Identification of miR156 Cleavage Targets in Lotus japonicus

The target transcripts for miR156 cleavage were predicted using the psRNATarget web server by following the default parameter (http://plantgrn.noble.org/psRNATarget/). In addition, the mature sequence of L. japonicusmiR156 was used to conduct a manual BLAST search against the L. japonicus genome database and the NCBI EST database. This eliminated any false candidates that could form potential precursors for LjmiR156. After the redundant sequences were manually excluded, potential targets were chosen as candidates for cleavage-site validation using a modified 5′-RACE method (Song et al. 2010 (link)). This technique was performed with a FirstChoice RLM-RACE Kit (Life Technologies) according to the manufacturer’s instructions, but with a slight modification. Instead of removing 5′PO₄, the adaptor was ligated directly to the RNA molecules, which were then subjected to reverse-transcription. Afterward, nested PCRs were run with outer/inner adaptor- and outer/inner gene-specific primers (Supplementary Table 1). The products were gel-purified and cloned into the pJET vector (Thermo Scientific, Waltham, MA, USA). Eleven (11) clones for TC70253, 12 clones for AU089181, and 16 clones for TC57859 were sequenced to determine the cleavage sites of the two candidate genes.