Comparative transcriptome analysis of genes involved in two peanut varieties under drought stress


 Background

Peanut is one of the most important world oil crops. Peanut qualities and yields are restricted dramatically by abiotic stresses particularly by drought. Therefore, it would be beneficial to gain a comprehensive understanding on regulatory mechanisms of the peanut genomic transcriptional activities responding to drought, and hopefully extracting peanut molecular drought-resistance mechanisms.
Results

In this study, two peanut varieties NH5 (resistant) and FH18 (sensitive) which showed significantly differential drought-resistance were screened from twenty-three main commercial peanut cultivars and used for physiological characterization and transcriptomic analysis. NH5 leaves showed higher water and GSH contents, faster stomatal closure and lower relative conductivity (REC) than FH18. Under the time-course of 0 h (CK), 4 h (DT1), 8 h (DT2) and 24 h (DT3), drought-treatments tent to exert repressive impacts on peanut transcriptomes since the number of down-regulated differential expressed genes (DEGs) increased with the progression of treatments in both varieties.
Conclusions

Nevertheless, NH5 seemed to maintain stabler transcriptomic dynamics than FH18. Furthermore, annotations of identified DEGs implicated that signal transduction, elimination of reactive oxygen species, maintenance of cell osmotic potential were key drought-resistance-related pathways. Last, examination of ABA and SA components suggested that the fast stomata closure in NH5 was likely to be mediated through SA rather than ABA signaling. In all, these results have not only provided us comprehensive pictures of peanut drought transcriptomic changes, but also laid a foundation for further identification of the molecular drought tolerance mechanism in peanut and other oil crops.


Results
In this study, two peanut varieties NH5 (resistant) and FH18 (sensitive) which showed signi cantly differential drought-resistance were screened from twenty-three main commercial peanut cultivars and used for physiological characterization and transcriptomic analysis. NH5 leaves showed higher water and GSH contents, faster stomatal closure and lower relative conductivity (REC) than FH18. Under the time-course of 0 h (CK), 4 h (DT1), 8 h (DT2) and 24 h (DT3), drought-treatments tent to exert repressive impacts on peanut transcriptomes since the number of down-regulated differential expressed genes (DEGs) increased with the progression of treatments in both varieties.

Conclusions
Nevertheless, NH5 seemed to maintain stabler transcriptomic dynamics than FH18. Furthermore, annotations of identi ed DEGs implicated that signal transduction, elimination of reactive oxygen species, maintenance of cell osmotic potential were key drought-resistance-related pathways. Last, examination of ABA and SA components suggested that the fast stomata closure in NH5 was likely to be mediated through SA rather than ABA signaling. In all, these results have not only provided us comprehensive pictures of peanut drought transcriptomic changes, but also laid a foundation for further identi cation of the molecular drought tolerance mechanism in peanut and other oil crops.

