Location: Corn Insects and Crop Genetics Research
Project Number: 5030-21220-005-03-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jan 18, 2012
End Date: Aug 31, 2015
1. An atlas of small RNAs will be created; 2. A website will be developed and publicly released for the visualization of small RNAs; 3. MicroRNAs will be identified, isolated, and evaluated; 4. Strand-specific, cleaved mRNA libraries will be used to identify targets of soybean microarrays.
Small RNA libraries will be constructed from tissues coordinated with ARS. Small RNA libraries will be generated in the University of Delaware lab using the improved Illumina TruSeq small RNA sample preparation kits, which eliminate all column purification and gel selection steps, are less time-intensive and more robust. These kits allow for decreased inputs of RNA; 500 ng total RNA is sufficient for library construction. Libraries will be bar-coded and four samples pooled and analyzed in a single lane on an Illumina HiSeq 2000 machine to yield single-end 50 base reads. We will quantitatively dissect the small RNA profiles across (a) tissues and (b) under stress treatments. Our proposed study will sequence far more deeply, with replicates, in a large array of tissues to give a comprehensive analysis of miRNA expression profiles in developing soybean tissues. By examining family member-specific PARE data, we will distinguish expression patterns between members of highly duplicated, conserved miRNA families. PARE data is semi-quantitative but any interesting expression differences will be validated by precursor-specific RT-PCR. Between the analysis of these conserved families and the many novel miRNAs, we will generate extensive data on miRNA tissue specificity, and these will be validated as described below. Analysis of differentially expressed siRNA clusters may identify loci subject to epigenetic regulation during development or stress, which is as yet poorly characterized in plants. The deep sequencing of matched PARE libraries will define small RNA targets and greatly enhance the information gained from other small RNA and RNA-seq projects. The deep sequencing of PARE libraries allows a systematic experimental analysis of small RNA targets. PARE is based on a modified 5'-RACE and generates libraries containing 3' cleavage products of mRNA, including those caused by small RNA-mediated cleavage. Computational tools are then used to compare PARE signatures to small RNA data to identify miRNA-target RNA pairs (see Aim 2). In addition to validating predicted targets, PARE signatures provide an important resource to validate new miRNAs. A database containing PARE signatures from diverse tissue samples will therefore be a tremendously valuable resource for the soybean community that will enhance the information gained from all small RNA sequencing data. PARE libraries will be generated from the same tissue samples indicated above, but because of the difficulty in generating these libraries, we will be able to make only a quarter as many PARE libraries as small RNA libraries.