher than that of Fine-wool Sheep, and the expression was opposite to that of miR-125b. miR-125b can down-regulate the levels of MXD4 and FGFR2. Dual-luciferase reporter gene assay showed that miR-125b could bind to the 3'-UTR region of target genes FGFR2 and MXD4, suggesting that MXD4 and FGFR2 were target genes of miR-125b. This study has shown that the growth and development of hair follicles in skin tissue of Fine-wool Sheep and Cashmere goats from the new regulatory levels of miRNAs, and clarified the mechanism of miR-125b and its target genes in the development of hair follicles in the skin.

Derivation of Arbas Cashmere Goat Induced Pluripotent Stem Cells in LCDM with Trophectoderm Lineage Differentiation and Interspecies Chimeric Abilities.

The Arbas cashmere goat is a unique biological resource that plays a vital role in livestock husbandry in China. LCDM is a medium with special small molecules (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) for generation pluripotent stem cells (PSCs) with bidirectional developmental potential in mice, humans, pigs, and bovines. However, there is no report on whether LCDM can support for generation of PSCs with the same ability in Arbas cashmere goats. In this study, we applied LCDM to generate goat induced PSCs (giPSCs) from goat fetal fibroblasts (GFFs) by reprogramming. The derived giPSCs exhibited stem cell morphology, expressing pluripotent markers, and could differentiate into three germ layers. Moreover, the giPSCs differentiated into the trophectoderm lineage by spontaneous and directed differentiation in vitro. The giPSCs contributed to embryonic and extraembryonic tissue in preimplantation blastocysts and postimplantation chimeric embryos. RNA-sequencing analysis showed that the giPSCs were very close to goat embryos at the blastocyst stage and giPSCs have similar properties to typical extended PSCs (EPSCs). The establishment of giPSCs with LCDM provides a new way to generate PSCs from domestic animals and lays the foundation for basic and applied research in biology and agriculture.

Effect of the FA2H Gene on cashmere fineness of Jiangnan cashmere goats based on transcriptome sequencing.

BACKGROUND: Cashmere goats are a heterogeneous hairy mammal. The fineness of cashmere can affect its economic value. Therefore, in this study, we used transcriptome sequencing techniques to analyze the gene expression profiles of the skin tissues of cashmere goats with different cashmere fineness. The selected candidate genes were functionally verified with the secondary hair follicle hair papillary cells of cashmere goats. RESULTS: We identified 479 DEGs, of which 238 mRNAs were up-regulated in the fine velvet group and 241 mRNA were down-regulated. Based on functional annotation and protein interaction network analysis, we found some genes that may affect the fineness of cashmere, including SOX18, SOX4, WNT5A, IGFBP4, KAP8, KRT36, and FA2H. Using qRT-PCR, Western blot, CCK-8 cell viability detection, EDU cell proliferation detection, and flow cytometry, we found that overexpression of the FA2H gene could promote the proliferation of secondary hair follicle DPCs in cashmere goats. At the same time, we proved that FA2H could regulate the expression levels of the FGF5 and BMP2 genes in DPCs. CONCLUSION: The results of this study provide a useful reference for the genetics and breeding of Jiangnan cashmere goats and goat genome annotation, and provide an experimental basis for improving cashmere quality of the cashmere goat.

Genome-wide association study of fleece traits in Inner Mongolia Cashmere goats.

Inner Mongolia Cashmere goat is a well-known local cashmere goat breed in China. It is famous for excellent fleece quality and a significant advantage in cashmere yield compared to other cashmere goat breeds. In this study, a genome-wide association study was used to investigate fiber length, fiber diameter, and cashmere yield of 192 Inner Mongolia Cashmere goats using the Illumina GoatSNP52K Beadchip panel. We discovered that four single nucleotide polymorphisms (SNPs) reached genome-wide significance levels. These SNPs were located in some genes, e.g. FGF12, SEMA3D, EVPL, and SOX5, possibly related to fleece traits in Inner Mongolia Cashmere goat. Gene ontology enrichment analysis revealed that these genes were enriched in several biological pathways that were involved in hair follicle development in cashmere goats. In summary, the identified significant SNPs and genes provide useful information to explore genetic mechanisms underlying the variation in fleece traits and genomic selection of Chinese cashmere goat.

