De novo assembly and comparative transcriptome analysis reveal genes potentially involved in changes of terpenoids in wheat germplasm resource, that defend against the wheat aphid Sitobion avenae (Fabricius)

Terpenoid volatiles play an important role in direct and indirect plant defense responses against herbivores, including in gramineous crops such as transgenic rice and corn. The conventional varieties of wheat, an important gramineous cereal crop, lack aphid-resistant genes. It is therefore, necessary to seek aphid-resistant genes by screening for potential terpenoid synthase genes in wheat germplasm resources. The result showed that aphid-damaged Octoploid Tirtitrigia emitted a higher amount of S-linalool, ent-kaurene, (+)-delta-cadinene, (3S,6E)- nerolidol compared to intact plant. In addition, (E)-β-caryophyllene, β‐Myrcene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) were new volatile terpenoids emitted by the damaged plant. Further olfactory responses tests showed that S-linalool significantly repelled Sitobion avenae (Fabricius). Using the Illumina sequencing platform, approximately 203.09 million high quality paired-end reads were obtained. After de novo assembly and quantitative assessment, a total of 182,348(74.8%) unigenes were annotated by alignment with public protein databases. Of these unigenes, 2,389 differentially expressed genes were identified between intact and damaged ears of Octoploid Trititrigia. The expression profile of 10 randomly selected TPSs was confirmed with RT-qPCR. Candidate genes involved in terpenes biosynthesis were identified showing significant transcript changes between intact and damaged plant ears of Octoploid Trititrigia. Also transcript abundances of terpenes biosynthetic pathway-related genes were positively correlated with the production of volatile terpenoids in ears. The unigenes of S-linalool synthase gene was mapped to the cloned cDNA WT008_M07 (AK333728) and WT013_P07 (AK335856) of the Chinese spring wheat cultivar. The predicted protein complete ORF sequence (TaLIS1/2) when compared with the S-linalool synthase gene genes in damaged plant ears were higher than those in intact plant ears, which was consistent with the volatile constituents of terpenoids. Our results could get insight into the mechanism of S. avenae infestation under different metabolic processes. This will enable the genetic manipulation of Octoploid Trititrigia to strengthen aphid resistance. with aphids. Every 2 the olfactometers were rotated by 90 to 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.

This assembled transcriptome of S. avenae-damaged Octoploid Trititrigia and intact ears can provide more molecular resources for future functional characterization analysis of genomics in volatile terpenoids involved in direct or indirect defenses. Our study describes the metabolic regulation mechanism of volatile terpenoids in gramineous crops, which provides support for both breeding and genetic modification of wheat varieties resistant to wheat aphids.

Background
Plants interact with the environment by producing a variety of chemical compounds. Plants can produce volatile compounds to protect themselves when attacked by herbivorous insects [1;2].
thaliana of wheat FPP synthase gene (FPS) could repellent the peach potato aphid (Myzus persicae Sulzer) [26]. Overexpression of P. lunatus TPS genes Pltps3 and Pltps4 in rice increased the attraction of transgenic rice plant to the natural enemies of the rice stemborer (Chilo suppressalis Walker) [15].
Transgenic Nicotiana tabacum Linn. plants overexpressing GhTPS12 gene, which produced relatively large amounts of (3S)-linalool, showed direct defense against herbivores [28]. Studies have shown that S-linalool is the most abundant volatile emitted by rice plants damaged by caterpillars of the fall armyworm (Spodoptera frugiperdaSmith & Abbot), and could significantly attract parasitic wasps [29].
S-linalool is a key component in the direct and indirect induced volatiles of rice. In the defense response against brown planthopper (Nilaparvata lugens (Stal)), rice adopts the "push-pull" strategy using S-linalool as the weapon to drive away the pest N. lugens and attracting the natural enemy, the parasitoid Anagrus nilaparvatae Pang & Wang [11]. Many TPSs are multiproduct enzymes, which produce volatile terpenoids that can play a direct or indirect defense against herbivory.
The English green aphid, Sitobion avenae (Fabricius), is considered one of the most important pests of wheat in Asia and Europe [30][31][32]. It can cause heavy economic damage to wheat, both as a phloem feeder and as a vector of plant viruses [33][34][35]. It was reported that the wheat (T. aestivum, AABBDD) gene Tafps1 and Tafps2 played important roles in induced responses to aphid infestation and in sesquiterpene synthesis [26]. The alarm pheromone for many pest aphids, the sesquiterpene (E)-βfarnesene, was successfully developed by genetic engineering of the hexaploid wheat cv. Cadenza.
The released pheromone showed intrinsic activity against aphid pests and attracted the natural enemy, the parasitic wasp Aphidius ervi Haliday in laboratory experiments. Although these studies show considerable potential for aphid control, there was no change in aphid numbers in field trials.
This was attributed to low insect numbers and erratic climatic conditions [36]. However, volatiles terpenoids play an important role in direct and indirect plant defense responses against herbivores, especially in transgenic gramineous crops such as rice and corn [15,24,38]. Moreover, studies have shown that the expression of terpene synthase genes in plants increased dramatically after pest infestation [15,39,40]. Conventional wheat varieties emit few volatile terpenoids which showed resistance to aphids. Our previous study showed that the wheat germplasm resource Octoploid Trititrigia was a promising candidate for breeding aphid-resistant wheat varieties. The quality and quantity of volatile terpenoids changed remarkably in aphid-damaged wheat plant compared with intact wheat plant. In order to regulate the synthase genes of volatile terpenoids which showed resistance to aphids, we conducted a de novo transcriptome analysis to compare RNA-seq profiles between intact and damaged wheat plant ears of Octoploid Trititrigia by using Illumina sequencing technology. The purposes of our study were to investigate differentially and specifically expressed genes which related to the biosynthesis of volatile terpenoids, revealing a greatly enriched pathway for the changes of volatile terpenoids between intact ears and damaged ears of Octoploid Trititrigia.
In addition, rely on the analysis of the transcript abundance of differentially expressed genes (DEGs), those candidate genes which involved in TPSs were also mined. The collected volatile terpenoids were analyzed with transcriptome data. The results could provide resources and candidate genes for