Background
The peanut (Arachis hypogaea L.) is one of the main human oil and protein food sources. Its rich nutritional values are especially bene cial to the human cardiovascular system. The plantation of peanuts is distributed widely across developing countries from semi-arid tropical to subtropical regions [40,46] Historically, peanuts have played important roles in the Chinese agricultural economy and are currently the top ranking exported-crops from China. The annual Chinese peanut output had reached 1.3 × 10 tons in 2008 [13]. Nevertheless, the quality and yield of peanuts are often seriously diminished by the drought. According to the public statistical data, the annual worldwide loss of peanut production caused by drought is about 6 million tons [41]. The frequency and severity of global drought are on the rise today and are expected to become severer in the next 30-90 years [13]. Future droughts have already exhibited the tendency of high frequency, long duration and wide range. Once struck by drought, the normal growth of crops will be prohibited then yield reduction and even no-grain harvest will be caused. Up to date, studies have shown that drought stresses affect various biological processes, including water physiology, nutrient absorption, enzyme activity, photosynthesis and assimilate transport [15,26,53]. Plants under drought stresses can adjust their morphological, physiological and metabolic processes by regulating gene expression patterns [10]. Firstly, the expression of transcription factors can be regulated through the plant hormone signaling transduction. Then, multiple stress-responsive genes are induced [43,49,60]. In detail, drought stresses usually up-regulate abscisic acid (ABA), ethylene (ETH) and salicylic acid (SA) signaling pathways thus directing plants to produce osmo-regulatory substances to maintain cell osmotic potentials and antioxidant enzymes to re-establish the oxidation balances [30,37,50]. In addition, plants can also close stomata, thicken cuticle and harden cell walls to increase their drought resistance.
Until recently, transcriptomic studies have been conducted attempting to gain insights on molecular mechanisms underlying various perspectives of peanut biology. For example, Chen et al. have used young pods of Yueyo7 peanut variety for transcriptome sequencing trying to answer the question why peanut young pods will not enter developmental programs in the air [11]. Also, Wu et al. have used leaf, stem and root tissues from different developmental stages of the Spanish botanical type peanut A. hypogaea L to investigate peanut development by transcriptomic analysis [58]. In addition, Cui et al. have conducted transcriptome sequencing with salt-stressed LH14 shoot and root tissues to understand impacts of salt-stress on peanut [12]. However, there are only a few reported transcriptomic studies aiming at drought-related molecular mechanisms in peanuts. Shen et al. have studied transcriptomes in peanut leaves which were drought-stressed for seven days from FH1 a drought-resistant variety. In this study, information on peanut molecular responses to long-term drought stresses was revealed [44]. Another transcriptomic study was conducted by Zhao et al. [65]. They speci cally studied peanut molecular responses to short-term drought (two-days) in root tissues from J1 another characterized drought-resistant peanut variety. Last, Brasileiro et al. have analyzed transcriptomes from wild-peanut tissues which were stressed for eleven-days [9]. Results from these three drought-transcriptome studies have demonstrated that drought stresses could induce the differential changes of expression levels in a suite of genes such as ABA-related, carbon metabolism-related, proline-related and photosynthesis-related genes. Despite above studies, molecular researches on drought-resistance mechanisms in peanut is still in a preliminary stage because of its huge-size allotetraploid genome.
Transcriptome sequencing technology has become an important tool for analyzing the molecular mechanism of drought-resistance in plants. At present, RNA-Sequencing (RNA-Seq) can provide increasing amount of information on differentially expressed genes, transcript structures, new transcripts and isomers, RNA alternative splicing and allele-speci c expression etc [57]. RNA-Seq has been successfully applied to cuckoo, Yerba Mate and cotton [1,10,21], as well as many crop plants such as lentils, buckwheat and millet [23,32,64], to analyze their molecular mechanisms of drought resistance.
These studies have supplied helpful information and improved our understanding of the molecular mechanism of plant resistance to drought stresses.
Transcriptome sequencing comparing varieties with signi cantly different resistance is proved to be an effective strategy for analyzing the stress-responsive molecular mechanism in a certain crop [55]. Since early drought-responses usually indicate the upstream regulatory events to the whole drought-responsive mechanism, it would be especially valuable to ll the blank of understanding on peanut drought-induced early molecular dynamic changes. Therefore, we chose two commercial peanut varieties which demonstrated differential drought-resistance in our screening as study materials (FH18 the sensitive type and NH5 the resistant type). PEG-6000 treatments were used to simulate drought stressful conditions during the seedling stage. Signature physiological indexes were further measured to monitor the physiological status of peanut seedlings under continuous drought stress. The RNA-Seq technology was used to sequence leaf transcriptomes of FH18 and NH5 at different stress time-points. The peanut transcriptomic spectrum under drought was studied, from which insights into the molecular mechanism of peanut drought resistance in the seedling stage were gained.

Result
Peanut drought-resistances The plantation acreage of peanuts in the Northeastern provinces of China has been constantly increasing during recent years. To evaluate the performances of our current peanut germplasms under drought and to search for suitable research materials for peanut drought biology, we examined twenty-three representative commercial peanut varieties for their drought-resistances. After 24 h of simulated drought stress, all tested varieties had exhibited differential relative fresh-weight (FW), wilting-index (WI), leaf water-loss and conductivity (Table S1). And the level of drought-resistance was represented by a calculated "the membership function" (described as in the "materials and methods"). By this approach, the most drought-resistant varieties were NH5 and HY22 with ratings of 0.884 and 0.833 respectively. The least drought-resistant varieties were FH18 and NH16 with ratings of 0.304 and 0.288, ~ 36% of NH5 ( Fig. 1). Showing synchronized paces of plant development, FH18 and NH5 were chosen as drought sensitive and drought-resistant peanut varieties for further analysis.

