A Combined Analysis Of MicroRNA And Transcriptome Sequencing In Gossypium Harknessii Cytoplasm Male Sterile Three Line Cotton

In hybrid cotton production,three lines with cytoplasmic male sterility(CMS),maintainer(B)and fertility restorer(R)materials has been widely used in hybrid production,which greatly reduced the production cost of artificial emasculation,and more conducive to the application of hybrid cotton varieties.The cytoplasmic male sterility(CMS)in flowering plants is a maternally inherited trait that lack functional pollen grains.CMS can be caused by the incompatibility between mitochondria and nuclear genome.However,the male fertility can be restored by nuclear genes known as restorer-of-fertility(Rf)genes.The CMS/Rf system is useful and convenient for economically producing commercially hybrid seed exhibiting heterosis,but the molecular mechanisms of CMS occurrence or fertility restoration are not clear in cotton until now.In this study,a combining small RNA sequencing and transcriptome analysis was completed in 3 mm floral buds of the Harknessii cytoplasm male sterile line(A),maintainer(B)and restorer line(R)to identify the known mi RNAs and novel mi RNAs by using high-throughput sequencing technology.Furthermore,to explore the molecular mechanism of CMS occurrence or fertility restoration,we identified the differentially expressed mi RNAs and its target genes in A-B and A-R the two compared groups and performed enrichment analysis,which will lay a foundation for the further usage of heterosis and commercially hybrid breeding in cotton.The mainly experimental results are as follows:1.Totally 370 known mi RNAs representing 62 known mi RNA families and 27 novel mi RNAs were identified.Of these mi RNAs,72 and 126 mi RNAs were differentially expressed during anther development in A-B and A-R groups,respectively.A total of 4686 target genes were predicted for all the mi RNAs,including transcription factor family such as SPL,MYBs and NAC and several functional gene familys like PPR gene,protein kinase gene family,which could have critical roles in gene regulation and anther development.2.According to the negative relationship between mi RNA and its corresponding targets,15 and 47 mi RNA-target pairs were identified in A-B and A-R two compare groups by combining small RNA sequencing and transcriptome data,and had RT-q PCR verified.3.According to the differentially expressed miRNA-target pairs in A-B and A-R two groups,a miRNA regulatory network that involved in cytoplasmic male sterility and fertility restoration was constructed.For example,hbr-mi R156-SPL10 pair may be involved in the regulation of CMS occurrence,and the gra-mi R7505b-PPR pair may be involved in the regulation of fertility restoration.4.Analysing the structure of differentially expressed PPR gene,and predicting the amino acid in specific site and specific nucleotide sequence.