Illumina sequencing and de novo assembly
We used the samples of the Octoploid Trititrigia ear, and produced 42.6 Gb of data with Illumina NovaSeq6000 high-throughput sequencing techniques. We obtained 243,718 high-quality reads for de novo assembly by trimming the adapter and primer sequences, fuzzy nucleotides, and low quality sequences. The total length of the transcribed sequence was 20,308,8986 bp, the average length was 833.29 bp, and the N50 size was 1041 bp with 47.09% GC content (Table 1). These assembled transcript sequence length was ranged from 300 to 3000 bp, and the dominate length of sequences was 300 to 400 bp (Fig. 2). In addition, the sequence length ≥3000bp transcripts numbers were 3863.
Candidate genes related to terpene biosynthesis typical conserved motifs and domains of specific terpene synthase (Fig. 9). TaLIS contained an aspartate-rich region DDxxD motif, which was described as a function involved in substrate binding to coordinate divalent metal ions [24].
TaLIS was clustered with angiosperm monoterpene synthases (TPS-g family) based on the phylogenetic relationship analysis (Fig.10). There was no difference in the phylogenetic relationship of the TaLIS gene family from that of the current plant. Furthermore, the S-linalool synthase of the close relatives almost clustered into a group, indicating that they have a closely evolutionary relationship in related plant species other than distant plant species.
Olfactory response of S. avenae to S-linalool In order to determine the response of S. avenae to S-linalool, its olfactory responses todifferent doses of S-linalool were investigated. The results showed that when S-linalool concentration was 100 μL·mL -1 , it had a significant repellent effect on aphids (W = -3.385, P < 0.05) (Fig. 11).

