Characterization of Hypoxia Induced Maize miRNAs Figure 1. Flowchart of the experimental design. Red octagonal shows all the wet lab experiments. Aqua diamond shows the data analysis method. Blue square shows the computational prediction experiment. T: tolerant line. M: mid-tolerant line. S: sensitive line. doi:10.1371/journal.pone.0039786.g001 miRNA loci) were selected as candidate genes for further study focusing on temporal and inbred specific expression in the three maize inbred lines under short-term waterlogging conditions. Figure 2. Short-term waterlogging phenotype-screening re- sults of all three inbred lines. T: Waterlogging treatment. C: Control. RL: Root Length. LL: Leaf Length. RW: Root dry Weight. LW: Leaf dry Weight. TW: Total dry Weight. Error bars show the standard deviation. Student t-test was performed on the tolerant line (Hz32) and mid- tolerant line (B73), comparing with sensitive line (Mo17). ** denotes the p-value below 0.01 and * denotes the p-value below 0.05. doi:10.1371/journal.pone.0039786.g002 Differential Expression of miRNAs in Response to Short- term Waterlogging SLRT-PCR was performed on the selected 24 miRNA signatures under three time point treatments (1 h, 2 h, 4 h) of waterlogging in each of the three inbred lines (Hz32, B73, Mo17) with three biological replicates. Cluster analysis of those miRNAs expression levels among the three inbred lines showed four major clusters (Figure 5). Cluster 1: miRNAs from families 159(a/b/f/j/k & c/d), 166, 167, and 396 were repressed in all inbred lines except in Hz32 after 4 h treatment. miRNAs from this cluster are involved in flowering or leaf development [12,37,38,39,40]. Cluster 2: miRNAs from families 156, 164, 168, 169, 171, 319, 390, 393 and 529 were induced in Hz32 and repressed in B73 and Mo17. This cluster is enriched in miRNAs involved in root or shoot development, as well as stress response and tolerance [12,37,41,42,43,44,45,46,47]. Cluster 3: miRNAs from families 159(h/i), 172, 398, 408, 528, and 1432 were repressed in Mo17 but were induced in B73 and Hz32. Most of the miRNAs from this cluster are directly involved in stress response [12,20,37,45,48]. Cluster of novel miRNAs: miRn6 and miRn7 showed a similar pattern among three inbred lines, which are induced at 1 h and 2 h and repressed at 4 h treatment suggesting that these 2 novel PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39786