Dataset Information

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression.

ABSTRACT: MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control.

SUBMITTER: Wang X

PROVIDER: S-EPMC5291412 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression.

Wang Xiu X Li Yisong Y Zhang Jing J Zhang Qingwen Q Liu Xiaoxia X Li Zhen Z

PloS one 20170203 2

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a t ...[more]

PMID: 28158242

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Project description:BACKGROUND:Invasive pest species have large impacts on agricultural crop yields, and understanding their population dynamics is important for ensuring food security. The oriental fruit moth Grapholita molesta is a cosmopolitan pest of stone and pome fruit species including peach and apple, and historical records indicate that it has invaded North and South America, Europe, Australia and Africa from its putative native range in Asia over the past century. RESULTS:We used 13 microsatellite loci, including nine newly developed markers, to characterize global population structure of G. molesta. Approximately 15 individuals from each of 26 globally distributed populations were genotyped. A weak but significant global pattern of isolation-by-distance was found, and G. molesta populations were geographically structured on a continental scale. Evidence does not support that G. molesta was introduced to North America from Japan as previously proposed. However, G. molesta was probably introduced from North America to The Azores, South Africa, and Brazil, and from East Asia to Australia. Shared ancestry was inferred between populations from Western Europe and from Brazil, although it remains unresolved whether an introduction occurred from Europe to Brazil, or vice versa. Both genetic diversity and levels of inbreeding were surprisingly high across the range of G. molesta and were not higher or lower overall in introduced areas compared to native areas. There is little evidence for multiple introductions to each continent (except in the case of South America), or for admixture between populations from different origins. CONCLUSIONS:Cross-continental introductions of G. molesta appear to be infrequent, which is surprising given its rapid worldwide expansion over the past century. We suggest that area-wide spread via transport of fruits and other plant materials is a major mechanism of ongoing invasion, and management efforts should therefore target local and regional farming communities and distribution networks.

Project description:BACKGROUND:The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. METHODOLOGY/PRINCIPAL FINDING:Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. CONCLUSION:Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics.

Project description:Deep shotgun sequencing on next generation sequencing (NGS) platforms has contributed significant amounts of data to enrich our understanding of genomes, transcriptomes, amplified single-cell genomes, and metagenomes. However, deep coverage variations in short-read data sets and high sequencing error rates of modern sequencers present new computational challenges in data interpretation, including mapping and de novo assembly. New lab techniques such as multiple displacement amplification (MDA) of single cells and sequence independent single primer amplification (SISPA) allow for sequencing of organisms that cannot be cultured, but generate highly variable coverage due to amplification biases.Here we introduce NeatFreq, a software tool that reduces a data set to more uniform coverage by clustering and selecting from reads binned by their median kmer frequency (RMKF) and uniqueness. Previous algorithms normalize read coverage based on RMKF, but do not include methods for the preferred selection of (1) extremely low coverage regions produced by extremely variable sequencing of random-primed products and (2) 2-sided paired-end sequences. The algorithm increases the incorporation of the most unique, lowest coverage, segments of a genome using an error-corrected data set. NeatFreq was applied to bacterial, viral plaque, and single-cell sequencing data. The algorithm showed an increase in the rate at which the most unique reads in a genome were included in the assembled consensus while also reducing the count of duplicative and erroneous contigs (strings of high confidence overlaps) in the deliverable consensus. The results obtained from conventional Overlap-Layout-Consensus (OLC) were compared to simulated multi-de Bruijn graph assembly alternatives trained for variable coverage input using sequence before and after normalization of coverage. Coverage reduction was shown to increase processing speed and reduce memory requirements when using conventional bacterial assembly algorithms.The normalization of deep coverage spikes, which would otherwise inhibit consensus resolution, enables High Throughput Sequencing (HTS) assembly projects to consistently run to completion with existing assembly software. The NeatFreq software package is free, open source and available at https://github.com/bioh4x/NeatFreq .

Dataset Information

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression.

Publications

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression.

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