Insects and plants
The colony of wheat aphid, S. avenae, were reared on seedlings of wheat lumai 21 in an environmental chamber (21℃, L16:D8 photoperiod, and 70% relative humidity), with new wheat seedlings provided once a week. Octoploid Tirtitrigia, the new intermediate type of wheat, which was breeding by introduced the genome of Thinopyrum intermedium (Elytrigia intermedia (Host) Nevski) into common wheat. Octoploid Tirtitrigia was provided by Dr. Xinfeng Li from National Wheat Improvement Center of China. We declare that the collection of plant materials comply with institutional, national, or international guidelines.
Treatments
Wheat Octoploid Tirtitrigia was sown in 10th October and covered by the insect-proof screen with 120 mesh. In the flowering stage, 100 adult wingless aphids of S. avanae were introduced in the ear of a plant to serve as treatment. Plants without aphids served as control. Both intact and damaged wheat ears were sampled after 72 h.
Volatile Collection and GC-MS Analysis
Sampling ears were collected in the field after 72 h infestation. the air entrainment (dynamic headspace collection) was carried out following standard procedures using the flowering ears of Octoploid Tirtitrigia. For each headspace collection, a ear was enclosed in a gas sampling bag (Polytetrafluoroethene, 2 L, E-Switch), open at the bottom, and with two collection ports at both sides (one for inlet of air and other for outlet), The bottom was closed without pressure around the plant stem, using seal the strip. Air, purified by passage through an activated charcoal filter, was pumped into the vessel through the inlet port at 600 mL.min–1 (flow rate was measured by a flow meter). Air was drawn out at 400 mL.min–1 through 50 mg Porapak Q (Alltech Associates, Carnforth, Lancashire, UK) in a 5 mm diam Tenax glass tube. The differences in flow rates created a slight positive pressure to prevent unfiltered air enter the system. All connections were made with polytetrafluoroethylene (PTFE) tubing with brass ferrules and fittings and closed with PITE tape. Porapak Q filled tubes were conditioned before use by washing with n-hexane (1 mL) and heating inside an oven (150 ℃) under a stream of nitrogen for a minimum of 2 h. VOCs were collected on Porapak Q tubes inserted into the collection port on the top of the vessel and were subsequently eluted with 500 μL freshly n-hexane. Air entrainment samples were analyzed by GC–MS. For air entrainment samples, aliquots of samples (1μL) were analyzed. All analyses were carried out using a DB5-MS column (30 m × 0.25 mm ID × 0.25 μm film thickness; Agilent Technologies) with a split/splitless injector (splitless mode, 220 °C). Helium was used as the carrier gas at 1 mL.min–1; the oven temperature was programmed to rise from 50 to 60°C (5 min hold) with a rate of 5°C min–1 and then raised to 250 °C with a rate of 10°C min–1 (5 min hold). The transfer line temperature was 250 °C; ion source temperature was 250°C. Ionization was by electron impact (70 eV), and the scan range was between m/z 50 and 650. Volatiles were identified by comparison of their GC retention times and mass spectra with authentic reference.
RNA isolation, cDNA library preparation and sequencing
Total RNA was extracted from three biological replicates of the flowering of earusing the Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions, and then treated with RNase-free DNase I (Invitrogen). The quality and concentration of the total RNA were determined using a NanoPhotometer® spectrophotometer (Implen, CA, USA) and the Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), respectively. The RNA integrity was confirmed using an Agilent 2100 Bio-Analyzer (Agilent Technologies, CA, USA). RNA samples with RIN ≥7 and 28S:18S ratio ≥1.5:1, total amount ≥3μg, were considered acceptable and used as input material to construct the sequencing library. Illumina paired-end library was prepared using the standard TruSeq RNA Sample Prep Kit (Illumina Inc.). Briefly, poly (A) mRNA was enriched from total RNA using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA) according to Illumina manufacturer’s instructions. Double-stranded cDNA was generated using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). After that, Pair-end RNA-seq libraries were constructed using the Illumina Paired End Sample Prep kit and were then sequenced on an Illumina HiSeqTM 2000 system. High-quality paired end reads with a length of 200bp were obtained by deleting low-quality reads with vague nucleotides and filtering adapters from the raw data.
De novo transcriptome assembly, functional annotation and classification
For transcriptome assembly, a strict Illumina pipeline was used for filtering the raw sequence reads. All reads with adapter sequences, unknown nucleotides comprising more than 10%, and low-quality reads (> 50% base with quality value Q≤5 in a read) were removed. De novo transcriptome assembly was accomplished from all these clean reads with the Trinity program. Only those sequences with perfect homology or not more than two nucleotide mismatches were used for conservative and accurate annotation.
