Transcriptome of the inflorescence meristems of the biofuel plant Jatropha curcas treated with cytokinin
© Pan et al.; licensee BioMed Central Ltd. 2014
Received: 21 June 2014
Accepted: 29 October 2014
Published: 17 November 2014
Jatropha curcas, whose seed content is approximately 30–40% oil, is an ideal feedstock for producing biodiesel and bio-jet fuels. However, Jatropha plants have a low number of female flowers, which results in low seed yield that cannot meet the needs of the biofuel industry. Thus, increasing the number of female flowers is critical for the improvement of Jatropha seed yield. Our previous findings showed that cytokinin treatment can increase the flower number and female to male ratio and also induce bisexual flowers in Jatropha. The mechanisms underlying the influence of cytokinin on Jatropha flower development and sex determination, however, have not been clarified.
This study examined the transcriptional levels of genes involved in the response to cytokinin in Jatropha inflorescence meristems at different time points after cytokinin treatment by 454 sequencing, which gave rise to a total of 294.6 Mb of transcript sequences. Up-regulated and down-regulated annotated and novel genes were identified, and the expression levels of the genes of interest were confirmed by qRT-PCR. The identified transcripts include those encoding genes involved in the biosynthesis, metabolism, and signaling of cytokinin and other plant hormones, flower development and cell division, which may be related to phenotypic changes of Jatropha in response to cytokinin treatment. Our analysis indicated that Jatropha orthologs of the floral organ identity genes known as ABCE model genes, JcAP1,2, JcPI, JcAG, and JcSEP1,2,3, were all significantly repressed, with an exception of one B-function gene JcAP3 that was shown to be up-regulated by BA treatment, indicating different mechanisms to be involved in the floral organ development of unisexual flowers of Jatropha and bisexual flowers of Arabidopsis. Several cell division-related genes, including JcCycA3;2, JcCycD3;1, JcCycD3;2 and JcTSO1, were up-regulated, which may contribute to the increased flower number after cytokinin treatment.
This study presents the first report of global expression patterns of cytokinin-regulated transcripts in Jatropha inflorescence meristems. This report laid the foundation for further mechanistic studies on Jatropha and other non-model plants responding to cytokinin. Moreover, the identification of functional candidate genes will be useful for generating superior varieties of high-yielding transgenic Jatropha.
The ever-decreasing crude oil reserves are insufficient to satisfy the increasing demand for petroleum as a transportation and heating fuel, and petroleum consumption also pollutes the environment. Liquid biofuels from plants and microalgae may help solve these problems. Jatropha curcas (hereafter referred to as Jatropha), a perennial deciduous shrub belonging to the family Euphorbiaceae whose seed content is approximately 30–40% oil, is an ideal feedstock for producing biodiesel and bio-jet fuels [1–3]. Because the quality parameters of Jatropha biodiesel are within the European EN 14214 specification and the emission parameters of sulfur and particulate matter are 80% lower than those of mineral diesel , Jatropha is emerging as a potential biofuel plant.
However, currently the seed yield of Jatropha is poor and insufficient for the biodiesel industry [5, 6]. Recently, researchers reported that applying plant growth regulators to Jatropha can improve seed yield [7–9]. Our previous study found that applying benzyladenine (BA, a synthetic cytokinin) to the inflorescence meristems of Jatropha significantly increased the flower number and the female to male flower ratio, which contributed to an increase in seed yield . Cytokinin is involved in plant development and growth and can be used to regulate many aspects of plant development in both practical and theoretical studies [8–16]. Endogenous levels of cytokinin content increased in Polianthes tuberosa and Litchi chinensis when they began to flower [17, 18]. In Arabidopsis, cytokinin treatment correlated with early flowering [19, 20]. Transgenic Arabidopsis plants overexpressing CYTOKININ OXIDASE (CKX), which degrades cytokinins, flowered late . These studies indicated that cytokinin stimulates flowering in these plants. Additionally, cytokinin increased the flower number in Arabidopsis and induced an aberrant floral phenotype including an increased number of flower organs [12, 22]. Exogenous cytokinin application and accumulation of endogenous cytokinin increased the flower number in several species [10, 13, 23–25]. In addition, the flower sex of Vitis vinifera[26, 27], Luffa cylindrical, Momordica charantia and Pinus densiflora was also affected by exogenous cytokinins.