Analysis of Drought Stress Responses
Since FH18 (sensitive type) and NH5 (resistant type) seedlings showed vigorous growth during the 4thleaf stage (Fig. 1), these seedlings were examined for phenotypic changes after being subjected to continuous simulated drought stresses. First, leaves of both varieties had exhibited an obvious wilting phenotype when the drought treatment prolonged but to a severer extent in FH18 than NH5 (Fig. 2). For example, FH18 leaves started drooping at DT1 (4 h), while no obvious change was observed in NH5 leaves at the same time-point. Furthermore, at DT2 (8 h), FH18 leaves signi cantly wilted but NH5 leaves only partially wilted (Fig. 2), indicating that NH5 could preserve a higher leaf water content than FH18 under drought conditions. Stomata are important gateways for plants to control carbon and water exchange between leaves and the atmosphere. Based on the above observations, different stomatal-closure patterns should be identi ed between FH18 and NH5 during the time-course of drought treatments. As expected, the stomata of both peanut varieties remained open at 0 h of drought stress (Fig. 3). NH5 showed stomatal closure at DT1 but not in FH18 (Fig. 3). For DT2 and DT3, the stomata in both peanut varieties were all closed ( Fig. 3). These results suggested that drought-induced quick stomatal closure in NH5 leaves compared to FH18 leaves, which might contribute to a relatively slower water-loss and a higher leaf water content as displayed by NH5 in Fig. 2.
Relative conductivity (REC) is an index which can be used to re ect the ability of osmotic adjustment to stresses in the plasma membrane. Under drought conditions, the lower the REC value correlates with the better abilities of adjusting osmotic balance and thus stronger drought tolerance. As shown in the Fig. 4a, the REC values of NH5 were lower than those of FH18 at DT1 and DT2 time points (relative REC increase compared with CK: 1.81% for NH5 and 7.36% for FH18 at DT1; 5.85% for NH5 and 16.36% for FH18 at DT2) (P < 0.01). These data indicated better osmotic adjustment ability in NH5 than in FH18.
Reduced glutathione (GSH) is one of the most effective scavengers for reactive oxygen species (ROS).
Next, the GSH contents in FH18 and NH5 were determined (Fig. 4B). Under the control conditions, there was no signi cant difference in the GSH content between these two peanut varieties. As the process of drought treatment progressed, the GSH content in both peanuts showed an increase trend but to different extent. Compared with the CK group, DT1, DT2 and DT3 of NH5 increased by 0.15 mol/g, 0.37 mol/g and 1.4 mol/g respectively, while DT1, DT2 and DT3 of FH18 increased by 0.15 mol/g, 0.26 mol/g and 0.52 mol/g respectively (P < 0.01). These results showed that stressed NH5 contained more GSH and therefore stronger ROS scavenging capabilities than FH18.

Transcriptome sequencing and assembly
Transcriptomes from the FH18 and NH5 seedlings which were stressed to different levels were sequenced using Illumina 2000, and totally twenty-four transcriptome libraries were constructed (three library repeats for each variety at every time-point). After removing the low-mass readings, 177.69 Gb of clean data were obtained. The clean data for each sample reached 5.90 Gb and the percentage of Q30 bases was 94.62% or more. The "Clean Reads" of each sample were sequenced with the designated reference genome, and the alignment e ciency ranged from 94.47% to 97.49%. Based on comparisons, alternative splicing prediction analysis, gene structure optimization analysis and discovery of new genes were carried out, and 6,940 new genes were discovered (Table S1).