To annotate the transcriptome, assembled sequences were further used as query sequences to blast with Nr (NCBI non-redundant protein sequences), SwissProt, KEGG (Kyoto Encyclopedia of Genes and Genomes database) and KOG (eukaryotic orthologous groups), Pfam (Protein family) and GO databases, respectively. The best hit of alignment was used to infer biological function of assembled transcripts. Additionally, GO (Gene Ontology) terms of assembled transcripts were extracted from the best hits against the Nr and Pfam using the Blast2GO. After acquiring the GO annotation for each assembled transcript, GO functional classification was achieved using WEGO software for all the transcripts. KEGG pathway annotations were retrieved from KEGG (http://www.genome.jp/kegg/) database.
Identification of differentially expressed genes
Using the de novo assembled transcriptome data as reference sequence, the clean reads of each sample were mapped to this reference sequence using Bowtie 2, allowing no more than two nucleotide mismatches. The gene expression levels were determined by the numbers of reads uniquely mapped to the specific gene and the total number of uniquely mapped reads in the sample and calculated using the RPKM method (reads per kb per million reads). Using the edgeR software, differentially expressed genes (DEG) were determined between the damaged and intact Octoploid Tirtitrigia ear libraries, respectively, and the DEGs were defined as significant based on a false discovery rate (FDR)≤0.05 and an absolute value of log2Ratio≥1 [46].
GO and KEGG enrichment analysis of DEGs
The differentially expressed genes (DEGs) were used for GO function and KEGG pathway enrichment analysis, and a Bonferroni-corrected p value≤0.05 was selected as a threshold level to determine significant enrichment of DEGs. GO enrichment was conducted using Blast2GO and WEGO. KEGG enrichment analysis was performed using KOBAS based on the comparative results between the identified genes and the background reference sequences. GO terms and pathways enriched in the set of DEGs were calculated by the hypergeometric test.
RT-qPCR validation of DEGs
Ten terpene synthases (TPSs) genes that were prominently and differentially expressed in our expression profile data were randomly chosen for validation by reverse transcription quantitative PCR (RT-qPCR). Ten differentially expressed candidate genes related to TPS were selected for validation through RT-qPCR, and gene-specific primers were designed by Primer Premier 5. All primer pairs for these qPCR were deposited in Additional (Additional file 9). The qPCR use Top Green qPCR SuperMix (TransScript) in 20 μL reactions according to the manufacturer’s instructions. The qPCR amplification was performed in thermocycler conditions starting with 30s at 94 °C, 5s at 94 °C, 40 cycles of 30s at 60 °C, and 10 s at 95 °C, followed by 1 min at 60 °C.The T. aestivum gene Taactin was taken as an internal reference. To calculate the expression abundances of target genes, the method 2-ΔΔCt for statistics was applied with three biological replicates.
Sequence analysis of Octoploid Trititrigia predicted S-linalool synthase gene
The theoretical isoelectric point (pI) and molecular weight (MW) of the Octoploid Trititrigia S-linalool synthase gene was analyzed by the ExPASy website (http://web.expasy.org/compute_pi/). The protein coding region of Octoploid Trititrigia S-linalool synthase gene was searched by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). Multiple sequence alignment of TPS protein sequences was performed by CLUSTALW. To reconstruct phylogenetic relationship, the neighbour-joining (NJ) tree was conducted using MEGA7.0 and 1 000 replicates of bootstrap analyses.
Four-arm olfactometer assays
Four-arm olfactometer were used to test the behavioural responses of aphids towards the S-linalool chemical compounds (Shyuanye, Shanghai, 98%). The olfactometer was made up of Plexiglas and possessed a 10.5 cm walking area that was similar to the one described by Vet et al. (1983). The four-arm olfactometer was connected to a vacuum pump to extract air, and the flow rate of each arm was 150 mL ˑmin–1. The olfactometer assays were conducted in a temperature of (21 ± 1) ℃ and a relative humidity of (70 ± 5) % in controlled room. Five amounts (0.01,0.1,1,10,100 μLˑmL–1) of S- linalool, were placed on a 1cm x 1cm piece of filter paper and offered to the tested aphids. Each aphid was placed at the center of the exposure chamber, which was observed during 15 min and record the time which was each insect in every olfactometer areas. Twenty replicates were performed with aphids. Every 2 observations, the olfactometers were rotated by 90 degrees to avoid directional bias. Between each treatment, the system was cleaned with pure ethanol and rinsed with distilled water. Test data was expressed as “Mean + Standard Deviation”, and IBM SPSS 20.0 was used for corresponding analysis and test. The nonparametric test (Wilcoxon signed rank sum test, and the test quantity was expressed by W) for the two correlated samples was performed for the aphid residence time in the treatment arm and 1/3 of the residence time in the control arm.