PMC:4979054 / 3540-13950 JSONTXT

{"project":"2_test","denotations":[{"id":"27600348-23593468-69481531","span":{"begin":877,"end":879},"obj":"23593468"},{"id":"27600348-23384169-69481532","span":{"begin":880,"end":882},"obj":"23384169"},{"id":"27600348-16081474-69481533","span":{"begin":3790,"end":3792},"obj":"16081474"},{"id":"27600348-10592173-69481534","span":{"begin":4060,"end":4062},"obj":"10592173"},{"id":"27600348-16712727-69481535","span":{"begin":8652,"end":8654},"obj":"16712727"},{"id":"27600348-17720982-69481536","span":{"begin":8655,"end":8657},"obj":"17720982"},{"id":"27600348-12184808-69481537","span":{"begin":10405,"end":10407},"obj":"12184808"}],"text":"2. Experimental Section\n\n2.1. Experimental Setup and Sampling\nA total of 16 tenera palms (Elaeis guineensis Jacq.; dura × pisifera) with similar genetic backgrounds (Serdang Avenue dura × AVROS pisifera) were selected from Sime Darby’s oil palm breeding program. These palms were selected from a total of more than 100 trees planted in the same field in a breeding trial located in East Estate, Carey Island, Malaysia, based on the homogeneity of vegetative and growth characteristics, but differentiated by different oil yields and bunch analysis traits that had been recorded over five years. No severe pest or disease issues were observed for any of the individuals during experimental period. Six different inflorescences of each of the 16 selected palms were open-pollinated over a two years period and harvested at 12, 14, 16, 18, 20 and 22 WAA, as described previously [19,20]. The full inflorescence was harvested and fresh oil palm fruits taken were randomized and selected for each time point. Fresh mesocarp tissue was separated from the randomized oil palm fruits immediately after the samples were taken in the field and frozen in liquid nitrogen before storing at −80 °C after transfer to the laboratory.\n\n2.2. Oil Palm Mesocarp RNA Extraction\nTotal RNA was extracted from the oil palm mesocarp tissue using the following RNA extraction method. The mesocarp tissue was first ground to a fine powder in liquid nitrogen. The extraction buffer contained 0.1 M Tris-HCl, pH 7.6, 0.1 M NaCl, 6% p-aminosalicylic acid, 1% SDS, and 0.35% β-mercaptoethanol. The buffer was added to frozen ground material at the rate of 4 mL/g plant tissue, vortexed thorough and extracted with phenol/chloroform. An additional chloroform:isoamylalcohol cleanup step was performed after the phenol/chloroform extraction steps to improve the purity of the RNA. The supernatant was precipitated using 3 M LiCl followed by an ethanol precipitation. The pellet was redissolved in DEPC-treated distilled water and again precipitated with 3 M LiCl followed by another round of ethanol precipitation. The precipitate was finally dissolved in DEPC treated distilled water and stored at −70 °C.\nThe concentration and purity of total RNA was determined by spectrophotometric quantification (Thermo Scientific, Nanodrop ND-1000, Wilmington, DE, USA). The AU 260/280 and AU 260/230 were measured and samples with a ratio of 1.8–2.0 were utilized. Gel electrophoresis was also performed on 1 µg of total RNA using a 1% agarose gel in TAE buffer to further determine the integrity and quality of the RNA. Samples that passed the initial quality tests were further analyzed using an Agilent Bioanalyzer (Agilent, Bioanalyzer 2100, Santa Clara, CA, USA), allowing the ratio of the 28S to 18S peaks to be determined. Samples with 28S to 18S ratios of greater than 2:1 and an RIN (RNA Integrity Number) score greater than 7 (out of 10) were utilized for further work.\n\n2.3. Building the Transcriptome, Custom Design of the Oil Palm Mesocarp Array and Agilent Commercial Array\nA consensus transcriptome sequence was built from reads generated from sequencing oil palm mesocarp tissues collected from samples harvested at different WAA. The reads generated by Roche 454 GS-FLX sequencers were assembled using the Newbler program V2.5 (Roche 454 Life Sciences, Branford, CT, USA). After sequence assembly, sequences shorter than 100 base pairs, as well as those originating from organelles and rRNA, were removed, leaving 31,804 sequences. The sequences have been deposited in the European Nucleotide Archive (ENA) (accession number(s): LM611910–LM643713). In order to undertake a global analysis of the mesocarp gene expression, the transcripts were compared to the Gene Ontology database using BLAST2GO (version 2.3.5) [21]. This process classifies the genes according to the molecular function, biological process or cellular component. Biological pathway analysis of the transcripts was carried out by comparison with plant sequences annotated to reference pathways in the KEGG database [22]. For the processes above, the E-value cutoff for the BLASTx program was set to be 10−5.\nThe custom oil palm mesocarp array probes were designed based on these 31,804 sequences with the annotations obtained by comparing isotig sequences to the Uniprot database [23]. The custom gene expression oil palm mesocarp array was designed using the Agilent eArray web-based application in a 2 × 105K format. Probes were designed using the Agilent internal design program through the eArray website [24]. Each of unique transcriptome sequences was represented by three distinct probes. Agilent 60-mer SurePrint technology was used for array printing.