Abstract Background Peanut is the world’s fourth largest oilseed crop that exhibits wide cultivar variations in cadmium (Cd) accumulation. To establish the mechanisms of Cd distribution and accumulation in peanut plants, eight cDNA libraries from the roots of two contrasting cultivars, Fenghua 1 (low-Cd cultivar) and Silihong (high-Cd cultivar), were constructed and sequenced by RNA-sequencing. The expression patterns of 16 candidate DEGs were validated by RT-qPCR analysis. Results A total of 75,634 genes including 71,349 known genes and 4484 novel genes were identified in eight cDNA libraries, among which 6798 genes were found to be Cd-responsive DEGs and/or DEGs between these two cultivars. Interestingly, 183 DEGs encoding ion transport related proteins and 260 DEGs encoding cell wall related proteins were identified. Among these DEGs, nine metal transporter genes (PDR1, ABCC4 and ABCC15, IRT1, ZIP1, ZIP11, YSL7, DTX43 and MTP4) and nine cell wall related genes (PEs, PGIPs, GTs, XYT12 CYP450s, LACs, 4CL2, C4H and CASP5) showed higher expression in Fenghua 1 than in Silihong. Conclusions Both the metal transporters and cell wall modification might be responsible for the difference in Cd accumulation and translocation between Fenghua 1 and Silihong. These findings would be useful for further functional analysis, and reveal the molecular mechanism responsible for genotype difference in Cd accumulation. Electronic supplementary material The online version of this article (10.1186/s12864-018-5304-7) contains supplementary material, which is available to authorized users. Keywords: Peanut, Root, Cadmium, Transcriptome, Gene expression Background Cadmium (Cd) is one of the most widespread and toxic heavy metal pollutants, whose contamination in arable soil from natural and anthropogenic sources has drawn wide public health concern [[37]1]. Although Cd is a non-essential element for plant growth, it is easily taken up by the root system and transferred to aboveground parts of plants [[38]2]. The excess amount of Cd in plants can trigger negative effects on many physiological processes such as inhibition of photosynthesis, interference of nutrient uptake and induction of oxidative stress [[39]3]. Moreover, Cd can accumulate in the human body via the food chain, posing various health problems including itai-itai, cancer, renal dysfunction, osteoporosis and cardiovascular diseases [[40]2]. It is very important to take effective measures to reduce Cd accumulation in edible parts of crops. For this purpose, screening and breeding low-Cd cultivars are expected to be one of the sustainable strategies [[41]4]. Peanut (Arachis hypogaea L.; AABB, 2n = 4x = 40) is an important oilseed crop that is cultivated extensively worldwide. China is one of the major exporters of peanut products, sharing around 40% of the world trade in peanut products [[42]5]. However, higher Cd enrichment capacity in the seeds of peanuts has become a serious problem for the peanut industry [[43]6–[44]9]. Seed Cd contents of peanuts in some regions of China such as Shandong and Liaoning have been shown to exceed the maximum level of the contaminant (0.5 mg/kg for Cd) following Chinese standard (GB 2762–2012) [[45]10]. Moreover, the higher Cd accumulation in peanut seeds has also been reported in other countries, such as Australia [[46]9, [47]11] and Argentina [[48]12]. Exposure of plants to 2 and 4 mg kg^− 1 Cd did not inhibit shoot biomass, seed yield, and oil content for most of the peanut cultivars [[49]13]. Thus, cultivation of peanuts in Cd contaminated soil may pose severe health risks for human beings. Peanuts exhibit a wide variation in Cd accumulation in vegetative organs and seeds among cultivars [[50]6–[51]8, [52]13, [53]14]. Seed Cd concentrations of peanuts are not associated with Cd concentrations in vegetative organs at the mature stage, but positively correlated with Cd concentrations in shoots of the seedlings [[54]6, [55]8]. The results suggest that shoot Cd concentrations at the vegetative growth stage may be important for determining cultivar differences in Cd accumulation