It is not economically viable to improve Jatropha seed yield by exogenous application of BA in large scale plantation. Generating transgenic Jatropha plants with increased female and/or bisexual flower number is critical for improving seed yield. The initial step of transgenic Jatropha study is to identify functional genes. Therefore, the identification of genes involved in flower development following BA treatment and the characterization of their expression profiles are two important prerequisites. Currently, next-generation sequencing technologies make it relatively inexpensive to study the transcriptome of a particular organism or tissue to gain insight into biological processes. In Cucumis stativus, the transcriptomes of flowers of different sexes were sequenced to determine the molecular mechanisms of plant sex determination . To study the genetic control of Fagopyrum floral development, the floral transcriptomes of two species that have the ability to self-pollinate, in contrast to the common Fagopyrum, were characterized . The floral transcriptome was also sequenced to evaluate self-incompatibility in Ziziphus celata, a highly endangered plant . Recently, transcriptome analysis was used to investigate global expression patterns of phytohormone-regulated transcripts in tomato leaves and roots [34, 35]. Therefore, transcriptome sequencing is a proven strategy for expression profiling of genes involved in various processes in plants.
Although the Jatropha genome has been sequenced by a combination of the conventional Sanger method and next-generation multiplex sequencing methods [36, 37], most of the molecular studies on Jatropha have focused on lipid metabolism in seeds. Global analysis of gene expression profiles in developing and germinating seeds were performed to assess differential gene expression and to discover genes involved in lipid metabolism [38–45]. For high-throughput discovery of novel Jatropha genes, de novo assembly and transcriptome analysis of different tissues of Jatropha were performed . However, molecular studies on flower development and/or flower developmental responses to phytohormone treatment in Jatropha are scarce.
Given that BA increased flower number and the female to male ratio and induced bisexual flowers in Jatropha, we conducted a time course study of gene expression profiles in inflorescence meristems of Jatropha exposed to BA. One 454 sequencing run was performed that generated a total of 294.6 Mb of transcript sequences. Differentially expressed genes involved in the biosynthesis, metabolism, and signaling of cytokinin and other plant hormones, flower development and cell division, which may be related to the phenotypic changes of Jatropha in response to cytokinin treatment, were further analyzed.
We expect that characterization of the transcriptome of Jatropha inflorescence meristems treated with BA will contribute not only to genetic engineering and breeding of Jatropha but will also provide insight into the mechanism of how cytokinin affects the flower development of Jatropha and other non-model plants.
Results and discussion
Effects of BA on Jatrophaflower development
454 sequencing and transcriptome assembly
Characteristics of raw data and assembly summary
Raw sequencing reads
Total number of raw read
Average length of raw read
High quality reads
Total number of high quality read
Average length of high quality read
Length range of high quality read
100 bp - 790 bp
Total number of singleton
Average length of singleton
Total number of contig
Assembled reads (of total number of high quality read)
Average length of contig
Transcriptome annotation summary
Average CDS length
Maximum CDS length
CDS with known function
CDS with KOG assignment
CDS with GO classification
Unigene sequences mapped to Jatropha genome
Functional annotation of CDSs was performed by searching against NCBI non-redundant protein database and KEGG protein database using BLASTP with E value 1e-3. Then the top hit protein (with the highest bit score) was chosen to count its origin (encoded by what organism). The distribution of sequences from various species closely related to those found in the Jatropha inflorescence meristem transcriptome is shown in Additional file 2: Table S1. More than half (58.4%) of Jatropha sequences are similar to sequences found in the close related castor bean (Ricinus communis) genome, which is consistent with the analysis of whole genome sequences of Jatropha[36, 37]. In contrast, the percentage of Jatropha sequences with top hit to sequences of the model plant Arabidopsis is as low as 0.81% (Additional file 2: Table S1). Given these results, studying the transcriptomes of Jatropha inflorescence meristems with and without BA treatment may shed light on the molecular mechanisms of cytokinin effects on the flower development of castor bean and other non-model plants.