Expression Analysis of Differential Genes
Gene expression patterns could be signi cantly affected by drought stresses. Therefore, differentially expressed genes (DEGs) were extracted according to their differential expression levels in different samples. Then functional annotation and enrichment analysis were carried out with these identi ed DEGs. DEGs at DT1, DT2 and DT3 of FH18 were identi ed as 7,989 (up-regulated 3,709/ down-regulated 4,280), 9,386 (up-regulated 4,052/down-regulated 5,334) and 11,218 (up-regulated 4,881/down-regulated 6,337), respectively. In contrast, 4,497 (up-regulated 2,448/down-regulated 2,049) DT1, 5,780 (up-regulated 2,673/down-regulated 3,107) DT2 and 5,762 (up-regulated 2,585/down-regulated 3,177) DT3 DEGs for NH5 were identi ed. It was obvious that at each time point DEGs of FH18 signi cantly outnumbered those of NH5, for example almost twice of NH5 DEGs at DT3. This difference indicated that drought stresses would induce more volatile transcriptomic dynamics in FH18 than in NH5. From another aspect, NH5 seemed to be able to maintain a stabler transcriptome under drought conditions. Furthermore, when carefully examined, the number of down-regulated FH18 DEGs was ~ 30% more than up-regulated DEGs at both DT2 and DT3. For NH5 DEGs, the ratios were ~ 15% at DT2 and ~ 20% at DT3.
These results suggested that drought stresses within 24 h tended to exert more of a down-regulation impact on peanut transcriptomes. Also the comparatively lesser extent of down-regulation in NH5 transcriptomes than FH18 might re ect and con rm previous physiological characterizations of NH5 as drought-resistant and FH18 as drought-sensitive. Next, cluster analysis was carried out with identi ed differential genes (Fig. 5b).

Functional Annotation of DEGs
Next, functional annotation was carried out for DEGs (refer to Table S2 for statistical numbers of genes annotated in each differential gene set). In order to determine subordinate categories of the responsive genes, we used GO classi cation for the DEGs in FH18 and NH5 respectively. And the matched DEGs were divided into three functional categories: biological processes, molecular functions and cell components ( Fig. 6a and b). In the category of biological processes, the most abundant genes belonged to metabolic processes and cellular processes. In the category of cell components, the number of genes in cell parts and cells was the highest. In the category of molecular function, DEGs mainly belonged to binding and catalytic activity subgroups. In order to identify active biological pathways enriched with DEGs in both peanut varieties, KEGG pathway database was used ( Figure S1). The results of KEGG enrichment analysis were shown in the following Figure with the rst twenty top-ranking pathways by smallest signi cant Q values ( Fig. 6C and D). Although FH18 and NH5 had shared similar pathwayenrichment results, the number of enriched genes and the expression levels of enriched genes were quite different (Table S3 and S4). The enriched-pathways included GSH-related glutathione metabolism, glycolysis, glyoxylic acid and dicarboxylic acid ester metabolism associated with pyruvic acid. Pathways of corneal and wax anabolism, fatty acid degradation related to stratum corneum, carbon xation, photosynthesis-antenna protein, photosynthesis, degradation of amino acids valine, leucine and isoleucine, and porphyrin and chlorophyll metabolism were also enriched. In addition, several pathways were only enriched in the drought-resistant variety NH5, including alanine metabolism, sulfur metabolism, sphingolipid metabolism, phenylpropane biosynthesis, isoquinoline alkaloid biosynthesis and biosynthesis of tropane, piperidine and pyridine alkaloid.

Peanut Drought Resistant-Related Genes and Pathways
In order to explore the drought-resistance mechanism of peanut, we examined transcriptional changes of potential drought-resistance genes in FH18 and NH5 with drought treatments. It was found that genes related to ABA and SA signal-transduction were signi cantly up-regulated, speci cally sixteen ABF genes and twenty-two TGA (TGACG motif-binding factor) genes (Table S5). Compared with FH18 transcriptomes, some genes were differentially expressed only in NH5. These NH5-speci c DEGs could be categorized into various biological pathways. Among them, fourteen genes were identi ed as ROSscavenging genes (Table S5), which belonged to glutathione metabolism and proline metabolism respectively. Thirty-three osmotic-potential-regulating genes (Table S5) were subordinate to the metabolism of arginine, proline, sucrose and starch. In addition, fourteen cell wall sclerosis-related genes and fourteen cutin and wax metabolism genes were also enriched from NH5 transcriptomes, which were believed to be able to affect water loss (Table S5). Another set of genes involved in peanut defenseresponses showed much higher expression levels in NH5 than in FH18. On the other hand, FH18-speci c differential genes were also identi ed, however their expression patterns indicated that these genes were suppressed by drought treatments. Furthermore, another 126 DEGs genes were identi ed to enrich in main drought-responsive metabolic pathways (Table S5) such as sphingolipid metabolism, photosynthesis, pyruvate metabolism, fatty acid degradation and tricarboxylic acid cycle. In conclusion, a diagram of interactions of above-described enriched-pathways was drawn and shown as in Fig. 7.

