Identification of miRNAs and their corresponding mRNA targets from chickpea nodules and functional characterization of candidate miRNAs by overexpression in chickpea roots

Abstract

O_LILegumes developed symbiotic associations with rhizobia to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. C_LIO_LIOne microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced to unravel the miRNA mediated regulation of symbiosis in chickpea. C_LIO_LISequencing of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. C_LIO_LIPhylogenetic analysis of the precursor sequences of the 91 miRNAs clearly indicated a clustering of two distinct miRNAs in the same clade representing a close ancestral precursor. C_LIO_LIFurther, biogenesis of miRNAs was predicted using the miRNAs identified from different legume genomes. C_LIO_LIThe miRNA reads from the nodule library were also mapped onto bacterial genomes from which bacterial small RNA were predicted. C_LIO_LIThe antagonistic expression of some of the miRNA-target pairs was investigated and the negative co-related expression profiling proved the validity of the libraries and the miRNA-target pairs. Four miRNAs were selected based on the antagonistic expression profiling and were ectopically expressed in chickpea roots by hairy root transformation. C_LIO_LIThe overexpression lines showed significant change in nodule numbers. The target of miR171f (NRK), miR394 (HP) and miR1509 (AK) are novel ones being reported for the first time. This analysis opens a wide arena for investigation of the novel miRNAs and target pairs, polycistronic miRNAs and the bacterial derived smRNAs predicted in this study. C_LI