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Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts.

ABSTRACT: BACKGROUND: The soybean aphid has significantly impacted soybean production in the U.S. Transcriptomic analyses were conducted for further insight into leads for potential novel management strategies. METHODOLOGY/PRINCIPAL FINDINGS: Transcriptomic data were generated from whole aphids and from 2,000 aphid guts using an Illumina GAII sequencer. The sequence data were assembled de novo using the Velvet assembler. In addition to providing a general overview, we demonstrate (i) the use of the Multiple-k/Multiple-C method for de novo assembly of short read sequences, followed by BLAST annotation of contigs for increased transcript identification: From 400,000 contigs analyzed, 16,257 non-redundant BLAST hits were identified; (ii) analysis of species distributions of top non-redundant hits: 80% of BLAST hits (minimum e-value of 1.0-E3) were to the pea aphid or other aphid species, representing about half of the pea aphid genes; (iii) comparison of relative depth of sequence coverage to relative transcript abundance for genes with high (membrane alanyl aminopeptidase N) or low transcript abundance; (iv) analysis of the Buchnera transcriptome: Transcripts from 57.6% of the genes from Buchnera aphidicola were identified; (v) identification of Arsenophonus and Wolbachia as potential secondary endosymbionts; (vi) alignment of full length sequences from RNA-seq data for the putative salivary gland protein C002, the silencing of which has potential for aphid management, and the putative Bacillus thuringiensis Cry toxin receptors, aminopeptidase N and alkaline phosphatase. CONCLUSIONS/SIGNIFICANCE: THIS STUDY PROVIDES THE MOST COMPREHENSIVE DATA SET TO DATE FOR SOYBEAN APHID GENE EXPRESSION: This work also illustrates the utility of short-read transcriptome sequencing and the Multiple-k/Multiple-C method followed by BLAST annotation for rapid identification of target genes for organisms for which reference genome sequences are not available, and extends the utility to include the transcriptomes of endosymbionts.

SUBMITTER: Liu S

PROVIDER: S-EPMC3440339 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts.

Liu Sijun S Chougule Nanasaheb P NP Vijayendran Diveena D Bonning Bryony C BC

PloS one 20120912 9

<h4>Background</h4>The soybean aphid has significantly impacted soybean production in the U.S. Transcriptomic analyses were conducted for further insight into leads for potential novel management strategies.<h4>Methodology/principal findings</h4>Transcriptomic data were generated from whole aphids and from 2,000 aphid guts using an Illumina GAII sequencer. The sequence data were assembled de novo using the Velvet assembler. In addition to providing a general overview, we demonstrate (i) the use ...[more]

PMID: 22984624

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Project description:Purpose: Soybean aphid (Aphis glycines Matsumura; SBA) is major pest of soybean (Glycine max) in the United States of America. One previous study on soybean, soybean-aphid interactions showed that avirulent (biotype 1) and virulent (biotype 2) biotypes can co-occur and potentially interact on resistant and susceptible soybean resulting induced susceptibility. The main objective of this research was to employ RNA sequencing technique to characterize the induced susceptibility effect in which initial feeding by virulent aphids can increase the suitability of avirulent aphids in both susceptible and resistant cultivars. Methods: The data in this submission come from the green house experiment using two genotypes of soybean: susceptible soybean cultivar was LD12-15838R and the resistant cultivar was LD12-15813Ra (with Rag1 gene) and two aphid populations: biotype 1 (avirulent) and biotype 2 (virulent biotype 2). RNA was extracted from the leave samples from resistant and susceptible cultivars treated with no aphids, biotype 2: biotype1 collected at day 1 and no aphids, biotype 2: biotype1 and no aphids: biotype1 at day 11 using PureLink RNA mini kit (Invitrogen, USA). RNA samples were treated with TURBOTM DNase (Invitrogen, USA) to remove any DNA contamination following the manufacturer’s instructions. Assessment of the isolated RNA integrity was performed by 1% agarose gel electrophoresis, and RNA concentration was measured by Nanodrop 2000 (Thermo Fisher Scientific, USA). Three replicates from these treatments in resistant and susceptible cultivars were pooled in equimolar concentration. RNAseq library construction was prepared using Illumina’s TruSeq Stranded mRNA Kit v1 (San Diego, CA). The libraries were quantified by QuBit dsDNA HS Assay (Life Technologies, Carlsbad, CA) and pooled in equimolar concentrations. The libraries were sequenced on an Illumina NextSeq 500 using a NextSeq 500/550 High Output Reagent Cartridge v2 (San Diego, CA) at 75 cycles. Results: A total of 10 RNA libraries were prepared and sequenced with the sequencing depth ranging from 24,779,816 to 29,72,4913. Total reads of 266,535,654 were subjected to FastQC analysis to determine the data quality using various quality metrics such as mean quality scores, per sequence quality scores, per sequence GC content, and sequence length distribution. The phred quality scores per base for all the samples were higher than 30. The GC content ranged from 45 to 46% and followed the normal distribution. After trimming, more than 99% of the reads were retained as the clean and good quality reads. Upon mapping these reads, we obtained high mapping rate ranging from 90.4% to 92.9%. Among the mapped reads, 85.8% to 91.9% reads were uniquely mapped. Conclusions: The objective of this study is to characterize the mechanism of induced susceptibility in soybean via transcriptional response study of soybean in presence of biotype 1 and biotype 2 soybean aphids using RNA-Seq. The data resulted from this study might provide insights into the interactions between soybean and soybean aphids and identify genes, their regulation and enriched pathways that may be associated with resistance or susceptibility to A. glycines.

Dataset Information

Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts.

Publications

Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts.

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