Summary: Purpose: Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae) and soybean cyst nematode, Heterodera glycines Ichinohe, (SCN) are the two most economically important pests of soybean, Glycine max (L.) Merr., in the Midwest. Although the soybean aphid is an aboveground pest and SCN is a belowground pest there is evidence that concomitant infestations result in improved SCN reproduction. This study is aimed to characterize the three-way interactions among soybean, soybean aphid and SCN using demographic and genetic datasets.Results: More than 1.1 billion reads (61.4 GB) of transcriptomic data were yielded from 47 samples derived from the experiment using whole roots of G. max. The phred quality scores per base for all the samples were higher than 30. The GC content ranged from 43 to 45% 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 73.8% to 94.3%. Among the mapped reads, 67.1% to 87.6% reads were uniquely mapped.Conclusions: The comprehensive understanding of these transcriptome data would help in understanding the molecular interactions among soybean, A. glycines, and H. glycines. The use of multifaceted bioinformatics approaches could facilitate finding candidate genes and their function that might play a crucial role in various pathways for host resistance against both soybean aphids and SCN. For differential gene expression analysis, EdgeR, limma, and DEseq2 could be used. Apart from standalone tools like iDEP, Galaxy (https://usegalaxy.org), CyVerse (http://www.cyverse.org), and MeV (http://mev.tm4.org) could also be used for both analysis and visualization of RNA- seq data.
Overall Design: Methods: Two genotypes of Glycine max [H. glycines susceptible (Williams 82 PI 518671), and H. glycines resistant (MN1806CN)] cultivars with three treatments/cultivar: soybean aphids (Biotype 1), SCN (HG type 0), or SCN: soybean aphids in a randomized complete block design (RCBD) with six blocks were used to evaluate the three-way interactions. The entire experiment was repeated eight times. The experiment was conducted in a greenhouse water bath using cone-tainers. Treatments receiving SCN were infested at planting with 2000 nematode eggs. Treatments with soybean aphids were infested at second trifoliate growth stage (V2) with 15 soybean aphids. Soybean aphid populations were counted 5, 15, and 30 days post-infestation (dpi). SCN eggs were sampled at 30 dpi. The whole roots were sampled from 5d and 30d time points post soybean aphid infestation for library construction and sequencing using Illumina Hiseq 3000.The whole roots were sampled from 5d and 30d time points post soybean aphid infestation for library construction and sequencing using Illumina Hiseq 3000. SRP178193, PRJNA514200
For this experiment, the soil-sand mixture was prepared by adding construction sand and clay soil including SCN infested clay soil in the ratio of 3:1. The 125 cc of the mixture was distributed in cone-tainers (diameter of 3.8 cm, a depth of 21 cm and a volume of 164 cc; Greenhouse Megastore, USA). For SCN included treatments, each cone-tainer received approximately 2,000 SCN eggs. The cone-tainers with three soybean seeds were arranged in a 2.0 U.S. gallon (7.57 liter) plastic buckets (Leaktite, USA) filled with construction sand (Quikrete, GA). These buckets were kept in a water bath for maintaining soil temperature between 26.7 °C and 28.9 °C to ensure the reproduction of SCN (i.e. ~30 days). The temperature of the water bathes were regularly monitored using thermometers. The plants were grown under 16:8 (L:D) in a greenhouse with a temperature of 28 °C and 45% relative humidity. The plants were thinned down to one plant per cone-tainer upon reaching the second vegetative growth stage (V2).
Treatment Protocol:
The V2-staged plants with the SBA included treatments were infested with 15 mixed age (i.e., fourth instar nymphs and adults) biotype 1 SBA using a 000 fine tip paintbrush (Winsor & Newton, England). The SBA were applied on the abaxial surface of the first trifoliate of V2-staged plants. All plants in each bucket were covered with a large no-see-um mesh net (Quest Outfitters, Sarasota, FL) to prevent inter-bucket movement of aphids. After SBA infestation, soybean plants were regularly checked to confirm the successful establishment of soybean aphids. Soybean aphid populations were counted at 5, 15, and 30 days post infestation (dpi). For SBA only treatment, the populations on the two soybean varieties were not significantly different, indicating that both lines were susceptible to SBA. SCN eggs were sampled at 30 dpi. The whole roots were collected on 5 and 30 dpi by snap freezing in liquid Nitrogen and stored at -80 °C for further analysis. The 5 dpi and 30 dpi root samples treated with each treatment were collected from Water bath I and Water bath II, respectively, representing each plant from three blocks (three biological replicates). The SCN soil and SCN infested roots were used for SCN cysts collection (except root samples collected for transcriptomic study) and the soil was examined for SCN counts.
Extract Protocol:
Frozen root samples from each treatment were grounded in liquid nitrogen with pestle and mortar to a fine powder followed by their processing for total RNA extraction using PureLink RNA mini kit (Invitrogen, USA). RNA samples were treated with TURBOTM DNase (Invitrogen, USA) to remove any DNA contamination using manual’s instructions.
Library Construction Protocol:
The cDNA libraries were constructed using NEBNext Ultra II RNA library 96 single index kit prep kit and sequenced using Illumina HiSeq 3000 (single read end utilizing a 100 –bp read length) at Iowa State University Sequencing Facilities.
Sequencing
Molecule Type:
poly(A)+ RNA
Library Source:
Library Layout:
SINGLE
Library Strand:
-
Platform:
ILLUMINA
Instrument Model:
Illumina HiSeq 3000
Strand-Specific:
Unspecific
Samples
Basic Information:
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Analysis:
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Publications
Transcriptome profiling of interaction effects of soybean cyst nematodes and soybean aphids on soybean.
