Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.     

Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.     

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setariaitalica)

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5′-RLM-RACE) method.     

Global analysis of non-coding small RNAs in Arabidopsis in response to jasmonate treatment by deep sequencing technology

In plants, non-coding small RNAs play a vital role in plant development and stress responses. To explore the possible role of non-coding small RNAs in the regulation of the jasmonate (JA) pathway, we compared the non-coding small RNAs between the JA-deficient aos mutant and the JA-treated wild type Arabidopsis via high-throughput sequencing. Thirty new miRNAs and 27 new miRNA candidates were identified through bioinformatics approach. Forty-nine known miRNAs (belonging to 24 families), 15 new miRNAs and new miRNA candidates (belonging to 11 families) and 3 tasiRNA families were induced by JA, whereas 1 new miRNA, 1 tasiRNA family and 22 known miRNAs (belonging to 9 families) were repressed by JA.     

Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2–13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.     

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 of miRNAs and Their Target Genes in Peach (Prunus persica L.) Using High-Throughput Sequencing and Degradome Analysis

MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development.