in the seed [[56]6, [57]8]. Although peanut seeds develop in the soil, their Cd concentration predominantly comes from the uptake of Cd by the root system [[58]7, [59]9, [60]15]. Cd immobilization in the peanut root system could inhibit Cd translocation in peanut plants, and reduce Cd accumulation in peanut seed [[61]16, [62]17]. Root morphology has been shown to significantly correlate with Cd accumulation in peanuts [[63]14]. Cultivars with longer fine roots have a high capacity of Cd accumulation [[64]14]. It seems that the root plays an important role in determining variation in Cd accumulation among peanut cultivars, however, the underlying mechanism is still unclear. Understanding the molecular mechanism of cultivar difference in Cd accumulation is essential for breeding low Cd cultivars of peanuts. The uptake and translocation of Cd in plant is controlled by some metal transporter proteins such as natural resistance associated macrophage proteins (Nramp) [[65]18, [66]19], zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) [[67]20], ATP-binding cassette transporters (ABC) [[68]21], and P[1B]-ATPases [[69]22, [70]23]. Furthermore, the translocation of Cd from roots to shoots was also determined by the capacity for retaining Cd in the roots, in which Cd binding to the cell wall and vacuolar Cd sequestration are involved [[71]24, [72]25]. Therefore, we hypothesize that cultivar differences in Cd accumulation in peanut may be resulted from the differential expression of genes encoding metal transporters and cell wall modification in the roots. In this study, a comparative transcriptome analysis was carried out on the roots of two peanut cultivars differing in seed Cd accumulation, Silihong (high Cd-accumulating cultivar) and Fenghua 1 (low Cd-accumulating cultivar) [[73]8], under Cd-free and Cd-treated conditions. The main aims are: (i) to reveal the gene expression patterns in the roots of Fenghua 1 and Silihong in response to Cd exposure; (ii) to identify the key genes involved in Cd uptake, distribution and translocation in peanut roots; and (iii) to elucidate the gene regulatory network that is responsible for the cultivar differences of peanuts. The results presented here would be important for further illustrating the mechanism of Cd uptake and translocation as well as for breeding low Cd cultivars of peanuts. Results Cd accumulation and translocation in plants When exposed to 2 μM Cd for seven days, the concentration of Cd in roots and shoots was 224.7 and 18.3 mg kg^− 1 dry weight for Fenghua 1, and 255.8 and 29.9 mg kg^− 1 dry weight for Silihong, respectively (Fig. [74]1). Shoot Cd concentration was significantly higher in Silihong than in Fenghua 1, while the root Cd concentration was similar between the two cultivars. Similar to the shoot Cd concentration, the translocation factor of Cd from roots to shoots was significantly high in Silihong (0.12) than in Fenghua 1 (0.08) (Fig. [75]1). The current results, in agreement with previous findings [[76]8], indicate that Fenghua 1 differed in the properties of Cd translocation and accumulation from Silihong. Fig. 1. [77]Fig. 1 [78]Open in a new tab Cd accumulation and translocation in plants of Fenghua 1 and Silihong exposed to 2 μM Cd for seven days. Asterisk (*) above error bars indicate values (mean ± SE, n = 4) are significantly different between the two peanut cultivars according to independent samples t-test at 0.05 the level RNA sequencing analysis of eight cDNA libraries Eight cDNA libraries, namely, F[CK]_1, F[CK]_2, F[Cd]_1, F[Cd]_2, S[CK]_1, S[CK]_2, S[Cd]_1 and S[Cd]_2, were constructed from Cd-free (CK) and Cd-treated (Cd) peanut roots of both cultivars and sequenced using a BGISEQ-500 platform. A total of 164.87, 167.18, 164.59 and 167.03 Mb raw reads were produced from two biological replicate libraries for F[CK], F[Cd], S[CK] and S[Cd], respectively. After removing the reads with adapters, low-quality and high content of unknown base (N), more than 63 million 100 bp paired-end clean reads with Q20 percentage over 97% remained in each cDNA library (Additional file [79]1: Table S1). The clean reads from each cDNA library were aligned to reference genome sequences of the two progenitors of the cultivated peanut (A. duranensis-AA and A. ipaensis-BB). The average genome mapping rate is 90.16%, and the average gene mapping rate is 86.05% (Additional file [80]2: Table S2). Analysis of gene expression levels A total of 75,634 genes including 71,349 known genes and 4484 novel genes were identified from A and B genome (Additional file [81]3: Table S3 and Additional file [82]4: Table S4). Furthermore, 24,024 novel isoforms and 5264 noncoding transcripts were identified in eight cDNA libraries (Additional file [83]3: Table S3). The expression levels of these genes from the mapped libraries were normalized as FPKM (Additional file [84]5: Figure S1). The gene expression distribution (FPKM) was similar among eight libraries. For instance, the low expression level genes with FPKM interval 0~10 account for 63.50, 64.62, 64.66, 63.41, 64.04, 63.36, 59.24 and 63.56% in F[Cd]_1, F[Cd]_2, F[CK]_1, F[CK]_2, S[Cd]_1, S[Cd]_2, S[CK]_1 and S[CK]_2 respectively. Identification of differentially expressed genes (DEGs) Pairwise comparison analysis for each gene were performed between Fenghua 1 and Silihong (F[CK]/S[CK] and F[Cd]/S[Cd]) or between Cd-free and Cd-treated samples in each cultivar (F[Cd]/F[CK] and S[Cd]/S[CK]). DEGs were identified by the threshold of |log[2] fold-change| ≥ 1 and P[adj]-value ≤ 0.05. A total of 6798 genes were differentially regulated in the four comparisons (Additional file [85]6: Table S5). Under control condition, 5297 DEGs were identified between two cultivars, while this value reached 2793 DEGs under Cd-treated condition (Fig. [86]2a). Of which, 1764 genes were common ones (Fig. [87]2c). Compared to Cd-free library, 331 genes were differentially regulated in Fenghua 1 in Cd exposure, including 278 up-regulated and 53 down-regulated genes. Whereas, 1302 Cd-treated DEGs were identified in Silihong, including 132 up-regulated and 1170 down-regulated genes (Fig. [88]2b). Among these genes, only 50 DEGs were common Cd-responsive genes in two cultivars (Fig. [89]2d), including 49 genes showed similar expression patterns and one gene showed opposing expression pattern (Additional file [90]6: Table S5). Fig. 2. [91]Fig. 2 [92]Open in a new tab Analysis of DEGs following Cd exposure in two peanut cultivars. (a) Number of DEGs between Fenghua 1 (F) and Silihong (S). (b) Number of DEGs between the control (CK) and Cd exposure (Cd). (c) Venn diagrams of DEGs between Fenghua 1 (F) and Silihong (S). (d) Venn diagrams of DEGs between the control (CK) and Cd exposure (Cd) Gene ontology (GO) analysis of DEGs GO assignments were used to classify the functions of DEGs, a total of 2860 genes including 157 (F[Cd]/F[CK]), 610 (S[Cd]/S[CK]), 2214 (F[CK]/S[CK]) and 941 (F[Cd]/S[Cd]) genes, were assigned into 46 GO terms consisting of 22 biological process, 13 cellular component and 11 molecular function subcategories (Additional file [93]7: Figure S2a-b). Among these GO terms, the top two abundant categories of biological process (‘metabolic process’ and ‘cellular process’), molecular function (‘catalytic activity’ and ‘binding’), and cellular component (‘membrane’ and ‘membrane part’) were similar in the four comparisons. Furthermore, the results of significantly enriched GO terms (corrected p-value < 0.01) are listed in Additional file [94]8: Table S6. For Cd-responsive DEGs, four GO terms such as response to inorganic substance, response to symbiont and response to metal ion in the biological process category and 16 GO terms such as zinc ion transmembrane transporter activity, divalent inorganic cation transmembrane transporter activity and transporter activity in the molecular function category were uniquely enriched in Fenghua 1. In Silihong, 11 GO terms such as transcription, DNA-templated, RNA biosynthetic process and nucleic acid-templated transcription in the biological process category and ten GO terms such as DNA binding transcription factor activity, transcription regulator activity and calcium ion binding in the