Gene Expression
Nebulas
Gene Expression NebulasSummary: The soybean aphid, a plant sap sucking insect, is an important soybean pest in the USA causing significant yield losses. The Rag2 gene of soybean provides resistance to soybean aphid biotypes I and II. Transcriptomic analyses were performed on near isogenic lines (NILs) with the Rag2 allele for aphid resistance or rag2 for susceptibility at the Rag2 locus. Soybeans were infested with soybean aphids and leaves were collected at 0, 4, 8, 24, and 48 hours after infestation. RNA were extracted and a high throughput RNA-seq approach was used to examine mRNA expression in Rag2 and rag2 soybean leaves. The expression of ~43,000 genes was detected in both the Rag2 and rag2 leaves. Statistical analysis identified 2361 genes significantly regulated between the resistant and susceptible lines at different times after aphid infestation. Genes found up-regulated in the Rag2 line were annotated as involved in the cell wall, secondary and hormone metabolism, as well as in stress, signaling and transcriptional responses. Genes found up-regulated in the rag2 line were annotated as involved in photosynthesis and carbon metabolism. Interestingly, mRNAs of 2 genes (unknown and mitochondrial protease) located within the Rag2 locus were expressed significantly higher in the resistant genotype. The expression of the putative NBS-LRR resistant gene present in the Rag2 locus was not different between the two soybean lines. However, another NBL-LRR gene located just at the border of the Rag2 locus was and, therefore, may be involved in the differential resistance to aphid infestation exhibited by the two NIL genotypes analyzed.
Overall Design: A total of 20 samples was analyzed. Two soybean genotypes, 5 time points and 2 biological replicates per condition were used.
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| Growth Protocol: | The seedlings of the NILs were grown in an environment controlled greenhouse. The experimental design was a randomized complete block with four replications. For each NIL, four seeds per replicate for each of the four sampling times (0, 8, 24, 48 hrs after aphid infestation) were planted in 4-cm diameter x 15-cm deep plastic conetainers in a USDA greenhouse at Ohio Agricultural Research and Development Center (OARDC), Wooster, OH and the best two seedlings per conetainer were kept after germination. The conetainers were arranged in a rack with 8-cm x 12-cm spacing on a greenhouse bench. The greenhouse was maintained at approximately 26/22˚C day/night temperatures with 15 h light daily. The biotype 2 soybean aphids used in this study was from a growth chamber colony maintained at OARDC, Wooster, OH 34 . At the V2 growth stage, each soybean seedling was manually infested with 15 adult soybean aphids by placing the aphids on the top leaves of the seedling. For the 0 hr samples, just before infestation of seedlings with soybean aphids, the youngest unfolded leaves and the tips of two seedlings representing each replicate of each NIL were collected in a 2 ml tube and were immediately frozen in liquid nitrogen. The samples for 8, 24, and 48 h after infestation, were collected in the same manner, except that the aphids were gently removed from the tissue by using a soft paint brush before tissue collection. The aphids continued to walk away from the resistant seedlings within 12 to 24 hours of placement on the seedlings, so more aphids were added to these seedlings every 12 hrs to have at least 15 aphids per seedling at all times. |
| Treatment Protocol: | Aphid infestation |
| Extract Protocol: | Total RNA were extracted using Trizol reagent according to manufacturer's instruction |
| Library Construction Protocol: | Complementary DNA libraries were built according to the manusfacturer’s instruction (mRNA sequencing, Sample preparation guide; Illumina, San Diego, CA) using 10 µg of RNA. Each library was labeled with a 4 nucleotide specific barcode ACGT, CGTT, GTAT and sequenced using a HiSeq 2000 (Illumina, San Diego, CA) |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | SINGLE |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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