Real-time qPCR Validation
In order to validate the accuracy of transcriptome data sets, the real-time qPCR technology was applied to analyze transcriptional levels ten genes which were randomly selected from drought-resistant-related pathways. The relative expression levels of genes were measured and calculated with ARAH1 as the internal reference gene. These ten genes were: pyruvate dehydrogenase; glutamate synthetase, agmatine deiminase isoenzyme X2, PXG, trehalose 6-phosphate synthase/phosphatase, inositol oxygenase 2, glutathione S-transferase, cinnamyl alcohol dehydrogenase, glycerol kinase and enoyl-CoA hydratase. RT-PCR results con rmed that the transcription changes of these ten genes were comparable with the foldchanges gained from our transcriptome analysis (Fig. 8).

Adaptation of Peanuts to Drought
Drought stress is one of the main limiting factors for crop growth and productivity. In general, plant drought tolerance involves the combination of a variety of physiological and biochemical changes based on coordinated expression of hierarchy of genes. This complex mechanism is the result of interaction between the plant heredity and changes in the external environment [20, 61,62]. In this study, we used PEG-6000 to simulate drought stresses in combination with the transcriptome sequencing technology to analyze the drought-resistance in two peanut varieties (FH18 and NH5). Compared to FH18, the droughtresistant variety NH5 showed stronger capabilities of adjusting osmotic-potential of the plasma membrane and scavenging reactive oxygen species (ROS). We also observed that the stomata of FH18 and NH5 closed to reduce water loss, and particularly the quicker stomatal closure in NH5 than in FH18.

Stratum Corneum Biosynthesis and Cell-Wall Sclerosis
The stratum corneum is a membrane structure composed of wax, cutin and polysaccharides. As a barrier against environmental stresses, its contents change under drought stresses and therefore play vital roles in reducing plant water loss [2,29,54]. Typical cutin is represented by epoxy C16/C18 fatty acids, which are crosslinked by ester bonds to form elastic polyester structures [5,16]. Waxes consist of various aliphatic molecules, mainly long-chain fatty acids (VLCFAs), containing more than 20 carbon atoms and their derivatives including primary alcohols, secondary alcohols, aldehydes, alkanes, ketones and wax esters [36]. In this study, we found that the transcriptional abundances of C18/C22 synthetic genes were induced by drought stress in both FH18 and NH5. It has been speculated that the drought-stressed peanut stratum corneum may be mainly composed of C18 fatty-acid cutin and docosan-acid wax. Our results showed that drought induced cutin and wax-related genes in FH18 faster than in NH5, however the induction levels were mostly higher in NH5 than in FH18.
Cell-wall hardening of leaves is considered as another main response of crops to drought stresses. Glucosyluronic acid kinase (GLCAK) participates in the precursor-synthesis of pectin and hemicellulose [59]. Plants harden cell walls by covalently combining lignin and hemicellulose molecules to form interwoven networks. Under drought stresses, the biosynthesis of lignin can be affected by regulating the phenylpropane biosynthesis pathway, thus the modi cation of cell walls. The phenylpropane biosynthesis would also affect the biosynthesis of anthocyanins through the formation of anthocyanins, thus promoting the formation of plant keratins [4]. It is known that plants contain lower water potential and higher level of cell-wall hardening under drought. The hardening of plant cell-walls will effectively lead toreduction in leaf growth and water transpiration. According to Xiao et al. arabidopsis GLCAK mutant (deletion mutant) has exhibited lower drought-resistance and soluble-sugar content than WT [59]. The observed drought-induction of GLCAK gene in this study may contribute to the hardening of plant cell-walls and the accumulation of soluble sugars to balance osmotic potential so to resist drought stresses. Additionally, the phenylpropane biosynthesis pathway, which is enriched only in NH5, might be another signi cant contributing factor why NH5 is more drought-resistant than FH18.