molecular function category were uniquely enriched (Additional file [95]8: Table S6). For DEGs between Fenghua 1 and Silihong, three GO terms including lignin metabolic process, phenylpropanoid metabolic process and lignin catabolic process in the biological process category and the two GO terms including hydroquinone: oxygen oxidoreductase activity and oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor (Additional file [96]8: Table S6) were significantly enriched under Cd-treated condition. KEEG metabolic pathway analysis of DEGs Here, DEGs were further annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG) database ([97]http://www.genome.ad.jp/kegg/) to elucidate the molecular interactions among the genes. A total of 266 (F[Cd]/F[CK]), 1073 (S[Cd]/S[CK]), 4164 (F[CK]/S[CK]) and 2115 (F[Cd]/S[Cd]) DEGs were respectively assigned to 66, 116, 133, and 132 pathways (Additional file [98]9: Table S7), which were grouped into five groups including metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems (Fig. [99]3). Among these pathways, the top two categories of them including metabolic pathway (59, 22.18%; 254, 23.67%; 960, 23.05%; 535, 25.30%) and biosynthesis of secondary metabolites (46, 17.29%; 193, 17.99%; 617, 14.82%; 337, 15.93%) were similar in the four comparisons (Additional file [100]9: Table S7). Followed by MAPK signaling pathway-plant, phenylpropanoid biosynthesis and ABC transporters in F[Cd]/F[CK]; plant-pathogen interaction, MAPK signaling pathway-plant and flavonoid biosynthesis in S[Cd]/S[CK]; Plant-pathogen interaction, MAPK signaling pathway-plant and plant hormone signal transduction in F[CK]/S[CK]; plant-pathogen interaction, RNA transport, protein processing in endoplasmic reticulum and phenylpropanoid biosynthesis in F[Cd]/S[Cd] (Additional file [101]9: Table S7). Fig. 3. [102]Fig. 3 [103]Open in a new tab Functional classification and pathway assignment of identified DEGs by KEEG in four comparisons. The DEGs were assigned pathways belonging to five main categories: Metabolism (a), Organismal systems (b), Cellular processes (c), Environmental information processing (d), and Genetic information processing (e) In addition, one (F[Cd]/F[CK]), six (S[Cd]/S[CK]), four (F[CK]/S[CK]) and two (F[Cd]/S[Cd]) pathways were identified as significantly enriched (Q ≤ 0.01) (Additional file [104]10: Table S8). For DEGs between Fenghua 1 and Silihong, circadian rhythm-plant pathway was shared enriched under both control and Cd conditions (Additional file [105]10: Table S8). Besides, the pathway of cyanoamino acid metabolism was specifically significantly enriched for DEGs between two cultivars under Cd-treated. Further, Cd-responsive DEGs in Fenghua 1 were specifically significantly enriched in ABC transporters. The pathways of alpha-Linolenic acid metabolism, plant-pathogen interaction, flavonoid biosynthesis, circadian rhythm-plant, zeatin biosynthesis and MAPK signaling pathway-plant were significantly enriched for Cd-responsive DEGs in Silihong. High expression level of DEGs between Fenghua 1 and Silihong under Cd exposure To obtain a general statistical overview of transcriptional gene regulation the difference of shoot Cd accumulation in two peanut cultivars, the high expression level of DEGs (an FPKM value > 10 in at least one of four cDNA libraries and the threshold of |log[2] (F[Cd]/S[Cd])| ≥ 3) including 109 up- and 96 down-regulated were identified in F[Cd]/S[Cd] (Additional file [106]11: Table S9). Based on functional analysis, some of them were involved in cell wall metabolism (i.e., polygalacturonase inhibitory PGI, chitinase-like protein and GDSL esterase/lipase), ion transport (i.e., ABC transporter D family member 1 ABCD1, V-type proton ATPase), transcription regualtion (i.e., transcription factor bHLH83, bZIP transcription factor 11 and auxin-induced protein AUX28) and other metabolic processes (i.e., serine/threonine-protein kinase, metallothionein-like protein). The results suggested that these DEGs might play crucial regulatory roles in the the difference of shoot Cd accumulation in two peanut cultivars. Here, the