Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

Background

A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes.

Results

M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees.

Conclusions

The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1125) contains supplementary material, which is available to authorized users.     

miRTar Hunter: A prediction system for identifying human microRNA target sites

MicroRNAs (miRNAs) are important regulators of gene expression and play crucial roles in many biological processes including apoptosis, differentiation, development, and tumorigenesis. Recent estimates suggest that more than 50% of human protein coding genes may be regulated by miRNAs and that each miRNA may bind to 300–400 target genes. Approximately 1,000 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. However, the targets for a majority of these miRNAs have not been identified due to the lack of large-scale experimental detection techniques. Experimental detection of miRNA target sites is a costly and time-consuming process, even though identification of miRNA targets is critical to unraveling their functions in various biological processes. To identify miRNA targets, we developed miRTar Hunter, a novel computational approach for predicting target sites regardless of the presence or absence of a seed match or evolutionary sequence conservation. Our approach is based on a dynamic programming algorithm that incorporates more sequence-specific features and reflects the properties of various types of target sites that determine diverse aspects of complementarities between miRNAs and their targets. We evaluated the performance of our algorithm on 532 known human miRNA:target pairs and 59 experimentally-verified negative miRNA:target pairs, and also compared our method with three popular programs for 481 miRNA:target pairs. miRTar Hunter outperformed three popular existing algorithms in terms of recall and precision, indicating that our unique scheme to quantify the determinants of complementary sites is effective at detecting miRNA targets. miRTar Hunter is now available at http://203.230.194.162/~kbkim.     

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.     

Shoot bending promotes flower bud formation by miRNA‐mediated regulation in apple (Malus domestica Borkh.)

Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down‐regulated and up‐regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering‐related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees.     

miRNA Genes of an Invasive Vector Mosquito,Aedes albopictus

Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10–14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA*) sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO) analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.     

MicroRNA Buffering and Altered Variance of Gene Expression in Response to Salmonella Infection

One potential role of miRNAs is to buffer variation in gene expression, although conflicting results have been reported. To investigate the buffering role of miRNAs in response to Salmonella infection in pigs, we sequenced miRNA and mRNA in whole blood from 15 pig samples before and after Salmonella challenge. By analyzing inter-individual variation in gene expression patterns, we found that for moderately and lowly expressed genes, putative miRNA targets showed significantly lower expression variance compared with non-miRNA-targets. Expression variance between highly expressed miRNA targets and non-miRNA-targets was not significantly different. Further, miRNA targets demonstrated significantly reduced variance after challenge whereas non-miRNA-targets did not. RNA binding proteins (RBPs) are significantly enriched among the miRNA targets with dramatically reduced variance of expression after Salmonella challenge. Moreover, we found evidence that targets of young (less-conserved) miRNAs showed lower expression variance compared with targets of old (evolutionarily conserved) miRNAs. These findings point to the importance of a buffering effect of miRNAs for relatively lowly expressed genes, and suggest that the reduced expression variation of RBPs may play an important role in response to Salmonella infection.     

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.