\nIn the cross-species study, the 4 × 44K Arabidopsis (V4) Gene Expression Microarray (G2519F-021169) and the 4 × 44K rice gene expression microarray (G2519F-015241) from Agilent Technologies were used for hybridization with the RNA from the 16 WAA samples. The array consists of 43,803 probes for both Arabidopsis thaliana and rice. The Arabidopsis microarray was designed based on various databases including; NCBI Reference Sequence Database (RefSeq) (July 2008), UniGene (May 2008), TAIR 8 cDNA (April 2008), TIGR (June 2006), TIGR Plant Transcript Assemblies (June 2006) and ATHI (Jan 2004) [25]. The Rice microarray was designed based on resources from National Institute of Agrobiological Sciences, RefSeq and GenBank 2007 [25].\n\n2.4. Synthesis of cRNA, Microarray Hybridization and Scanning\nTotal RNA samples from mesocarp were individually treated and labeled with a one-color (Cy3) dye according to the Low Input Quick Amp Labeling protocol (version 6.0; December 2009) provided by Agilent. A total of 100 ng of total RNA was used to synthesize cRNA labeled with Cy3 dye. Total RNA of 100 ng in 1.5 µL was mixed with 2 µL of Agilent One-Color Spike Mix. T7 promoter primer was added into the mixture and the reaction made up with Nuclease-free water to 5.3 µL. The reaction mixture was incubated at 65 °C for 10 min using a thermal cycler (BIO-RAD, C-1000, Hercules, CA, USA). After incubation, the reaction mixture was incubated on ice for 5 min. To reverse transcript the mRNAs to cDNA, a total of 4.7 µL cDNA Master mix was added to the previous reaction mixture. This consisted of 2 µL of 5X first strand buffer, 1 µL of 0.1 M DTT, 0.5 µL of 10mM dNTP mix and 1.2 µL of AffinityScript RNase Block mix. The reaction mixture was mixed thoroughly by pipetting up and down. After briefly centrifuging, the reaction mixture was incubated at 40 °C for 2 h, followed by 70 °C for 15 min using a thermal cycler (C1000, BIO-RAD). After the cDNA synthesis process, the reaction mixture was kept on ice for 5 min, before 6 µL of transcription mixture was added giving a total volume of 16 µL. This step was performed to transcribe cDNA to cRNA and incorporated the Cyanine 3-CTP dye to cRNA during the transcription process. The transcription mix consist of 3.2 µL of 5X transcription buffer, 0.6 µL of 0.1 M DTT, 1 µL of NTP mix, 0.21 µL of T7 RNA Polymerase Blend, 0.24 µL of Cyanine 3-CTP, made up with nuclease-free water to a volume of 6 µL. The reaction was mixed and incubated at 40 °C for 2 h using a thermal cycler. Labeled cRNAs were then purified using Qiagen’s RNeasy mini kit (Hilden, Germany) as recommended by Agilent. The quality and quantity were assessed using the Spectrophotometer ND-1000 (Thermo Scientific). A total of 1.65 µg of purified labeled cRNA was used for hybridization. The purified cRNA was mixed with 25 µL of 10X blocking Agent, 5 µL of 25X Fragmentation buffer and made up to a volume of 120 µL with nuclease-free water. The reaction was mixed and incubated at 60 °C for 30 min to fragment the cRNA and cooled on ice immediately after fragmentation. A total of 125 µL 2X GE Hybridization Buffer HI-RPM was mixed with the fragmented cRNA and hybridized onto the arrays at 65 °C for 16 h in a rotating hybridization oven. After hybridization, 2 steps of washing were performed with wash buffer 1 and 2 for 1 min each. The array was then air-dried for a few seconds before proceeding with image scanning using the Agilent microarray slide scanner (SG11350602). The slides were scanned using the green dye channel with scanning resolution of 5 µm at 20 bit of dynamic range.\n\n2.5. Data Extraction, Normalisation and Comparisons\nRaw microarray data were extracted from scanned images by using the Feature Extraction software (version 10.7.31; Agilent) [26]. Background normalization was carried out using the normexp algorithm whereas normalization between the samples was carried out using quantile normalization [27,28]. Both normalization steps were implemented using the R package of limma (Linear Models for Microarray Data, version 2.13.1) [29]. The remaining parameters in limma were set to default value. For an expression difference to be determined as statistically significant, the log fold change must be ≥0.6 and false discovery rate (FDR) ≤ 0.05. Comparisons of selected candidates between oil palm mesocarp, rice and the Arabidopsis microarray were carried out based on the normalized signal intensities produced.\n\n2.6. Quantitative Real-Time PCR\nValidation of the microarray expression data was performed using quantitative real-time PCR (qPCR). The first strand cDNA prepared from pooled biological replicates of palms at each stage of maturation was used. Two micrograms of total RNA from each sample was used in a reverse transcription reaction using Omniscript Reverse Transcriptase with standard conditions as recommended by manufacturer (QIAGEN). The first strand cDNA synthesis was primed by random hexamer primers. Specific primers were then designed based on the in-house mesocarp transcript database at Sime Darby using the Primer Premier 5.0 software [30]. The qPCR reaction mix consists of 5 μL of a 5X dilution of cDNA, 0.8 μL of forward and reverse primers (10 mM), 10 μL of BIO-RAD iTaq™ Fast SYBR GREEN Supermix with ROX and topped up with distilled water to 20 μL. The PCR cycling conditions were based on optimized conditions suggested by BIO-RAD with 95 °C (1 min) for 1 cycle and followed by 95 °C (15 s) and 55 °C (35 s), for 40 cycles. Relative expression of each transcript was analyzed using qBase Plus 2.0 [31] and normalized against multiple reference genes. In this study, Cyp2 and GRAS were used as previously published [32,33].\n"}