DEGs were involved in the cell wall metabolism and ion transport as our main further investigation. DEGs involved in heavy metal transport According to GO functional annotation, 183 DEGs were identified to have high similarity with diverse transporters such as ATP-binding cassette (ABC) transporters (e.g. ABCAs, ABCBs, ABCCs and ABCGs), natural resistance-associated macrophage proteins (e.g. Nramp5 and Nramp6), ZIP transporters (e.g. IRT1, IRT3, ZIP1, ZIP4, ZIP5 and ZIP11), yellow stripe-like transporters (e.g. YSL3 and YSL7), MATE efflux family proteins (e.g. protein DETOXIFICATIONs DTXs) and metal tolerance proteins (e.g. MTP4 and MTP9) (Additional file [107]12: Table S10). These transporters were classified into seven categories based on their expression patterns (Additional file [108]13: Table S11). The classes from first to fourth were Cd-responsive DEGs. Of these, two genes in the first class were up-regulated, and one gene in the second class was down-regulated in both Fenghua 1 and Silihong after Cd exposure; the 22 genes in the third class and five genes in the fourth class were up-regulated in Fenghua 1 and Silihong roots under Cd exposure, respectively. Furthermore, the fifth and sixth classes were DEGs between two cultivars. The fifth class contained 58 genes whose expressions were higher in Fenghua 1 than those in Silihong, whereas, the sixth class had 61 genes expressed lower in Fenghua 1 than those in Silihong. The 34 genes in seventh class were Cd-responsive DEGs that is also differentially expressed between Fenghua 1 and Silihong. We also found that the DEGs encoding heavy metal-mediated transporters, such as ABCs, IRTs, ZIPs, YSL3, Nramp5, MTP9, pleiotropic drug resistance proteins (PDRs) and copper-transporting ATPase HMA5 (HMA5), were all up-regulated by Cd in Fenghua 1 (Additional file [109]12: Table S10). In S[Cd]/S[CK], besides the DEGs encoding transporters including ABCB15, ABCG20, HMA5, Nramp6, DTX40 and two pore calcium channel protein 1 (TPCN1), the remaining 22 DEGs encoding transporters such as heavy metal-associated isoprenylated plant proteins (HIPPs), calcium-transporting ATPase 2 (Ca^2+-ATPase2), MATE efflux family protein (MATE), and ABCG11, were down-regulated after Cd exposure (Additional file [110]12: Table S10). For the F[Cd]/S[Cd], genes encoding ABC transporters (e.g. ABCB28, ABCC4/8/15, ABCD1 and ABCG15), oligopeptide transporter 7 (OPT7), DTX43/49, IRT1, PDR1, YSL7 and MTP4, were higher in Fenghua 1 than in Silihong. However, ABCA1, ABCB1/11, ABCC10, ABCG7, HIPP35, ZIP1, Nramp2, Ca^2+-ATPase12, cation/H(+) antiporter (CHX18/20), DTX16/43/48/51 and MATE expressed higher in Silihong than in Fenghua 1 (Additional file [111]12: Table S10). DEGs related to cell wall modification and vacuolar sequestration Plant cell wall has been considered as the critical site for retention of Cd, especially in roots. Our study suggested that a total of 260 DEGs were homologous with cell wall metabolism-related genes, including 108 involved in cell wall degradation (75, 23 and five were involved in pectin-, hemicellulose- and cellulose-degrading process, respectively; five were other cell wall degrading-related enzymes), 143 involved in cell wall synthesis (53 in cellulose biosynthesis, 49 in suberin biosynthesis, 24 in lignin biosynthesis, 11 in hemicellulose, four in pectin, and two in Casparian strip formation). Furthermore, three encoding expansin proteins, five encoding COBRA proteins and one encoding extension 2 like protein were involved in cell wall organization (Additional file [112]14: Table S12). For the 108 cell wall degradation DEGs, six were Cd-responsive DEGs including one upregulated in Fenghua 1 and five downregulated in Silihong, and 87 were DEGs between Fenghua 1 and Silihong (Additional file [113]14: Table S12). The remaining 15 genes were Cd responsive DEGs, which also expressed differentially between two cultivars, and 13 of them were down-regulated by Cd in Silihong (Additional file [114]14: Table S12). Furthermore, among 108 DEGs, only three upregulated were found in F[Cd]/F[CK]. Remarkably, 32 of 108 DEGs were expressed higher in Fenghua 1 than in Silihong. Regarding the 143 cell wall synthesis DEGs, 19 were Cd-responsive DEGs and 97 were DEGs between Fenghua 1 and Silihong. For the 19 Cd responsive DEGs, 15 were up-regulated by Cd in Fenghua 1, and four were down-regulated by Cd in Silihong. The remaining 27 genes were Cd responsive DEGs, which also expressed differentially between two cultivars. 