ROS Scavenging and Steady Osmotic Potential
The regulation of plant osmotic potentials is considered as a defensive mechanism against drought stresses [28]. Under drought conditions, osmotic-adjusting substances will accumulate in plants, maintaining the balance of cell osmotic potential, turgor pressure and cell volume [3]. Proline is a protective agent for osmotic regulation. High levels of proline can reduce the cell water potential and enhance the ability of removing ROS by antioxidants [39,51]. Sucrose, a soluble sugar, also plays an important role in plant osmotic regulation. The accumulation of soluble sugars can enhance the cell water absorption to protect cells [4,34]. Glutamine can be another osmotic regulator to help plants with resisting drought stresses [48]. The results from the present study have suggested that peanuts could maintain the balance of osmotic potential under drought stresses by inducing the expression of synthetic genes of proline, sucrose and glutamic acid.
Drought stressed plants tend to accumulate reactive oxygen species and thus peroxidize plasma membrane, which will lead to cell death in severe cases [22]. Glutathione reductase (GR) and dehydroascorbate reductase (DHAR), as antioxidant enzymes, can effectively scavenge free radicals in cells and protect plant organisms [7,8]. GR can reduce oxidized-glutathione (GSSH) to reducedglutathione (GSH) which is the scavenger for free radicals and particular organic peroxides [19,63]. In this study, GR and DHAR genes were up-regulated under drought stress conditions. Although the content of GSH in both FH18 and NH5 varieties showed an increasing trend with the progress of drought treatments, the transcription of GSH in the resistant variety NH5 was higher than that in the sensitive variety FH18. Since the metabolism of glutathione and ascorbic acid are important protective mechanisms against drought stresses, our ndings also indicated their vital involvements as peanut drought-resistance mechanisms.
The Roles of ABA and SA Signal Transduction Pathways Usually, plants respond to external stimuli by activation of signaling cascades in order to modify downstream gene expression patterns, nally to realize physiological and metabolic adaptations [33]. Abscisic acid (ABA) and salicylic acid (SA) signaling pathways were signi cantly induced by drought in this study. ABA and SA are two well-known plant hormones whose biosynthesis and signaling play key roles in drought-stress responses [17,31,35]. The core factors of ABA signal transduction pathway include ABA receptors (PYL/PYR), protein phosphatase 2C (PP2C), SNF1-related kinase (SNRK2) and ABA response-element-binding-factors (ABFs). Under drought stresses, ABA binds to PYLs/PYRs to inhibit PP2C which will lead to the promotion SnRK2. Then SnRK2 activates ABFs to regulate downstream transcription factors and to initiate ABA signal responses [18,36]. Drought stresses often induce an elevated ABA level in plant which will cause the binding of ABI1 (Abel son interactor protein 1) to PYL/PYR receptors. Once ABI1 binds to PYLs/PYRs, the inhibition of SLAC1 kinase by ABI1 will be released, which in turn will result in the closure of anion channels and eventually stomatal closure [66,67]. In the present study, the transcription of an ABA-biosynthesis-related gene NCED in both FH18 and NH5 was found to be signi cantly induced under all drought treatments. On the other hand, the ABAreceptor PYL/PYR-related genes were repressed by all drought treatments in NH5, while in FH18 they were partially repressed by 4 h and 8 h treatments. Taken together the observation that NH5 but not FH18 showed stomatal closure under the 4 h treatment, it was reasonable to postulate that this fast stomatal closure response might not be mediated through PYLs/PYRs. Furthermore, our results showed that the negative ABA signaling regulator PP2C was also induced and the positive component SNRK2 was repressed by drought treatments, suggesting decreased ABA-sensitivities. However, the SNRK2 targets ABFs transcription factors showed signi cant induction pattern in drought-stressed leaves. These seemingly different or even contradicting results were exactly the evidence for the complicate and intricate involvement of ABA signaling in peanut drought-resistance mechanisms.
The synthesis of SA in peanut is the phenylalanine pathway mediated by phenylalanine ammonia lyase (PAL). Previous research has shown that drought stress can promote the increase of SA content by increasing PAL activity, thus improving plant drought-resistance [6]. As Miura et al. have pointed out SA can promote stomatal closure and induce defense-genes [31]. In this study, PAL and TGA genes were expressed at high levels indicating that SA signal transduction participated in peanut drought stress responses. Although SA might dominate the peanut stomatal closure, some TGA genes were only induced in NH5 which could explain the observation that NH5 stomatal closure was faster than FH18. All these ndings on the drought-induction of ABA and SA related genes strongly implicated that the signal transduction under drought stress in peanuts was initiated by both ABA and SA hormones, thus comprised a highly complex drought-combating molecular mechanism in peanuts.

