Differential Expression Of MicroRNA In Cotton Male Sterility And Defence Response To Verticillium Dahliae

MicroRNAs(miRNAs) are a class of 19- to 25-nucleotides endogenous non-coding small RNAs. Plant miRNAs play key roles in flower development and developmental timing in response or tolerance to biotic and abiotic stress. Thus, the research on the regulation mechanism of male sterile miRNAs and stress-regulated mi RNAs could help us gain a better pathway of utilization of male sterility in heterosis, and provide new information for disease resistance breeding.In this study, we performed miRNA microarray, high throughput sequencing and experimental technology to identified the miRNAs and their targets and.discuss the regulation mechanism of GMS and stress-regulated miRNAs in cotton. The profiling of these miRNAs may provide important clues on the functional mechanism of miRNAs in the regulation of anther development and stress-defence responses, and the main results were as follows:1.Morphological comparison of flower between“21A”male sterile and fertile plants was made and found that the flowers of male sterile plants were smaller than those of fertile plants.There was no pollen or a little in the mature anthers of male sterile plants, and no seed set by self-crossing. The microsporocyte development of 21 A plant was abnormal at meiosis, and anther abortion occurred mainly at the pollen grain stage.2.Developing anthers at the sporogenous cell, pollen mother cell and pollen grain stages were separately harvested for miRNA microarray analysis. Microarray data indicated that 504, 369 and 401 miRNAs were detected perfectly at the sporogenous cell, pollen mother cell and pollen grain stages of GMS line 21 A anther development, respectively. And 146,286 and 343 miRNAs were detected in three stages of its maintainer line anther development.There are 54 miRNAs expressed differently between the cotton GMS and its maintainer line. In addition,using a bioinformatic analysis, we identified 35 potential miRNAs target genes involved in cotton growth and development, signal transduction and metabolism pathways.3.To better understand the expression and functions of genes related to anther development, we performed a digital gene expression(DGE) analysis between an upland cotton GMS line and its maintainer line. Compared with the maintainer line, we identified 916 up-regulated and 826 down-regulated genes in the GMS line, including 12 transcription factors. Transcriptome profiling analysis showed that many key genes were involved in pentose and glucuronate interconversions, monoterpenoid biosynthesis, sesquiterpenoid biosynthesis, starch and sucrose metabolism, galactose metabolism, alpha-linolenic acid metabolism,and peroxisome,etc. MYB and AP2(targets of miR159, miR319 and miR172) involved in gene transcription, hormone signal transduction and other metabolic regulation pathways were significantly differentially expressed between the fertile and sterile libraries. 4.In order to study the roles of male sterility related mi RNAs in disease resistance reaction, four small RNA libraries and two degradome libraries from mocked and infected roots of G. hirsutum and G. barbadense were constructed for solexa sequencing and bioinfomatic analysis. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among all the identified miRNAs, a lot of mi RNAs were differentially expressed between libraries. In G. Hirsutum and G. barbadense roots, a total of 26 and 19 miRNAs were identified to be Verticillium dahliae-responsive miRNAs, respectively. In addition, it was shown that a total of 35 and 38 mi RNAs had a species-specific expression in mock-inoculated treatments and Verticillium-inoculated treatments, respectively. Among them, miRNAs(miR156, miR172, miR399, miR414) which play an important regulated roles in the process of pollen development were involved in the reaction process of resistance to cotton verticillium wilt.5.Degradome analysis of the cotton plants(hai7124 and Yi11) infected Verticillium dahliae showed that a total of 107 genes were found to be the miRNA targets. Among the identified targets, SBP and SPL transcription factor, NAC domain-containing protein, Class III HD-Zip protein, AP2-like factor, F-box/RNI-like superfamily protein, growth-regulating factor(GRF), MYB transcription factor, leucine-rich repeat(LRR)-containing protein, LRR and NB-ARC domain-containing disease resistance-like protein, etc. playing important roles in stress response and signaling conduction and the regulation of Verticillium defence responses in cotton.