46 of 143 DEGs were expressed higher in Fenghua 1. In addition, the formation of Cd complexes with glutathione (GSH), metallothionein (MT), phytochelatin (PC) and nicotianamine (NA) is another way used by plants to sequestrate Cd in vacuolar. In this study, two (107459596, 107612042) and one (107465178) nicotianamine synthase (NAS) genes were up-regulated by Cd in Fenghua 1 and Silihong, respectively. Moreover, NAS (107612042) and MT2 (107639467) were expressed higher in Fenghua 1 than in Silihong (Additional file [115]14: Table S12). However, no significant difference in GSH and PC biosynthesis-related genes expression were observed in each comparison. RT-qPCR validation To verify the differential expression patterns of DEGs identified in our transcriptome data, a total of ten candidate transport- and six cell wall-related genes were selected for RT-qPCR analysis. The results indicated that the expression patterns of 15 out of 16 DEGs showed good agreement with the RNA-Seq-based gene expression patterns, and only one gene (ABCC36) was not well consistent with the results of sequencing (Fig. [116]4; Additional file [117]6: Table S5). For example, several Cd-responsive up-regulated DEGs also indicated relatively high expression level in Fenghua 1 (ZIP4, Nramp5/6, and ABCC3) and Silihong (Nramp6) under Cd exposure in RT-qPCR analysis (Fig. [118]4). For DEGs between Fenghua 1 and Silihong, the expression level of ABCC4, PDR1, DTX43, β-Gal8, XYL1, EG10 genes were higher in Fenghua 1, whereas LAC11 was highly expressed in Silihong under Cd exposure (Fig. [119]4). These results suggested that the data detected by RNA-Seq analysis of this study provide reliable inference. Fig. 4. [120]Fig. 4 [121]Open in a new tab RT-qPCR analyses of 16 candidate DEGs under the control and Cd treatment in roots of two peanut cultivars. Each bar represents the mean ± SD of triplicate assays. The values with different letters indicate significant differences at P < 0.05 according to Duncan’s multiple range tests Discussion Comparison of gene expression in roots between Fenghua 1 and Silihong There are clear physiological differences in shoot Cd concentrations between Fenghua 1 and Silihong following Cd exposure [[122]8]. However, the molecular mechanism underlying the difference in Cd accumulation among peanut cultivars has not been fully elucidated. RNA sequencing (RNA-seq) analysis has been used for revealing the molecular mechanisms of differential Cd uptake, accumulation and translocation in many plants [[123]26–[124]28]. However, the comprehensive identification of the DEGs involved in root uptake, accumulation and transport of Cd in peanut remained unexplored. Based on peanut genome [[125]29], a total of 6798 genes including 331 (F[CK]/F[Cd]), 1302 (S[CK]/S[Cd]), 5297 (F[CK]/S[CK]) and 2793 (F[Cd]/S[Cd]) genes, were identified significant differentially expression in pairwise comparisons (Additional file [126]6: Table S5). Of them, 183 DEGs encoding transporter proteins and 260 DEGs encoding cell wall metabolism-related proteins were identified. Most Cd-responsive DEGs (278/331, 83.99%) in Fenghua 1 were up-regulated by Cd, while the majority of Cd-responsive DEGs (1170/1302, 89.86%) in Silihong were down-regulated by Cd (Additional file [127]6: Table S5). The results suggested that the two cultivars differed in the molecular mechanisms in response to Cd exposure. To our knowledge, this is the first research to identify and dissect extensive Cd-responsive genes in peanuts, and insight into molecular mechanisms responsible for the differential shoot Cd accumulation in two cultivars. Heavy metal transporters involved in differential root uptake and translocation