| Wumeng crested chicken has a cluster of slender feathers on its head,and the underlying skull region exhibits an obvious tumor-like protrusion.This is the typical skull structure of crested chickens.The associated regulatory genes are located on autosomes and are incompletely dominant.This trait is related to brain herniation,but the genetic mechanisms of its formation and development are unclear.In this experiment,Wumeng crested chicken was used as the research object,using Whole Genome Bisulfite Sequencing(WGBS)and transcriptome sequencing(RNA-sequence,RNA-seq)technology to find the control of the characteristics of the crested head Key candidate genes to explore the genome-wide changes in gene expression levels,changes in DNA methylation status and the regulatory mechanism of DNA methylation on the expression of crested-related genes during the growth of Wumeng crested chicken crested chickens,and explore the regulatory mechanism of Wumeng crested chicken crested chickens The growth and development laws and the development mechanism of crested traits provide the theoretical basis for the breeding and production of Wumeng crested chickens.The results of the study are as follows:1 RNA-seq analysis was conducted on 6 skull tissue samples from 3 Wumeng crested chickens with prominent skull protrusions and 3 without a prominent skull protrusion phenotype.A total of46,376,934-43,729,046 clean reads were obtained,the percentage of uniquely mapped reads compared with the reference genome was between 89.73%-91.00%,and 39,795,458-41,836,502 unique reads were obtained.Among different genomic regions,the highest frequency of sequencing reads occurred in exon regions(85.44-88.28%).Additionally,a total of 423 new transcripts and26,999 alternative splicings(AS)events were discovered in this sequencing analysis.This study identified 1089 differentially expressed genes(DEGs),among which 485 were upregulated and 604 were downregulated.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses indicated that the DEGs were enriched in terms related to signal transduction,cell development,cell differentiation,the lysosome,serine,and threonine metabolism,and the interaction of cytokines with cytokine receptors.Based on the comprehensive analysis of DEGs combined with reported quantitative trait loci(QTLs),the expression of BMP2,EPHA3,EPHB1,HOXC6,SCN2 B,BMP7,and HOXC10 was verified by real-time quantitative polymerase chain reaction(qRT-PCR).The qRT-PCR results were consistent with the RNA-Seq results,indicating that the above 7 genes may be candidate genes for regulating crested traits2 WGBS sequenced the skulls of Wumeng crested chickens with or without crested heads,and obtained a total of 181.57 G of valid data.4 244 differentially methylated regions(DMRs)and 1806 differentially methylated genes(DMGs)were screened.Under the CG sequence environment,GO enrichment analysis found that DMRs differential genes were enriched to 3 111 GO terms,among them,cell component GO terms,biological process GO terms,and molecular function GO terms were significantly enriched in 2,39,and 9 GO terms respectively(Corrected＿P-value<0.05).Hypermethylation genes are significantly enriched in 20 or 2 GO terms in biological processes and molecular functions(Corrected＿P-value<0.05).The biological process of hypomethylation genes was significantly enriched to 23 GO terms(Corrected＿P-value<0.05).DMGs KEGG was enriched in 135 signal pathways,7 signal pathways were significantly enriched(Corrected＿P-value<0.05),namely adhesion junction,Wnt signaling pathway,focal adhesion,calcium signaling pathway,insulin signaling pathway,Hedgehog signaling pathway,Erb B signal path.Hypermethylation genes were enriched in 123 signal pathways,and 5 signal pathways were significantly enriched(Corrected＿P-value <0.05),namely adhesion junction,focal adhesion,Wnt signaling pathway,tight junction,and regulation of actin cytoskeleton.Hypomethylated genes were enriched in 106 signal pathways,and there was no significant enrichment(Corrected＿P-value >0.05)pathway.According to the comprehensive analysis of DMGs,combined with the reported QTLs,the intracellular expression pattern after RG108 treatment showed that RG108 inhibited the methylation level of intracellular genes,SCN2 B,BMP2,HDAC7,LARP7,LARP4,EPHA2,COX10,HOXC11 gene is regulated by methylation,indicating that the above 8 genes may be candidate genes for regulating crested traits.3 The combined analysis results of WGBS and RNA-seq showed that there were 133 genes related to DMRs and DEGs,and 84 negatively related genes,among which 59 genes were highmethylation level and down-regulated genes,and 25 genes were low-methylation level and upregulated genes.A total of 84 negatively-related genes were enriched in 1 260 GO terms,biological processes were enriched to 721 GO terms,cell components were enriched to 220 GO terms,and molecular functions were enriched to 319 GO terms.There was no significant enrichment.The GO terms of the set(Corrected＿P-value >0.05).High methylation level and expression down-regulated negatively correlated genes enriched to 18 KEGG signaling pathways,low methylation level and expression up-regulated negatively correlated genes enriched to 10 KEGG signaling pathways,1significantly enriched KEGG pathway transforming growth factor-β signal pathway(Corrected＿Pvalue <0.05).The two sets of studies were combined to finally screen the genes SCN2 B,BMP2,and transforming growth factor-β signaling pathway that may be negatively related to DNA methylation and expression levels that may regulate crested traits. |