MiR-27a regulates WNT3A and KITLG expression in Cashmere goats with different coat colors.

MicroRNAs(miRNAs) regulate and control gene expression at the post-transcriptional level by base pairing with its target gene 3'UTR, resulting in degradation of the target mRNA or inhibition of its translation. The previous high-throughput sequencing results indicated that miR-27a was involved in coat color regulation. However, the mechanism of action is not still illuminated. In this research, using western blotting and real-time quantitative polymerase chain reaction(qRT-PCR), the expression of miR-27a, WNT3A and KITLG were examined in the skin of Cashmere goats with white and brown, and human embryonic kidney 293 T cells (HEK-293T cells) which over-express miR-27a. Targeting relationship between miR-27a and WNT3A or KITLG was examined by the luciferase reporter gene system. The qRT-PCR detection showed that miR-27a was more highly expressed in white Cashmere goats skin than that in brown Cashmere goats skin. Furthermore, WNT3A and KITLG mRNA and protein expression were down-regulated by miR-27a in vitro and in vivo. A dual-luciferase reporter gene indicated that miR-27a negatively correlates with WNT3A or KITLG. In a word, our research demonstrated that the expression of miR-27a was evidently differential in the white and brown Cashmere goats skin and WNT3A or KITLG was negatively regulated by miR-27a.

Synchronous profiling and analysis of mRNAs and ncRNAs in the dermal papilla cells from cashmere goats.

BACKGROUND: Dermal papilla cells (DPCs), the "signaling center" of hair follicle (HF), delicately master continual growth of hair in mammals including cashmere, the fine fiber annually produced by secondary HF embedded in cashmere goat skins. Such unparalleled capacity bases on their exquisite character in instructing the cellular activity of hair-forming keratinocytes via secreting numerous molecular signals. Past studies suggested microRNA (miRNAs) and long non-coding RNAs (lncRNAs) play essential roles in a wide variety of biological process, including HF cycling. However, their roles and related molecular mechanisms in modulating DPCs secretory activities are still poorly understood. RESULTS: Here, we separately cultivated DPCs and their functionally and morphologically distinct dermal fibroblasts (DFs) from cashmere goat skins at anagen. With the advantage of high throughput RNA-seq, we synchronously identified 2540 lncRNAs and 536 miRNAs from two types of cellular samples at 4th passages. Compared with DFs, 1286 mRNAs, 18 lncRNAs, and 42 miRNAs were upregulated, while 1254 mRNAs, 53 lncRNAs and 44 miRNAs were downregulated in DPCs. Through overlapping with mice data, we ultimately defined 25 core signatures of DPCs, including HOXC8 and RSPO1, two crucial activators for hair follicle stem cells (HFSCs). Subsequently, we emphatically investigated the impacts of miRNAs and lncRNAs (cis- and trans- acting) on the genes, indicating that ncRNAs extensively exert negative and positive effects on their expressions. Furthermore, we screened lncRNAs acting as competing endogenous RNAs (ceRNAs) to sponge miRNAs and relief their repressive effects on targeted genes, and constructed related lncRNAs-miRNAs-HOXC8/RSPO1 interactive lines using bioinformatic tools. As a result, XR_310320.3-chi-miR-144-5p-HOXC8, XR_311077.2-novel_624-RSPO1 and others lines appeared, displaying that lncRNAs might serve as ceRNAs to indirectly adjust HFSCs status in hair growth. CONCLUSION: The present study provides an unprecedented inventory of lncRNAs, miRNAs and mRNAs in goat DPCs and DFs. We also exhibit some miRNAs and lncRNAs potentially participate in the modulation of HFSCs activation via delicately adjusting core signatures of DPCs. Our report shines new light on the latent roles and underlying molecular mechanisms of ncRNAs on hair growth.

Differential expression of miR-let7a in hair follicle cycle of Liaoning cashmere goats and identification of its targets.