of Cd between Fenghua 1 and Silihong It has been widely proven that Zn^2+, Ca^2+, Fe^2+, Mn^2+, Cu^2+ and Mg^2+ transporters could transport Cd into plant root cells, and affect root-to-shoot Cd translocation [[128]30]. The physiological data indicated that Fenghua 1 showed lower shoot Cd concentrations than Silihong, for which the root-to-shoot translocation of Cd might be responsible [[129]8]. In this study, we identified 183 DEGs encoding ion transporter proteins, among which 30 were Cd-responsive DEGs. We also found that 34 Cd-responsive DEGs encoding ion transporter proteins were expressed differentially between the two cultivars (Additional file [130]12: Table S10). Compared with the control, Cd up-regulated the Cd-responsive DEGs in Fenghua 1, whereas most of DEGs in Silihong were down-regulated by Cd. Only four DEGs, 107471962 and 107623484 (HMA5), 107477738 (DTX40) and 107491040 (HIPP2), were induced by Cd treatment in both cultivars. These results indicate that the two peanut cultivars differed in the expression of ion transporter that may contribute to the difference in Cd uptake and translocation. We found that six DEGs encoding metal transporters related to Cd influx, including IRT1, ZIP4 and Nramp5/6 (Table [131]1). AtIRT1 [[132]31], NcZNT1/AtZIP4 [[133]20], SaNramp6 [[134]32] and OsNramp5 [[135]33] have been shown to mediate cellular metals (Zn, Fe, Mn and Cd) uptake across the plasma membrane. IRT1, a Fe-regulated transporter, is expressed in the root epidermis [[136]34]. Overexpressing AtIRT1 in Arabidopsis leds to increased accumulation of Cd and Zn in the transgenic lines under Fe starvation [[137]31], which indicates that AtIRT1 can affect the Cd uptake capacity. Therefore, under Cd exposure, the higher expression of IRT1 in the roots of Fenghua 1 compared with Silihong may be involved in the difference of root Cd absorption between Fenghua 1 and Silihong (Figs. [138]5 and [139]6). Overexpression of AtZIP4 and SaNramp6 in Arabidopsis increased accumulation of Cd in transgenic plants [[140]20, [141]32]. AtNramp6 is also thought to be a major role in the intracellular distribution of Cd [[142]35]. Our results showed that ZIP4, Nramp5 and IRT1 were up-regulated by Cd in Fenghua 1, while in Silihong, they were unchanged (Fig. [143]5; Table [144]1). Nramp5 (107606033, 107470537 and 107460699) shows a high degree of homology to the AtNramp6 from Arabidopsis. However, Nramp6 (107618045) was upregualted in Silihong, while in Fenghua 1, it was unchanged (Fig. [145]5; Table [146]1). ZIP4 and Nramp5/6 were not DEGs as judged by our criteria between the two cultivars. These results suggested that ZIP4 and Nramp5/6 may be related to Cd-response in roots of two peanut cultivars. However, the functions of these genes in peanuts need further investigated. Table 1. Critical DEGs involved in Cd transport in roots of two peanut cultivars Gene ID Log[2] fold-change Description Gene name Functional analysis^b Reference F[Cd]/F[CK] S[Cd]/S[CK] F[Cd]/S[Cd] F[CK]/S[CK] Plasma membrane-localized transporters  107606033 1.80^a 1.13 −0.03 − 0.78 metal transporter Nramp5 Nramp5 Transport Cd into cytoplasm [[147]19, [148]33]  107470537 2.01^a 1.15 0.20 − 1.11 metal transporter Nramp5-like Nramp5 Transport Cd into cytoplasm [[149]19, [150]33]  107460699 1.63^a 0.91 − 0.02 − 0.80 metal transporter Nramp5-like Nramp5 Transport Cd into cytoplasm [[151]19, [152]33]  107618045 0.79 1.25^a − 0.65 − 0.25 metal transporter Nramp6 Nramp6 Transport Cd into cytoplasm [[153]32]  107461206 1.17^a 0.57 0.03 − 0.61 zinc transporter 4, chloroplastic ZIP4 Transport Cd into cytoplasm [[154]20]  G003600 1.05^a 0.60 − 0.13 −0.62 zinc transporter 4, chloroplastic ZIP4 Transport Cd into cytoplasm [[155]20]  107637744 3.10^a 0.12 4.03^a 0.20 Fe (2+) transport protein 1 IRT1 Transport Cd into cytoplasm [[156]31, [157]34]  107614121 1.09^a 1.13 −0.01 0.04 zinc transporter 1 ZIP1 Export Cd out of cell [[158]46]  107458727 0.34 0.61 − 1.28^a −1.05 zinc transporter 1 ZIP1 Export Cd out of cell [[159]46]  107482454 1.42^a 0.89 −0.42 −0.97 zinc transporter 1; zinc transporter 5 ZIP1/ZIP5 