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RNA-seq analysis of two genealogically linked South African Diuraphis noxia biotypes

ABSTRACT: RNA was extracted from all instar (insect developmental) stages for both D. noxia biotypes SA1 and SAM with the purpose to capture as many expressed transcripts as possible. South African D. noxia biotype SA1 is known to be the least virulent aphid, while its offspring, the South African D. noxia biotype SAM is the most virulent. The overall purpose of the experiment was to establish a baseline availability of transcripts to the aphids as well as help improve on current genome assemblies. Three biological replicates of 100 aphids each was collected from both biotypes SA1 and SAM that were respectively reared on preference host cultivars. Whole aphids were flash frozen in liquid nitrogen, ground to a powder with micro pistils and RNA was extracted making use of a Qiagen RNeasy kit. Library preparation for sequencing was performed using an Illumina TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide, Part # 15031047 Rev. E protocol. The replicate samples from the SAM biotype yielded between 120 – 140 million 100bp PE reads and the replicate samples from the SA1 biotype yielded between 113 – 137 million 100bp PE reads (with a Q20 phred score above 98% for all replicates) after sequencing on the NovaSeq6000 system. De novoassembly was performed making use of the Trinity package.

ORGANISM(S): Diuraphis noxia

PROVIDER: GSE143502 | GEO | 2020/12/31

REPOSITORIES: GEO

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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.

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RNA-seq analysis of two genealogically linked South African Diuraphis noxia biotypes

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