Identification And Analysis Of Mirnas Releated To Pollen Abortion In CMS-C Maize

The Cytoplasmic male sterility?CMS?has been found in various plants and widely used for hybrid seed production in plants.Therefore,studying the abortion and recovery mechanism of maize cytoplasmic male sterility has a great meaning for maize breeding.miRNAs,an important regulators in plant,are involved in all stages and tissues of plants.Many studies suggest that some miRNAs are abundant in pollen,indicating that the miRNA may be related to flower development or male sterility.Nevertheless,there has more reports about fertility-related miRNAs in model plant,like Arabidopsis thaliana,rice etc,and fewer studies on C-type cytoplasmic male sterility in maize.In this study,miRNAs of maize cytoplasmic male sterile line C48-2 and its maintainer N48-2 from pollen mother cell stage anthers and mononuclear stage anthers were excavated and analyzed for preliminary functional analysis in order to identify the relationship between fertility and miRNA using High-throughput sequencing.At the same time,the further prediction of target genes involved in fertility-related miRNAs was explored,and the biological processes that miRNAs might be involved in this experimentwere discussed.Now the main results of this experiment are summarized as follows:1.A total of 321 known miRNAs belonging to 29 miRNA families and 724 novel miRNAs were obtained by High-throughput sequencing analysis.2.The bioinformatics analysis showes that 15 known miRNAs and 9 novel miRNAs are significant difference expressed in uninuclear microspore between the male sterile line C48-2 and the maintainer line N48-2,8 known miRNAs between pollen mother cells stage and uninuclear microspore stage in C48-2,8 known miRNAs and 8 novel miRNAs are differentially expressed in uninuclear microspore between C48-2 and N48-2,and 11 known miRNAs and 8 novel miRNAs are differentially expressed in pollen mother cells between C48-2 and N48-2.Further analysis found that miR159h-3p,miR167h-3p,miR171-5p,miR3449-3p,miR5141,novel_mir₄1 were significantly different between the two stages of the maintainer line N48-2,and also significantly different expressed between uninuclear microspore in C48-2 and N48-2.Whether these five known miRNAs and one new miRNA are related to maize C-type cytoplasmic male sterile line fertility remains to be further studied.3.We validate the six miRNAs above that be predicted by bioinformatics via miRNA poly?A?qRT-PCR.The results showed that the expression model are consistent with that of prediction between uninuclear microspore in C48-2 and N48-2 except miR5141.Six novel miRNAs?mir₈3,mir₈1,mir₂25,mir₂39,mir₁82,mir₁75?that significantly differentially expressed in the N48-2 and C48-2 mononuclear phases,as well as the miRNAs?including miR2111-3p and miR444b.2?reported that may be associated with anther development has also been validated via the same method.The results showed the miRNAs were identical with bioinformatics prediction,except that the expression of miR2111-3p was the opposite.4.We predict the target of 14 miRNA above using psRobot and TargetFinder.Analysis found that the target are involved in many biological process of plant growth and development,like the energy metabolism,hormone transfer and transcription regulation.5.The target of 14 sterility-ralated miRNAs were also analyzed by qRT-PCR.mir₁82 were found to regulate their candidate target genes,of which mir₁82 was significantly higher than that in CU,and the target gene was significantly lower in CU than in NU,whereas some miRNAs,like miR159h-3p,miR167h-3p,miR171-5p,miR3449-3p,mir₄1,mir₂25,miR2111-3p,miR444b.2,have no intuitive targeting relationship with their candidate target genes.There are two possible reasons,one is candidate genes are false positive,and secondly,miRNA may co-regulate its target gene with other factors to affect anther development.6.The miR171-5p and miR444b.2 precursor overexpression vector were constructed and introduced into wild-type Arabidopsis thaliana in our study.We found that the fertility were reduced in overexpressed lines,and then we speculate that miR171-5p,miR444b.2 may be associated with CMS-C male sterility in maize.