Jatropha

Tag libraries of the samples of seeds in the seven developmental stages were prepared in parallel using an Illumina gene expression sample preparation kit and sequenced using the Illumina GAII platform at BGI-Shenzhen without replications (http://en.genomics.cn/navigation/index.action) (Methods S1). A preprocessed database of all possible CATG+17 nucleotide tag sequences was created using our genome reference database. Further information on the genomic sequences and the predicted protein-encoding genes is available at ftp://Jatropha:9uebluesrjd7@ftp.genomics.org.cn and at the NCBI nucleotide database (Project ID: 63485). For annotation, all tags were mapped to the reference sequences, allowing no more than one nucleotide mismatch per tag. All the tags that mapped to reference sequences from multiple genes were filtered and the remaining tags were defined as unambiguous tags. As a result, we generated between 3.26 and 6.18 million raw tags for each of the seven samples (Table S5). After removing the low quality reads, the total number of tags per library ranged from 3.03 to 6.07 million and the number of tag entities with unique nucleotide sequences ranged from 83,820 to 167,765 (Table S5). For gene expression analysis, the number of expressed tags was calculated and then normalized to TPM (number of transcripts per million tags) [27] (link).
A 3×3 self-organizing map (SOM) of the gene expression data was constructed using GeneCluster 2.0 [84] (link) (http://www.broadinstitute.org/cancer/software/genecluster2/gc2.html) with a variation filter (Max/Min≥5) to eliminate genes whose expression did not change significantly across samples, and normalization of the means and variance (mean  = 0 and variance = 1). The SOM algorithm was executed with the desired cluster range of 3–9 and the rest of the parameters left unchanged. They are 50000 iterations, seed range of 42, initialization of centroids to random vectors, bubble neighborhood, initial and final learning weights of.1 and .005, and initial and final sigmas determining the size of the update neighborhood of a centroid set to 5 and .5, respectively.

For Northern blotting, 15 μg of RNA was isolated from immature jatropha seeds and leaves and fractionated on 1.2% agarose-formaldehyde denaturing gel (Qiagen RNeasy Mini handbook). The RNA was blotted onto Hybond-N⁺ nylon membranes (Amersham Pharmacia) and stained for visualization of the RNA bands [17 (link)]. The KAR cDNA probe generated using RT-PCR was labelled with [32P]-dCTP (GE Healthsciences). Pre-hybridization was for 3 hours and hybridization was for 16 hours at 65°C (Techne, Staffordshire UK). Filters were washed first (20 min) in buffer A (2 × SSC + 0.1% SDS) and then Buffer B (20 min) in (1 × SSC + 0.1% SDS) and lastly (30 min) in buffer C (0.5 × SSC + 0.1% SDS) at 65°C. The bound probe was detected by exposing filters to KODAK Biomax MS Autoradiography Film using exposure cassettes at -80°C.

Total lipid was extracted from 100 mg fresh Jatropha leaves as previously described [8 (link)]. The outer seed coat was removed from dried Jatropha seeds. The seeds were surface sterilized for 1 minute with 75% (v/v) ethanol, immersed in 10% (v/v) H₂O₂ for 1 hour, rinsed with sterile water two times, and finally immersed in sterile water overnight at 28°C in darkness for 24 hours. The seed endosperm was carefully separated from the embryo. The dry endosperm part was ground to fine powder and the lipids were extracted with hexane three times. The combined supernatant was transferred to a glass vial and the hexane was evaporated with a flow of dry nitrogen gas at 50°C. The weight of the raw oil was determined and the oil content was recorded as the ratio of raw oil to dried endosperm weight.
About 10 mg of lipid was transmethylated with 3N methanolic-HCl (Sigma, St. Louis, MO, USA) plus 400 μL 2,2, dimethoxypropane (Sigma). The resultant FAMEs were separated by GC and detected using GC Agilent 6890 (Agilent, Santa Clara, CA, USA) employing helium as the carrier gas and DB-23 columns for components separation. The GC analysis was performed at 140°C for 50 seconds and 30°C per minute ramp to 240°C, and the final temperature was maintained for 50 seconds. Peaks were identified based on their retention times compared with a FAME reference mixture (Sigma). The fatty acid composition value included in the analyses was calculated based on the peak area percentage of total fatty acids in three biological replicates. The data were presented as means ± standard deviations.

Chitosan of high molecular weight and degree of deacetylation of ~80% was provided by the Northeastern Biomaterials Evaluation and Development Laboratory–CERTBIO (Campina Grande, PB, Brazil). Gelatin from porcine skin, phosphate-buffered saline (PBS), lysozyme and genipin were purchased from Sigma-Aldrich^®, Merck Group (Darmstadt, Germany). Acetic acid and absolute ethyl alcohol were purchased from Neon^® (Suzano, –SP, Brazil). The latex of Jatropha mollissima (Genetic Heritage No. AD5B98B) was collected at Sítio Valente, located in the city of Boa Vista–PB, Brazil (Lat 7° 20’ 18.29” S Long 36°14’ 17.11” W).

Jatropha seeds were surface-sterilized with 1% CuSO₄ and placed on six layers of wet filter papers to germinate in dark for five days. Then the sprouted seeds were planted in matrix soil mixed with perlite, vermiculite and humous at the ratio of 1:1:1. The soil-grown J. curcas was cultivated in a growth chamber at 26 ± 1 °C under a light/dark regime of 16/8 h. 0.1 mg leaf pieces were clipped from Jatropha seedlings with 2–4 emerging true leaves and used to extract genomic DNA as previously described [26 (link)].
This study used the K326 tobacco cultivar. Tobacco culturing was maintained in a growth chamber at 25 ± 1 °C under a light/dark regime of 16/8 h.

JcTAW1 gene sequence was retrieved with the query of rice TAW1 protein from the whole-genome shotgun contigs (WGS) of Jatropha via the tblastn tool of NCBI. Similar to the rice TAW1 gene, JcTAW1 has no introns, so it can be amplified directly from Jatropha genomic DNA extracted as previously described [26 (link)]. JcTAW1 gene was amplified by two rounds of nested PCR using Phusion high-fidelity DNA polymerase (NEB). The first round of PCR used the flanking gene primers JcTAW-Fw, JcTAW-Rv and Jatropha genomic DNA as the template. The crude product was 100-fold diluted as a template for the second round of PCR with the gene-specific primer pair JcTAW1-5Kn/JcTAW1-3Sc. Then the purified amplicon fragment was cloned into the plant binary vector pBI121 (Clontech) by KpnI and SacI (NEB) digestions to generate plant expression vector pBI121 (JcTWA1). All primers used for vector construction are listed in Table S1.