Transcript clustering by expression signatures
Compared to Arabidopsis orthologs, a number of Jatropha genes showed different expression patterns in inflorescence meristems exposed to cytokinin. The difference may result from different treatment methods and/or different concentrations of BA used for treatment. Most treatments of Arabidopsis are on seedlings rather than on inflorescence meristems employed in this study for Jatropha. And the concentration of BA usually was less than 20 μM in treatments of Arabidopsis, whereas the concentration of BA used in this study was 1 mM.
Gene ontology enrichment analysis of eight clusters
GO enrichment analysis of differential transcripts in eight clusters (p <0.05)
axon extension involved in development
developmental cell growth
ubiquitin-dependent protein catabolic process
carbohydrate metabolic process
cellular aromatic compound metabolic process
glutamine family amino acid metabolic process
molecular transducer activity
nitrogen compound biosynthetic process
signal transducer activity
positive regulation of signal transduction
sensory perception of light stimulus
base pairing with mRNA
carboxylic acid metabolic process
sodium ion binding
cellular ketone metabolic process
triplet codon-amino acid adaptor activity
long-chain fatty acid metabolic process
cellular response to hormone stimulus
signal transducer activity
negative regulation of cell death
transmembrane receptor activity
regulation of cell activation
ferric iron binding
fatty acid binding
threonine-type endopeptidase activity
sodium:hydrogen antiporter activity
regulation of cell size
histone acetyltransferase activity
regulation of hormone secretion
lysine N-acetyltransferase activity
calcium ion homeostasis
carboxylic acid catabolic process
transmembrane receptor activity
gene silencing by RNA
fatty acid transporter activity
glucose catabolic process
copper ion binding
signal transducer activity
positive regulation of signal transduction
translational initiation in response to stress
translation regulator activity
negative regulation of translation
protein transporter activity
glutathione metabolic process
antigen processing and presentation of exogenous antigen
translation initiation factor binding
arginine metabolic process
cadmium ion binding
cellular aromatic compound metabolic process
nitrogen compound biosynthetic process
signal transducer activity
positive regulation of cell differentiation
The enriched terms represented critical biological processes in inflorescence meristems treated with BA and indicated the cross-talk of cytokinin signaling with other biological processes, such as circadian rhythms [14, 60], glucose catabolism and nitrogen compound biosynthesis [52, 61].
Metabolic pathway analysis
KEGG pathway enrichment analysis of differentially expressed transcripts in eight clusters (p <0.05)
Steroid hormone biosynthesis
Arginine and proline metabolism
Alanine, aspartate and glutamate metabolism
Citrate cycle (TCA cycle)
Calcium signaling pathway
Steroid hormone biosynthesis
Metabolism of xenobiotics by cytochrome P450
Genes involved in cytokinin biosynthesis, metabolism and signaling
The differentially expressed genes that were annotated to function in cytokinin biosynthesis, metabolism and signaling were further analyzed.
