Expression profiling by high throughput sequencing
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
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