One target of Can miR 06 is the growth regulating factor gene, which is also tar geted by miR396, indicating that multiple miRNAs may regulate the same gene family. MiRNA profile changes during grain filling To study the expression patterns of miRNAs during grain development, 17-AAG side effects we generated miRNA chips contain ing 546 probes, and comprising 254 known miRNAs from miRBase version 13. 0, the 11 newly identified can didates, and 50 controls. Small RNAs isolated from grains at the milk ripe stage, the soft dough stage, and the hard dough stage were hybri dized to the miRNA chips. The raw signal values are provided in Additional file 6. As shown in Figure 3, 190, 168, and 187 miRNAs were detected above background levels in G1, G2, and G3, respect ively.

Among them, 143 miRNAs were expressed in all three filling stages, whereas 26, 12, and 30 were specific ally expressed in G1, G2, and G3, respectively. Most of the phase specific miRNAs were newly identified, in the 1 10 DAF rice grain library, and another 26 reported by Xue et al. in a 3 12 DAF rice grain li brary, only nine were detected in our library. These included miR1862d and miR1862e with relatively abun dant expression levels of 181 and 122 reads, respectively, whereas the others were detected with expression levels of only one to five reads. The lack of shared novel miRNAs could be, 1 due to our using indica cultivar Baifeng B whereas all previous studies were with subspe cies japonica, 2 because the majority of the rice specific miRNAs are expressed at very low levels, they might not have been detected at our sequencing depth.

Targets of novel miRNAs were predicted and some appeared to be involved in the grain filling process. For example, Can miR 07 was pre dicted to target starch synthase II, which is preferentially expressed in the endosperm at the middle to later stages of grain filling and plays an important role in elon gation of 1,4 amylase chains. Can miR 04 and Can miR 08 may target a ubiquitin protein ligase gene such as Can miR 11, which is expressed at G1 and G2, Can miR02 and Can miR03, which are expressed at G2 and G3, and Can miR04 and Can miR11, which are detected only at G3. Using a relative intensity change of 2 fold or above be tween consecutive filling stages, the expression patterns of miRNAs were clustered.

As shown in Figure 4, 13 miRNA families included 18 members that were differentially expressed across the three filling stages. Nine members of seven miRNA families were up regulated. The expression of miR1862 and miR1874 increased from G1 to G2, but remained largely un changed from G2 to G3, whereas miR159, miR164 and miR1850 underwent rapid increases from G2 to G3. In contrast, nine members AV-951 of six miRNA families were down regulated. Among them, the expression of miR160, miR166, and miR171 declined rapidly from G1 to G2, whereas miR167, miR396, miR444 and miR530 gradually declined with advancing grain filling.