In order to improve the production and quality of Chinese cashmere, the research of hair follicle development has aroused more and more attention; the regulation mechanism of miRNA in hair follicle development has become a hot spot. A survey of transcriptome profiling screened 10 hair follicle-related miRNAs that were differentially expressed, including miR-let7a. In this study, the expression of miR-let7a was lower in anagen of hair follicle of cashmere goats than that in catagen of hair follicle of cashmere goats (p < 0.01). Results were in accordance with transcriptome data. The expression patterns of miR-let7a target genes (IGF-1R, C-myc, and FGF5) were verified by qRT-PCR, which were consistent with the results of Western blot and showed a downward trend. The dual-luciferase reporter gene system was used to verify the correlation between the expression of miR-let7a and its target genes, and it showed that miR-let7a negatively correlates with C-myc and FGF5. Present study offers new information on miRNAs and their related target genes in the regulation of hair follicle development mechanism.

Differential responses of rumen and fecal fermentation and microbiota of Liaoning cashmere goats after 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester supplementation.

The 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi), a rumen protective methionine, has been extensively studied in dairy cows and beef cattle and has been shown to regulate gastrointestinal microbiota and improve production performance. However, knowledge of the application of HMBi on cashmere goats and the simultaneous study of rumen and hindgut microbiota is still limited. In this study, HMBi supplementation increased the concentration of total serum protein, the production of microbial protein in the rumen and feces, as well as butyrate production in the feces. The results of PCoA and PERMANOVA showed no significant difference between the rumen microbiota, but there was a dramatic difference between the fecal microbiota of the two groups of Cashmere goats after the HMBi supplementation. Specifically, in the rumen, HMBi significantly increased the relative abundance of some fiber-degrading bacteria (such as Fibrobacter) compared with the CON group. In the feces, as well as a similar effect as in the rumen (increasing the relative abundance of some fiber-degrading bacteria, such as Lachnospiraceae FCS020 group and ASV32), HMBi diets also increased the proliferation of butyrate-producing bacteria (including Oscillospiraceae UCG-005 and Christensenellaceae R-7 group). Overall, these results demonstrated that HMBi could regulate the rumen and fecal microbial composition of Liaoning cashmere goats and benefit the host.

N6-methyladenosine (m6A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter.

Objective: The objective of this study was to identify the m6A-circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m6A-circHECA and promoter methylation of its host gene (HECA). Methods: The validation of circHECA m6A sites was performed using Methylation Immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m6A-circHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSP-PCR) technique. Results: The m6A-circHECA was confirmed to contain four m6A modification sites including m6A-213, m6A-297, m6A-780, and m6A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m6A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m6A-circHECA. Conclusion: The m6A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m6A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

CircERCC6 Positively Regulates the Induced Activation of SHF Stem Cells in Cashmere Goats via the miR-412-3p/BNC2 Axis in an m6A-Dependent Manner.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m6A modified sites (named m6A-685, m6A-862, and m6A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m6A-modified circERCC6 (m6A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m6A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m6A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.

Combined Genome-Wide Association Study and Haplotype Analysis Identifies Candidate Genes Affecting Growth Traits of Inner Mongolian Cashmere Goats.

In this study, genome-wide association analysis was performed on the growth traits (body height, body length, chest circumference, chest depth, chest width, tube circumference, and body weight) of Inner Mongolian cashmere goats (Erlangshan type) based on resequencing data. The population genetic parameters were estimated, haplotypes were constructed for the significant sites, and association analysis was conducted between the haplotypes and phenotypes. A total of two hundred and eighty-four SNPs and eight candidate genes were identified by genome-wide association analysis, gene annotation, and enrichment analysis. The phenotypes of 16 haplotype combinations were significantly different by haplotype analysis. Combined with the above results, the TGFB2, BAG3, ZEB2, KCNJ12, MIF, MAP2K3, HACD3, and MEGF11 functional candidate genes and the haplotype combinations A2A2, C2C2, E2E2, F2F2, I2I2, J2J2, K2K2, N2N2, O2O2, P2P2, R1R1, T1T1, W1W1, X1X1, Y1Y1, and Z1Z1 affected the growth traits of the cashmere goats and could be used as molecular markers to improve the accuracy of early selection and the economic benefits of breeding.