Cytokinin-regulated genes involved in the metabolism and signaling of other phytohormones
Following BA treatment, Jatropha orthologs of genes encoding a GA receptor, GA INSENSITIVE DWARF1 (JcGID1), and a DELLA protein, RGA LIKE 2 (JcRGL2), were up-regulated (Figure 6). A previous study of cytokinin response genes in Arabidopsis by Brenner et al.  also reported the induction of DELLA protein-encoding genes by cytokinin and reported that cytokinin treatment caused reduced expression of GA20 oxidase. However, in our study, BA did not cause changes in the transcript abundance of a Jatropha ortholog of GA20 oxidase (Figure 6). Earlier studies showed feedback regulation between GA content and GID1 gene expression , and the amount of DELLA proteins was found to be inversely related to the amount of bioactive GA . A Jatropha ortholog of another DELLA protein-encoding gene, REPRESSOR OF ga1-1 (JcRGA1), was slightly up-regulated (Figure 6). The induction of these GA signaling genes by cytokinin in Jatropha inflorescence meristems suggests that cytokinin reduces GA activity by up-regulating suppressors of GA signaling genes and supports previous findings that GA and cytokinin exert antagonistic effects on many aspects of plant development [52, 85].
Jatropha orthologs of two ABA biosynthesis genes, ABA DEFICIENT 1 (JcABA1) and JcABA2, were found to not be affected by BA treatment (Figure 6). However, we identified three Jatropha orthologs of ABA receptors, PYR1-LIKE (PYL) , JcPYL1, JcPYL4 and JcPYL8, and one of them (JcPYL8) was significantly repressed by BA, whereas mRNA levels of JcPYL1 and JcPYL4 showed no clear response to BA treatment (Figure 6). In addition, a Jatropha ortholog of protein phosphatase 2C (JcPP2C), whose activity was inhibited by PYLs in response to ABA in Arabidopsis[86, 87], responded to BA with an increase of transcript abundance (Figure 6). Conversely, the expression of a Jatropha ortholog of calcium-dependent protein kinase 4 (JcCDPK4), which is an important positive regulator in CDPK/calcium-mediated ABA signaling pathways at the whole-plant level in Arabidopsis, was down-regulated significantly by BA treatment (Figure 6). The BA responsiveness of Jatropha orthologs of genes involved in ABA signaling indicated an antagonistic interaction between cytokinin and ABA, which is in line with earlier studies revealing that cytokinins inhibit ABA production [89, 90].
Cytokinin also interacts with ethylene . Decreases were identified in the transcript abundance of the Jatropha orthologs of ethylene receptor 1 (JcETR1) and the gene encoding mitogen-activated protein kinase 6 (JcMPK6) (Figure 6), both of which are negative regulators of ethylene signaling [92, 93]. The Jatropha orthologs of two additional genes, ethylene-responsive transcription factor 1 (JcERF1) and ethylene insensitive protein 3 (JcEIN3), were also repressed by BA treatment (Figures 5 and 6). In Arabidopsis, ethylene-insensitive protein 2 (EIN2) and EIN3-binding F-box 1 (EBF1) are induced by ethylene [94, 95]. The Jatropha orthologs of both of these genes were up-regulated by BA treatment in the present study (Figure 6), indicating that positive regulators of the ethylene signaling pathway were induced by BA treatment. The expression profiles of these genes suggested that cytokinin may act partially by influencing ethylene metabolism and signaling genes .
Four Jatropha orthologs of genes involved in brassinosteroid (BR) signaling were identified. The expression levels of two of them, BRASSINOSTEROID-INSENSITIVE 1 (JcBRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (JcBAK1), which work together as a receptor kinase pair initiating the signal transduction cascade [97, 98], were not significantly altered by BA treatment (Figure 6). The Jatropha ortholog of BRASSINOSTEROID-SIGNALING KINASE 8 (JcBSK8), which is in a small family of kinases that activate BR signaling downstream of BRI, was induced by BA (Figure 6). Furthermore, BA treatment inhibited the expression of the Jatropha ortholog of BRASSINAZOLE RESISTANT 1 (BZR1) (Figures 5 and 6), which is a transcriptional repressor with dual roles in BR homeostasis and growth responses . These findings indicate positive crosstalk between the cytokinin and BR signaling pathways in Jatropha.
