Small RNA Sequencing And Transcriptome Analysis Of Anther Development Stages In Cotton Genic Male Sterile Line Dong A

Cotton is one of the most important economic crops worldwide.It has been the main goal of breeders to breed cotton varieties with high fiber quality,lint yield and multi resistance.The utilization of cotton heterosis can greatly improve fiber quality and lint yield.Genic male sterility(GMS)has been one of key strategies to take advantage of heterosis in cotton.Therefore,it is very important to reveal the molecular mechanism of GMS.Recently,the rapid development of high-throughput sequencing technology and the completion of genome sequencing of tetraploid upland cotton which provide us conveniences to study the molecular mechanism of GMS in cotton.MicroRNA(miRNA),an important endogenous,non-coding regulatory small RNA,plays crucial roles in the regulation of plant development and stress response.However,limit is known about the roles of miRNA in cotton male sterility.Here,we performed small RNA sequencing and trancriptome sequencing to systematically explore differentially expressed mi RNAs and coding RNAs in the developing anther cells of the genic male sterile line ‘Dong A' and fertile cotton.Aiming to screen and identify potential functional miRNAs and genes related to cotton male sterility,and attempted to uncover their functions during this process.In total,through high-throughput sequencing,approximately 80 million clean reads were obtained from six small RNA libraries constructed from fertile and sterile cotton anther cells at three anther development stages(meiosis stage,tetrad stage and uninucleate stage).Based on the genome sequences of Gossypium hirsutum TM-1,we identified 80 known and 220 novel miRNAs.Comparative expression analysis of the known and novel mi RNAs between the GMS and fertile cotton revealed that totally 71 miRNAs were shown to be differentially expressed during anther development.A further degradome sequencing revealed a total of 117 candidate target genes cleaved by 16 known and 36 novel miRNAs.Furthermore,we verified the existence and expression patterns of 8 differentially expressed miRNAs in fertile and GMS cotton anthers by quantitative real-time PCR(qRT-PCR).Indicating that regulatory networks between miRNAs and their corresponding targets may involved in the regulation of cotton male sterility.Through the bioinformatics analysis of transcriptome sequencing data,we detected 3343,5480 and 7159 differentially expressed genes at meiosis stage,tetrad stage and uninucleate stage of anther development between GMS and fertile cotton,respectively.These results showed more differentially expressed genes were down-regulated than up-regulated at tetrad stage and uninucleate stage of GMS anther development,which indicated that expression of the majority of genes was suppressed in these two stages of anther development.Simultaneously,q RT-PCR were performed to investigate the expression patterns of 6 differentially expressed genes in fertile and GMS mutant cotton anthers and the results were consistent with transcriptome sequencing data.Based on the analysis of GO and KEGG pathways functional enrichment,the majority of differentially expressed genes were most likely involved in sucrose and starch metabolism,carbohydrate metabolism and plant hormone signal transduction and played functional regulation roles in cotton male sterility.Moreover,it was shown in degradome data that GhAP2,GhARF17 and GhMYB33 were targeted by ghr-miRNA172,nGhmiR68/69 and nGhmiR92,respectively.And the expression levels of GhAP2,GhARF17 and GhMYB33 shown in transcriptome sequencing were negative correlation with the expression levels of ghr-miRNA172,nGhmiR68/69 and nGhmiR92 by small RNA sequencing.Therefore,there may exist a regulatory interaction between these miRNAs and their target genes.In total,our results provided new insight for further functional investigations of the miRNA-mediated regulatory network involved in cotton genetic male sterility.