Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

Characterization of microRNAs from sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

Identification and Analysis of Powdery Mildew‐Responsive miRNAs in Wheat

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most consistently damaging diseases of common wheat worldwide and greatly affects crop productivity. Recently, several plant microRNAs (miRNAs) have been reported as gene expression regulators related to various adverse environments. However, up to now, less is known on the roles of miRNAs in powdery mildew infection response of wheat. In this study, miRNA expression patterns were investigated for identifying Bgt‐responsive miRNAs in wheat leaves using a plant miRNA microarray platform. A total of 79 miRNAs from 24 families were detected in wheat leaves. Among those, seven miRNAs were further validated to be involved in wheat powdery mildew response and two of them have never been reported. In addition, their target expression profiles showed a negative correlation with that of the seven miRNAs in mock‐ and Bgt‐infected samples furtherly proved, which in turn as the robust evidence, that those seven powdery mildew‐responsive miRNAs are highly reliable. These findings could extend the current view about miRNAs as ubiquitous regulators under stress conditions.