Genes encoding the jasmonate ZIM-domain (JAZ) proteins, which are key regulators of jasmonate signaling , have been reported to repress transcription of jasmonate-responsive genes . The transcription level of the Jatropha ortholog of JAZ1 (JcJAZ1) increased upon BA treatment (Figures 5 and 6). The Jatropha ortholog of ENHANCED DISEASE SUSCEPTIBILITY 5 (JcEDS5) was up-regulated, whereas the Jatropha ortholog of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 4 (JcNPR4) showed no response to BA treatment (Figure 6). Both of EDS5 and NPR4 are involved in salicylate signaling . These results imply that jasmonate and salicylate signaling may involve crosstalk with cytokinin in Jatropha.
Genes involved in flower development
The ABCE model was formulated from the analysis of floral homeotic mutants with organ identity defects in two adjacent whorls of the flower, and similar classes of mutants were described in both Arabidopsis and Antirrhinum, suggesting that the regulation of organ identity was highly conserved in evolution [112–114]. In this study, Jatropha orthologs of A-function genes APETALA 1,2 (JcAP1,2), B-function gene PISTILLATA (JcPI), C-function gene AGAMOUS (JcAG) and E-function genes SEPALLATA1,2,3 (JcSEP1,2,3) were significantly repressed, with an exception of one B-function gene JcAP3 that was shown to be up-regulated by BA treatment (Figure 7A,B). Along with the altered expression of these ABCE model genes, bisexual and asexual flowers were induced, but no abnormal floral organ development was found in Jatropha treated with BA (Additional file 1: Figure S1, ). In Arabidopsis, exogenous BA application resulted in abnormal flowers that resemble the phenotypes of mutants, clv1, ap1, ap2, and ap3[22, 115]. However, the increased mRNA levels of AP1, PI and AG in transgenic Arabidopsis, which resulted from the expression of AtIPT4 under control of AP1, also caused abnormal flower and floral organ development . The different phenotypic changes in Arabidopsis[22, 115] and Jatropha occurring upon BA treatment (Additional file 1: Figure S1, ) likely resulted from the differential responses of flowering-related genes as revealed in this study and previous studies on Arabidopsis. It is noteworthy that the relative expression of several genes involved in flower development were found to be significantly reduced after BA treatment by qRT-PCR analysis (Figure 7B), whereas their expression did not show significant difference compared to the control in transcriptome sequencing analysis (Figure 7A). This discrepancy probably resulted from the low background expression of these genes and/or the insufficient transcriptome sequencing depth. Normalizing the cDNA library and enhancing the depth of transcriptome sequencing may help to solve this problem.
Cytokinin-regulated genes involved in cell division
In this study, a time-course experiment was conducted to characterize activated and repressed genes in the inflorescence meristems of Jatropha following cytokinin treatment using transcriptome sequencing by a 454 GS FLX Titanium instrument. This approach produced 703,755 high-quality reads ranging from 100 bp to 790 bp, with an average length of 364 bp (Table 1). Up-regulated and down-regulated annotated and novel genes were identified (Figure 3, Additional file 3: Table S2), resulting in an unprecedented view of the regulatory activities of cytokinin in Jatropha inflorescence meristems.
Abundant physiological data suggest that plant hormones interact with each other to regulate various aspects of development [132–135]. In this study, the cytokinin signaling pathway was found to crosstalk with other signals, mainly through pathways converging on or through transcriptional factors or other signaling components. For example, interactions with GA can occur through induction of the negative regulators JcGID1 and JcRGL1. The expression profiles of genes involved in other hormone signaling pathways indicated that: 1) there are multiple agonistic and antagonistic effects between cytokinin and auxin; 2) cytokinin may reduce plant responsiveness to GA and ABA by repressing GA and ABA metabolism- and signaling-related genes and 3) cytokinin may act synergistically with ethylene and BR in controlling Jatropha inflorescence meristem development.