Conclusion
In conclusion, we rst characterized the phenotype and physiology of drought-treated peanuts. Then we obtained peanut transcriptome data sets of different genetic materials by the RNA-Seq technology, in order to explore the drought-related key genes and metabolic pathways. Our results showed that the signal transduction pathways of ABA and SA hormones were activated in peanut under simulateddrought stress. The expression patterns of genes related to stratum corneum biosynthesis, cell wall hardening, ROS clearance and osmotic potential were also changed in favor of resisting drought stress.
All these ndings expanded our knowledge of mechanisms of peanut drought-resistance and could facilitate future breeding of elite peanut germplasms.

Materials and growth
A total of twenty-three major commercial peanut varieties in the Northeastern China were obtained from Shenyang Agricultural University. Sixteen of them were undertaken the formal identi cation by national and local approval committee, respectively, and the others are under review. The more detail information were listed in Table S8. Peanut seeds were pre-soaked in de-ionized water and germinated in the dark for 24 h in a 28 °C incubator. Germinated seeds were planted in sand and grew under 16 h/8h light cycle, 60% humidity and 28 °C supplemented with ½ Hoagland solution every other day. Seedlings at the 4th true-leaf stage with similar height were washed, dried and then root-cultured in Hoagland solution for another three days. Addition of 20% PEG6000 to Hoagland solution was adopted as the simulateddrought condition and the untreated Hoagland solution was the control condition.

Drought-resistance screen:
After 24-hours of treatment, stressed (S) and control (CK) seedlings were collected for the following measurements. All measurements were performed with three independent biological replicates if not speci ed.
Determination of Water loss rate (RWL): The second compound leaf (1.0 g) was detached from plants and weighed immediately for FW 1 . Then detached leaves were placed in the yarn net and air-dried for 2 hours (kept from the wind and direct sunlight). Next, the air-dried leaves were weighed for FW 2 . Then leaves were dried in the oven at 80 °C to constant weight (DW). The oven-drying time-duration was represented as (t1-t2). RWL was calculated using the following equations: RWL(mg·g − 1 ·min − 1 )= FW 1 -FW 2 /DW t 1 -t 2 .
Determination of relative plant fresh weight (RFW): rst, the average fresh weights of seedling of droughtstressed and CK groups were respectively measured and calculated using three randomly-chosen seedlings as independent biological replicates for each group. Relative plant fresh weight RFW was calculated as the following: RFW = average fresh-weight of drought-treated plants/ average fresh-weight of CK plants.Conductivity: The conductivity was measured using a conductivity meter (model, maker) at room temperature (24 °C) and calculated as described by Xu et al. [62].
Determination of wilt index: the peanut wilt index grades were visually evaluated. Peanut seeds were germinated as described above. Germinated seeds were planted in 15 cm-diameter owerpots with the same amount of sand and under 16 h/8h light cycle, 60% humidity and 28 °C. Seedlings were supplemented with ½ Hoagland solutionevery other day. Once reaching the 3rd true-leaf stage, watering was stopped and the soil was allowed to dry naturally. When the soil reached 75% relative water content, digital pictures of peanut plants were taken every day. Namely: grade 0: the peanut leaves were naturally expended and were bright and glossy; the culm was rm as well. Grade 1: the leaves began to lose water; the leaves were dull and the top one or two leaves were slightly drooping. Grade 2: the plants continued to lose more water; the drooping of leaves was aggravated. Grade 3: some leaves were dry, hard and curly. Grade 4: all leaves were drooping and shrinking, and turned yellow. Grade 5: leaves were completely dry and hard, and the plants died. If the wilting degree was between two levels, it would be treated as a grade and half Calculation of comprehensive index: The relative drought tolerance of peanuts was determined by the method of average "membership function" [68].
Formula for "membership function" was: µ xj =(X j -X min )/(X max -X min ) For a certain variety, µ xj was the "membership function" for the "J" trait; x j is the value of the "J" trait; X max and X min were respectively the maximum and minimum values for the trait among all considered varieties. In order to avoid errors caused by variety-differences, X j , X max and X min were all calculated "relative values" instead of "measured values". Relative value = the measured value under stress/ the measured value under the control.
Drought-treatment time-course FH18 and NH5 seedlings were prepared and treated as described in "materials and growth". The droughttreatment time-course was composed of a series of treatment time points: 0 h (CK), 4 h (DT1), 8 h (DT2) and 24 h (DT3). The secondcompound leaves of seedlings were respectively collected at each time point which were frozen in liquid nitrogen then stored in a refrigerator at -80 °C for further analysis.

