SsCBP1 is a target of the monocot-specific miR528. (A) Stem-loop pulsed RT-PCR to detect miR528 transcripts in tissues of sugarcane, sorghum, maize, rice, and Arabidopsis. The highly conserved miR156 was used as an internal control. (B) Mapping of SsCBP1 mRNA cleavage sites by RNA ligase-mediated 5'RACE. Top panel shows the 4% agarose gel containing the expected 160 nt amplicon using specific reverse primers of SsCBP1 and RNA ligase-mediated 5'RACE primers. Bottom panel depicts the miR528-complementary site in the target mRNA and the miRNA. Watson-Crick pairing (vertical dashes) is indicated. Arrows indicate the 5' termini of mRNA fragments isolated from sugarcane, as identified by cloned 5'RACE products, with the frequency of clones shown. (C) Inferred phylogenetic relationships among SsCBP1 orthologous. p-distances were calculated from amino acid alignments of conserved blocks, and tree topology was inferred with the Neighbor-Joining method. The red branches show the monocot group of CBP genes containing the predict target site for miR528. This monocot group of genes belongs to a possible group of angiosperm orthologs (PoGO A). None of the eudicot sequences within this PoGO contain miR528 predicted target site. The 10 most closely related sequences out of PoGO A were included in this analysis. Only bootstraps values higher than 50% are shown for 1000 replicates. Sequence references are given in Methods.