Transcriptome Analysis Of MRNA And MiRNA Of Green Eggshell Character In YouXian Duck

Egg-shell colour has got heated attention in the poultry industry.Some researchers have studied related genes about this character,however,they were just confined to the single gene,which made the regulation mechanism of egg-shell clour formation being still unclear.Even though YouXian ducks were the same species or the same group,we found that their egg-shell colour was probably different.Therefore,we reckoned that the green egg-shell colour formation was regulated by many genes and only by transcriptomics can we obtain more genes related with this character,which provide theoretical basis for illuminate its mechanism.This experiment is based on the contrast of shell glands between green egg-shell colour(D)and white egg-shell colour(W)in YouXian duck,and the RNA-Seq technology was used to indentify the differentially expressed genes and miRNAs.These identified differentially expressed genes and miRNAs were enciched to GO terms and KEGG pathways,which can be researched the function of differentially expressed genes in green eggshell colour formation.In addition,we also predicted the target genes of differentially expressed miRNAs,and constructed the regulated network between differentially expressed mRNAs and miRNAs.These results make up the shortcomings of the traditional research of single gene,enrich the factors influencing the laying of green egg-shell colour in poultry,and further contribute to the exploration of the regulation mechanism of egg-shell colour formation in YouXian duck.The sequencing results of mRNA and miRNA were validated by the qRT-PCR,and the result shows that the transcriptome results were more reliable.The results of this study are as follws:(1)Transcriptome analysis of shell gland between D and WRNA-Seq technology was applied to sequencing three libraries of D and three libraries of W,42.81Gb Clean Data was obtained after filtering unqualified sequences;Q30 of each samples was no less than 90.75 and the average ratio of sequences compared to the reference genome of mallard ducks was 58.07%.On average,245525 and 260338 SNP loci was obtained in D and W,respectively;while InDel number was 22986 and 24556,respectively.Theses results provide polymorphism markers for molecular breeding in this species.5 kinds of alternative splicings were analyzed by JC.only and JC+readsOnTarget,among which the percentage of SE(skipped exon)was biggest(93.7%).We found 14298 genes were expressed in W,while 14090 genes were expressed in D.But 13671 genes were expressed in W and D.Moreover,13991 novel genes were identified with 10804 known genes studied for structural optimization.In additon,W being the control group and padj<0.05 being the screening criteria,124 differentially expressed genes were identified in D,with 79 up-regulated and 45 down-regulated genes.The differentially expressed genes were further annotated and pathway analyzed,especially ABC transporters and solute carrier family possibly related with colour formation and so on.qRT-PCR was applied to validate the RNA-Seq data,revealing a strong reliability of transcriptome.(2)miRNA sequencing analysis of shell gland between D and W6 miRNA libraries were constructed in this study and sequenced.The results showed that the length of sRNA was between 21 nt-23nt;260 and 195 known miRNAs were mapped to mature and hairpin of miRNA,respectively;while 199 and 221 novel miRNA sequence were mapped to mature and hairpin of miRNA,respectively.In addition,the target genes of 262451 known miRNA and 210281 novel miRNA were predicted,which has important significant on finding new miRNA and studing its functions.The analysis of miRNA expression suggested that gga-miR-148a-3p was the most biggest expression miRNA in both D and W.With the W group being the control group and padj<0.05 being the screening criteria,31 differentially expressed miRNAs were identified,which was consist of 18 up-regulated miRNAs and 13 down-regulated miRNAs,among which the fold change of gga-miR-144-3p was biggest(1.54)and the smallest padj was gga-miR-203a and the second was gga-miR-214.The analysis of GO and KEGG enrichment of their target genes was researched.Moreover,qRT-PCR was applied to validate the data of miRNA,revealing a strong reliability of transcriptome.(3)Correlation analysis of differentially expressed miRNAs and mRNAsThe correlations of 124 differentially expressed mRNAs and 31 differentially expressed miRNAs were analyzed,and the enrichment of GO and KEGG was researched in this study.The correlation results showed that 81 differentially expressed genes can be regulated by 29 differentially expressed miRNA,and the netwok interaction was constructed.46 up-regulated and 35 down-regulated mRNAs were concluded in differentially expressed mRNAs,while 17 up-regulated and 12 down-regulated miRNAs were contained in differentially expressed miRNAs.