Like most of the published reports of RNA-seq data [31, 32, 137, 138], due to the high cost of 454 sequencing at the commencement of this study, only a single biological replicate was included for transcriptome analysis, which prevented proper statistical testing on identification of differential expressed genes. Nonetheless, different transcript abundance of the annotated genes in control and BA-treated inflorescences revealed by 454 sequencing in this study provide a valuable data source for selecting putative BA-regulated Jatropha genes for further verification by qRT-PCR with sufficient biological replicates. In this study, we did each qRT-PCR experiment with three biological and three technical replicates per biological replicate. Our results, as shown in Figures 4, 5, 6, 7 and 8, indicated a high correlation of expression levels between 454 sequencing data and qRT-PCR, but the congruence of statistically significant differential expression revealed by the two approaches was low. This observation is consistent with previous work showing lack of congruence between 454 sequencing and qRT-PCR results regarding genes predicted as significantly differential expression .
To our knowledge, this is the first report on the transcriptional regulation and identification of genes that are differentially expressed in the inflorescence meristems of Jatropha exposed to cytokinin. We have identified a set of cytokinin-regulated genes in Jatropha inflorescence meristems through expression profiling. Some of them correspond to previously identified genes in Arabidopsis, and others show different expression patterns from their Arabidopsis orthologs. Further analyses of these genes with different expression patterns are needed to elucidate their roles in the cytokinin responses of Jatropha inflorescence meristems. Although transcriptional analysis is only an initial step and does not identify functional relevance as it may or may not relate to changes in the level and/or activity of the corresponding proteins, the potential cytokinin-responsive transcripts identified in this study will provide a good starting point for investigations into the molecular mechanisms of Jatropha responses to cytokinin.
Plant material and BA treatment
Cuttings from one Jatropha plant were propagated into individual plants and used as experimental plants. BA treatment was performed when the plants were undergoing the flowering stage in the following year. In total, 230 inflorescence meristems with a diameter of approximately 0.5 cm were selected as experiment subjects. First, each meristem was wrapped around by a piece of absorbent cotton weighing 10 mg. Then, 200 μl of a 1 mM BA solution containing 0.05% Tween-20 was applied to the cotton using a pipette.
Before BA treatment, 50 meristems were collected as control. Two hours after treatment, all cotton pieces wrapped around the inflorescence meristems were removed, and 50 meristems were sampled, which were identified as T (BA treatment for 2 hours). Fifty meristems were sampled at 4 and 22 hours post BA treatment, which were identified as T +4 H and T +22 H, respectively. The remaining 30 meristems were kept for phenotypic analysis. The fifty inflorescence meristems sampled at each time point, which were pooled together as one sample for 454 sequencing, were immediately frozen in liquid nitrogen and stored at −80°C.
To investigate the effect of cytokinin on branching of inflorescence, BA working solution (0.5 mM) with 0.05% (v/v) Tween-20 was sprayed onto each inflorescence meristem (about 0.5 cm in diameter) and the surrounding leaves using a hand sprayer. Control inflorescence meristems were sprayed with distilled water containing 0.05% (v/v) Tween-20. Spraying was conducted once. Thirty inflorescence meristems were used for each treatment.
RNA extraction, cDNA synthesis and 454 sequencing
Each frozen sample was ground in a mortar with liquid nitrogen, and total RNA was isolated using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) following the standard protocol. An Agilent 2100 instrument was used to check the RNA quality (RIN >0.7), and a NanoDrop spectrophotometer (ND-2000C, Thermo Fisher Scientific, USA) was used to quantify RNA concentration. Messenger RNA was further purified using a MicroPoly(A) Purist Kit (Ambion) according to the protocol. Double-stranded cDNA was synthesized from mRNA according to Ng's full-length cDNA synthesis protocol  with some modifications  and then fragmented to 300–800 bp. The prepared cDNAs were transformed into single-stranded template DNA (sstDNA) libraries using the GS DNA Library Preparation kit (Roche Applied Science). sstDNA libraries were clonally amplified in a bead-immobilized form using the GS emPCR kit (Roche Applied Science) and sequenced on the 454 Genome Sequencer FLX instrument.