Physiological index measurements and stomatal observation
In order to compare the different effects by drought stress on FH18 and NH5, the second-compound leaf of seedlings were randomly selected from the treatment group and the control group, and then selected physiological indexes were measured. The conductivity was measured as described above. The reduced glutathione was measured by using a kit (Suzhou Keming Biotechnology Company) following manufacture's protocols. Observation of peanut Stomata after the simulated-drought treatments were carried out on a uorescence positive microscope by Zeiss [47]. All measurements were performed with three independent biological replicates.
RNA extraction and RNA-seq RNA samples were prepared from 24 harvests (4 treatments × 2 genotypes × 3 biological replicates) of peanut plants. Total RNA was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer instructions. The quality of RNA was assessed by Agilent 2100. The transcriptional group was sequenced using the method described by Wang et al [56]. Even if via magnetic beads with Oligo (dT), the mRNA of the samples were enriched. Brie y, a single stranded and double stranded cDNA was synthesized from the mRNA using random hexamers and AMPure XP beads (Beckman Coulter, Beverly, CA, USA), respectively, and nally, PCR enrichment was performed to obtain nal cDNA libraries. In order to separate the cDNA fragments with a length of 240 bp, the library was puri ed by AMPure XP and the library quality was evaluated in Agilent Bioanalyzer 2100 system. Finally, Illumina 2000 was used to PCR amplify the library.

Data analysis
The built-in software "perl scripts" was used to clear the inferior quality readings from the original data DEseq was used for differential expression analysis and genes with p < 0.01 were assigned as differentially expressed genes (DEGs). In order to analyze the functional relations of DEGs, we performed the GO and KEGG enrichments based on GOseq R language pack and pathways in the KEGG database [52]. Hypergeometric test was used to test the enrichment-signi cance of enriched pathways against the whole genomic background.

QRT-PCR veri cation
To verify the accuracy of RNA-seq sequencing, ten putative drought-tolerance-related differential genes were randomly selected for qRT-PCR veri cation. The Arah 1 gene was used a reference gene and the genes-speci c primers of the selected DEGs were designed using PRIMER5. QRT-PCRs were performed on an ABI Stepone plus platform with three reactions for each biological replicate and a total of three biological replicates for each gene. Availability of data and material The datasets generated and/or analysed during the current study are not publicly available due [These data are being used for the next part of the research] but are available from the corresponding author on reasonable reques. Figure 1 Comprehensive evaluation of drought resistance of peanut under drought stress   The schematic diagram of predicting the main process of peanut response to drought stress shows that peanut can resist drought stress by regulating the expression of stress genes by ABA and SA under drought stress. Thermography showed that FH18 and NH5 response genes were up-regulated (red) and

Figures
down-regulated (green) under drought stress.