Study of the Influence of Non-Genetic Factors on the Growth and Development Traits and Cashmere Production Traits of Inner Mongolia White Cashmere Goats (Erlangshan Type).

The purpose of this study was to investigate the effects of non-genetic factors on the growth and development performance of Inner Mongolia white cashmere goats (Erlanghan type), such as birth weight (BW), weaning weight (WW), 6-month weight (6 WT), 12-month weight (12 WT), body height (BH), and body length (BL), and wool production performance, such as cashmere fineness (CF), cashmere thickness (CT), and cashmere yield (CY). The research objects were 4654 kids produced by 45 buck goats and 2269 doe goats in the Erlang Mountain Ranch of Beiping Textile Co., Ltd., Inner Mongolia, from 2020 to 2023. Based on the generalized linear model, ANOVA was used to analyze the effects of non-genetic factors, such as birth year (Y), birth month (M), sex (S), birth type (T), birth herd (H), assay flock (F), age at measurement (MA), and the age of doe goats at lambing (DLA), on growth and development traits and cashmere traits. The results show that the birth weight (BW), weaning weight (WW), 6-month weight (6 WT), 12-month weight (12 WT), body length (BL), body height (BH), chest depth (CD), chest width (CW), chest circumference (CC), cannon circumference (CNC), wool length (WL), and cashmere yield (CY) of buck goats were significantly higher than those of doe goats (p < 0.01), and the fineness of the cashmere produced by doe goats was significantly finer than that produced by buck goats (p < 0.01). The birth weight, weaning weight, and 6-month weight of single kids were significantly higher than those of multiple kids (p < 0.01), but the effect on the 12-month weight was not significant (p > 0.05). The age of doe goats at lambing had significant effects on birth weight, weaning weight, and 6-month weight (p < 0.01). Assay flock and age at measurement had significant effects on cashmere fineness, cashmere thickness, and cashmere yield (p < 0.01). This study will provide a basis for the scientific breeding and management of cashmere goats and lay a foundation for the setting of fixed effects in the genetic evaluation model of Inner Mongolia white cashmere goats (Erlangshan type).

Impacts of reference population size and methods on the accuracy of genomic prediction for fleece traits in Inner Mongolia Cashmere Goats.

Introduction: Inner Mongolia Cashmere Goats (IMCGs) are famous for its cashmere quality and it's a unique genetic resource in China. Therefore, it is necessary to use genomic selection to improve the accuracy of selection for fleece traits in Inner Mongolia cashmere goats. The aim of this study was to determine the effect of methods (GBLUP, BayesA, BayesB, Bayesian LASSO, Bayesian Ridge Region) and the reference population size on accuracy of genomic selection in IMCGs. Methods: This study fully utilizes the pedigree and phenotype records of fleece traits in 2255 individuals, genotype of 50794 SNPs after quality control, and environmental data to perform genomic selection of fleece traits. Then GBLUP and Bayes series methods (BayesA, BayesB, Bayesian LASSO, Bayesian Ridge Region) were used to perform estimates of genetic parameter and genomic breeding value. And the accuracy of genomic estimated breeding value (GEBV) is evaluated using the five-fold cross validation method. And the analysis of variance and multiple comparison methods were used to determine the best method for genomic selection in fleece traits of IMCGs. Further the different reference population sizes (500, 1000, 1500, and 2000) was set. Then the best method was applied to estimate genome breeding values, and evaluate the impact of reference population sizes on the accuracy of genome selection for fleece traits in IMCGs. Results: It was found that the genomic prediction accuracy for each fleece trait in IMCGs by GBLUP method is highest, and it is significantly higher than that obtained by Bayesian method. The accuracy of breeding value estimation is 58.52% -68.49%. Also, it was found that the size of the reference population has a significant impact on the accuracy of genome prediction of fleece traits. When the reference population size is 2000, the accuracy of genomic prediction for each fleece trait is significantly higher than other levels, with accuracy of 55.47% -67.87%. This provides a theoretical basis for design a reasonable genome selection plan for Inner Mongolia cashmere goats in the later stag.