Sequence assembly and annotation
The 454 transcriptome sequencing reads were first handled by a 454 GS FLX system, which cut off the adapter and low-quality bases. The reads were then filtered by an in-house-developed program to remove low-quality reads. The qualified reads were then assembled by CAP3 using the default parameters . Open reading frames were identified using an in-house-developed program based on ‘GetORF’ from EMBOSS , and the annotation was performed through BLAST searches against the Swiss-Prot and GenBank databases with an E-value cutoff of 1E-3. Gene ontology analysis was performed using GoPipe through BLASTP against the Swiss-Prot and TrEMBL databases using an E-value cutoff of 1E-3 . The metabolic pathway was constructed based on the KEGG database by the BBH (bi-directional best hit) method .
Analysis of differentially expressed genes
To estimate gene expression, the read number for each gene was first transformed into RPKM (reads per kilobase per million reads) , and differentially expressed genes were identified by the DEGseq (identifying differentially expressed genes from gene expression data) package using the method MARS (MA-plot-based method with random sampling model) . We use p-value <0.05 and the absolute value of log2Ratio >1 as the threshold to judge the significance of contig expression difference.
We perform BLASTX (NT query to AA database) in TAIR WU Blast against TAIR10 Arabidopsis proteins using NO filters to identify the most highly similar genes involved in plant hormone signaling, flower development and cell division (Additional file 5: Table S4). The members of the two-component signaling pathway in Jatropha was named following the nomenclature reported by Heyl et al. .
Where G is any gene from the transcriptome database and RPKMcontrol is the RPKM of control.
We identified enriched GO terms and pathways for the differentially expressed genes using DAVID (the Database for Annotation, Visualization and Integrated Discovery). DAVID is a web-based bioinformatics application that systematically identifies enrichment for biological annotations based on large gene lists derived from high-throughput genomic experiments [50, 51].
Quantitative real-time PCR (qRT-PCR) confirmation
The expression profiles of 15 genes involved in plant hormone signaling, 14 genes associated with flowering and 4 genes involved in cell division were investigated by qRT-PCR to confirm the transcriptome data. The RNA samples used for qRT-PCR were isolated from tissues collected from the experiment used for constructing the 454 libraries, and from inflorescence meristems collected in a replicate experiment. cDNA for each sample was synthesized from total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan) according to the instructions. The primers used for qRT-PCR are listed in Additional file 6: Table S5. The PCR products were sent to BGI (Shenzhen, China) for sequencing using specific primers for sequence confirmation. Relative gene expression levels were detected using the SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara, Japan) according to the manufacturer’s instructions on a LightCycler 480 II (Roche, USA) instrument. The relative expression of each gene was calculated using the 2-ΔΔCT method . All quantitative PCR experiments were repeated with three biological and three technical replicates per biological replicate.
All of the sequences of the unigenes greater than 200-bp in length obtained from the transcriptome sequencing of inflorescence meristems in Jatropha have been deposited in the Transcriptome Shotgun Assembly (TSA) database, http://www.ncbi.nlm.nih.gov/bioproject/265802 (Accession: PRJNA265802;ID: 265802). Unigenes less than 200-bp in length were listed in Additional file 7: Table S6.
We thank Dr. Changning Liu for helping in sequence data submission. This work was supported by funding from the Top Science and Technology Talents Scheme of Yunnan Province (2009CI123), the Natural Science Foundation of Yunnan Province (2011FA034), the National Natural Science Foundation of China (31370595) and the CAS 135 program (XTBG-T02) awarded to Z.-F. Xu. The authors gratefully acknowledge the Central Laboratory of the Xishuangbanna Tropical Botanical Garden for providing research facilities.
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