Discussion
The results of this study showed that the S. avenaecan induce biosynthesis of volatile terpenoids such as S-linalool, DMNT, (E)-β-caryophyllene with significant ramifications for plant-insect interactions.
Specifically, higher levels of S-linalool were detected in Octoploid Trititrigia ears infested byS. avenae compared with uninfested ears. Previous studies have shown that S-linalool was the most abundant volatile emitted from pest-damaged rice and cotton plants, and showed direct or indirect defense against herbivores [28,29]. Also, A. thaliana plants genetically engineered to release linalool, repelled aphids [7]. Additionally, field studies conducted with OsLIS-silenced rice plants showed that inducible (3S)-linalool attracted predators, parasitoids as well as chewing herbivores, but repelled the rice brown planthopper Nilaparvata lugens (Stal)) [11]. The olfactory response of S. avenae indicated that the high concentration of S-linalool (100 μLˑmL -1 ) had remarkable repellent effect on S. avenae.
Further investigation would be needed to test the ecological functions of S-linalool on the aphid natural enemies such as Aphidius gifuensis Ashmaed and Aphidius avenae Haliday. Apart from Slinalool, the amount of ent-kaurene, (+)-delta-cadinene were also significantly higher in S. avenaedamaged plant ears than intact ears. Likewise, the infested ears emitted new HIPVs such as β-Myrcene, (E)-β-caryophyllene and DMNT. Previous studies had shown that DMNT, (E)-β-caryophyllene, (+)-delta-cadinene could act as a direct or indirect defense substances against pests on plants [11,23,37,38]. The functional significances of the other volatile terpenoids to aphids remain to be determined.
Some of the structural genes of TPSs have been identified, and even successfully used to increase direct plant defense [8,28] or indirect plant defense by attracting natural enemies of the herbivores [9][10][11]41]. Many structural genes involved in TPSs have been discovered in field crops in the past few years [15,36,42,43], which has greatly promoted understanding of the modulation of terpene compounds biosynthesis. Meanwhile, many studies have reported that the expression levels of TPSs and cytochrome P450 enzyme were significantly correlated with pest defense in plants [15,28,38].
The emission of volatile terpenoids were higher in S. avenae-treated Octoploid Trititrigia ears than untreated ears. However, little has been known about the molecular mechanisms controlling the TPSs biosynthesis in wheat. Thus, a comprehensive identification of DEGs and modulating pathways related to the TPSs was profiled using de novo transcriptome data via comparison between intact and aphid damaged Octoploid Trititrigia ears.
TPSs and their volatile compounds, volatile terpenoids, play an important role in direct and indirect plant defense responses against herbivores [10,44,45]. Volatile terpenoids are generally released until plants have been damaged by herbivorous insects for some time [4,35]. In this work, changed transcript abundances of the TPSs gene were detected in damaged Octoploid Trititrigia ears compared to intact Octoploid Trititrigiaplant ears. Based on RT-qPCR results, the expression profiles of the 10 genes were identical by Illumina sequencing or RT-qPCR analysis, but the accurate folding change of the two methods was biased. This suggested a relative rationality and accuracy of the transcriptome analysis in the present study.
In this study, TPSs and cytochrome P450 enzyme related genes displayed significant expression changes between the intact and aphid-damaged Octoploid Trititrigia tissues, which is in accord with previous findings about changes of volatile terpenoids in other plants after pest infestation [27]. Such information would help provide a deeper understanding of how changes in gene expression are related to the volatile terpenoids changes in the Octoploid Trititrigia ears.
The GC-MS analysis of volatile terpenes was combined with transcriptome data. The results showed that the quality and quantity of terpenoids volatile compounds in S. avenae-damaged ear changed compared to intact ear. Linalool was the most abundant volatile in S. avenae damaged compared to intact Octoploid Trititrigia (Fig.1). The transcripts regulating S-linalool synthase genes were also the most abundant. Most of the unigene of S-linalool synthase can be mapped to the cloned cDNA WT008_M07 (AK333728) and WT013_P07 (AK335856) of Chinese spring wheat cultivar, and the predicted protein sequence can be compared with the S-linalool synthase gene of other species.
Phylogenetic tree results showed that the TaLIS1 and TuNES1werehighly homologous. TPSs are multifunctional enzyme [23]. S-linalool synthase genes at the same time also can control (3S,6E)nerolidol (DMNT precursor substances) [15]. Therefore, the two T. aestivum synthase genes may also Furthermore, it has been identified that the transcription levels of the TPSs family genes in damaged plant ears were higher than those in intact plant ears, which was consistent with the volatile constituents of terpenoids. Our results could get insight into the mechanism of S. avenae infestation under different metabolic processes. This will enable the genetic manipulation of Octoploid Trititrigia to strengthen aphid resistance. 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.

Insects and plants
Ionization was by electron impact (70 eV), and the scan range was between m/z 50 and 650. Volatiles 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

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 fourarm 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 Declarations Ethics approval and consent to participate Not applicable. Neither human or animal subjects, human or animal materials nor human or animal data were used on this manuscript. The authors declared that experimental research works on the plants described in this paper comply with institutional, national and international guidelines.

Consent for publication
Not applicable.

Availability of data and materials
Part of the data generated or analysed during this study are included in this published article and its supplementary information files. The datasets generated transcriptomic analysis data during the current study are not publicly available due research period but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Length distribution of the assembled transcript sequences of Octoploid Trititrigia ears