Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing.

BACKGROUND: MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events. RESULTS: We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study. CONCLUSIONS: We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N2-fixing symbiotic nodules.

Cloning of stress-responsive microRNAs and other small RNAs from plants.

In plants, small RNA (microRNAs and other endogenous small RNAs)-guided target gene expression is vital for a wide variety of biological processes including adaptation to stress conditions. Identification of stress-regulated microRNAs or other classes of endogenous small RNAs advances our understanding of post-transcriptional gene regulation important for plant stress tolerance. This chapter describes a detailed step-by-step protocol for cloning of small RNAs. Following 5' and 3' adapter ligation to the purified small RNAs, cDNA will be synthesized using reverse transcription, which will be further amplified using a polymerase chain reaction. The resulting small DNA fragments can be either cloned and sequenced using traditional sequencing method or subjected to direct high-throughput pyrosequencing or sequencing-by-synthesis technology.