Export Cd out of cell [[160]46]  107638316 1.37^a 0.64 −0.15 − 0.96 zinc transporter 5 ZIP5 Export Cd out of cell [[161]46]  107460374 1.33^a 0.70 0.76 0.08 metal-nicotianamine transporter YSL3 YSL3 Export Cd out of cell [[162]43]  107605860 1.51^a 0.81 0.93 0.19 metal-nicotianamine transporter YSL3 YSL3 Export Cd out of cell [[163]43]  107460375 1.01^a 0.68 0.33 −0.04 metal-nicotianamine transporter YSL3 YSL3 Export Cd out of cell cytoplasm [[164]43]  107629798 0.44 N/A 6.18^a 6.36^a metal-nicotianamine transporter YSL7 isoform X1 YSL7 Export Cd out of cell [[165]40]  G001484 0.36 − 1.57 −0.18 − 2.31^a ABC transporter G family member 36 ABCG36 Export Cd out of cell [[166]41]  107617325 0.03 −1.75 0.00 −1.93^a ABC transporter G family member 36-like ABCG36 Export Cd out of cell [[167]41]  G001485 0.04 −1.61 −0.44 − 2.28^a ABC transporter G family member 36-like ABCG36 Export Cd out of cell [[168]41]  107459920 −0.15 − 0.38 2.20^a 1.94^a pleiotropic drug resistance protein 1 PDR1 Export Cd out of cell [[169]41, [170]42]  107647851 2.76^a −0.20 0.11 − 3.44^a pleiotropic drug resistance protein 1 PDR1 Export Cd out of cell [[171]41, [172]42]  107491711 3.49^a −0.04 −0.52 −4.52^a pleiotropic drug resistance protein 1-like PDR1 Export Cd out of cell [[173]41, [174]42]  107614075 −0.43 0.06 1.90^a 2.45^a pleiotropic drug resistance protein 1-like PDR1 Export Cd out of cell [[175]41, [176]42]  107472005 1.18^a 0.68 −0.24 −0.77 metal tolerance protein 9 MTP9 Export Cd out of cell [[177]38]  107622321 1.24^a 0.86 −0.38 − 0.81 metal tolerance protein 9 MTP9 Export Cd out of cell [[178]38]  107614236 0.06 0.11 −1.00^a − 0.97 protein DETOXIFICATION 43 DTX43 Export Cd out of cell [[179]44, [180]45]  107462988 0.91 − 0.55 1.49^a − 0.38 protein DETOXIFICATION 43-like X1 DTX43 Export Cd out of cell [[181]44, [182]45] Tonoplast-localized transporters  107481980 −0.06 −1.12 1.50^a 0.36 ABC transporter C family member 15 ABCC15 Sequestrate Cd into vacuole [[183]21]  107632015 −0.27 −0.61 1.38^a 1.03 ABC transporter C family member 15 ABCC15 Sequestrate Cd into vacuole [[184]21]  107496267 −0.18 −0.34 −2.11 − 2.83^a ABC transporter C family member 3 ABCC3 Sequestrate Cd into vacuole [[185]21]  107496250 1.63 −1.25 − 0.86 −4.15^a ABC transporter C family member 3-like ABCC3 Sequestrate Cd into vacuole [[186]21]  107496160 1.65^a − 0.62 − 0.15 −2.52^a ABC transporter C family member 3-like ABCC3 Sequestrate Cd into vacuole [[187]21]  110262356 2.02^a − 0.29 −0.01 − 2.56^a ABC transporter C family member 3-like ABCC3 Sequestrate Cd into vacuole [[188]21]  107606454 1.58^a − 1.08 − 0.87 −3.66^a ABC transporter C family member 3-like ABCC3 Sequestrate Cd into vacuole [[189]21]  107496270 0.95 − 0.52 − 0.29 − 1.92^a ABC transporter C family member 3-like ABCC3 Sequestrate Cd into vacuole [[190]21]  107606407 0.34 − 1.08 −0.99 −3.49^a ABC transporter C family member 3-like ABCC3 Sequestrate Cd into vacuole [[191]21]  107630594 −0.05 0.74 4.48^a 5.84^a ABC transporter C family member 4 X3 ABCC4 Sequestrate Cd into vacuole [[192]36]  107466097 −0.26 −0.10 3.46^a 3.94^a ABC transporter C family member 4 X3 ABCC4 Sequestrate Cd into vacuole [[193]36]  107475935 0.83 −0.28 −0.37 −1.55^a ABC transporter C family member 4-like ABCC4 Sequestrate Cd into vacuole [[194]36]  107626139 0.88 −0.33^a − 0.26 − 1.55^a ABC transporter C family member 4-like ABCC4 Sequestrate Cd into vacuole [[195]36]  107636396 0.78 0.54 2.70^a 2.42^a metal tolerance protein 4 MTP4 Sequestrate Cd into vacuole [[196]37] Endomembrane-localized transporters  107461527 1.33^a 1.14 −0.03 − 0.25 zinc transporter 1 ZIP1 Sequestrate Cd into vesicles [[197]28]  107462023 1.37^a 0.33 0.89 − 0.21 zinc transporter 11 ZIP11 Sequestrate Cd into vesicles [[198]28]  107617536 1.55^a 0.93 0.97 0.33 zinc transporter 11 ZIP11 Sequestrate Cd into vesicles [[199]28]  107618045 0.79 1.25^a −0.65 − 0.25 metal transporter Nramp6 Nramp6 Sequestrate Cd into vesicles [[200]35] [201]Open in a new tab ^aThe gene differentially expressed between the two groups. ^bFunction analysis of DEGs was based on references listed in the same row