Computational protocol: The arbuscular mycorrhizal status has an impact on the transcriptome profile and amino acid composition of tomato fruit

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[…] Solanum lycopersicum cv.Micro-Tom tomato seeds were sterilized with a series of washes: 3 min in 70% ethanol, to which 3-4 drops of tween 20 were added, 13 min in a 5% bleach solution and 3 washes of 10 min each in sterile water. The seeds were then placed in a 0.6% agar medium (5 seeds per petri dish). The petri dishes were kept for 5 days in the dark, followed by 4 days in the light. The germinating seedlings were then transferred to pots with sterile quartz sand. For mycorrhization, the fungus Glomus mosseae Gerd. & Trappe (BEG 12) was purchased from Biorize (Dijon, France). A mixture of sand (70%) and fungal inoculum (30%) was used. The mycorrhizal and control plants were grown in a growth chamber under a 14 h light (24°C)/10 h dark (20°C) regime, and watered, 125 ml/plant twice a week with water, and once a week with a modified Long-Ashton solution containing a low phosphorus concentration (3.2 μM Na2HPO4·12H2O) []. The inoculated and control plants were observed during their development and the traits related to plant growth, fruit production and phenology, such as flowering time [] and fruit maturation, were measured. For phenologial data, a statistical analysis was conducted using the Statistica 6 software, applying the one-way ANOVA and Tukey test adopting a probability level of p < 0.05. The statistical analysis of fruit production and fruit weight was conducted using the Kruskal-Wallis non-parametric test.The fruit was collected when it reached the required ripening stage i.e. turning, as depicted in Figure . The seeds were discarded and the pericarp was cut into small pieces, put in 2 ml reaction tubes, frozen immediately in liquid nitrogen and stored at -80°C until required. The fruit pericarps were freeze-dried overnight and stored at -80°C.At the end of the experiment, the plant roots were cut and the fungal colonization was assessed according to the Trouvelot five-class system [] using MYCOCALC software. Twelve plants were considered for the root mycorrhization intensity assessment and five microscope slides were analyzed per plant, each slide containing 20 root pieces of 1 cm. [...] The TOM2 microarrays were obtained from the Center for Gene Expression Profiles (CGEP; Cornell University, Ithaca, NY, USA). Each microarray contains 11769 oligonucleotide probes, whose design was based on gene transcript sequences from the Lycopersicon Combined Built # 3 unigene database (http://www.SGN.cornell.edu). Three biological replicates were analysed and a 'dye swap' approach was adopted. Total RNA (500 ng) was used to generate direct fluorescently labelled cRNA using the Low RNA Input Linear Amp Kit (Agilent) according to the manufacturer's instructions. Slides were treated following the prehybridization protocol provided by the manufacturer (http://ted.bti.cornell.edu/cgi-bin/TFGD/array/TOM2_hybridization.cgi). Microarray hybridization was performed using the Gene Expression Hybridization kit (Agilent). Post-hybridization was performed following the manufacturer's instructions with slight modifications as described in Fiorilli et al. []. The slides were then scanned using an Agilent microarray scanner (G2565BA) at a resolution of 10 μm and laser power set to 90%. The fluorescence data were processed using IMAGENE software (version 5.6; BioDiscovery Inc.; http://www.biodiscovery.com). Normalization and analysis of the microarray data were performed using the LIMMA software package (BIOCONDUCTOR package) []. The values of all the spots on the arrays were per spot and per chip intensity dependent (Lowess) normalized. Significant up- or down- regulated genes were filtered for a false discovery rate of < 0.05 and for greater or lower normalized expression ratios than 1.5- or 0.67-fold, respectively. Gene ontology (GO) term annotation was obtained using the BLAST2GO software []. [...] The DNA extraction was performed on 100 mg of leaves using the DNA Plant Mini. Kit (Qiagen) according to the manufacturer's instructions. Sequence data of the differentially regulated transcripts were obtained from the Tomato Functional Genomics Database http://ted.bti.cornell.edu/ using the microarray unigene ID.Specific primers were designed with Primer3 software (http://frodo.wi.mit.edu/primer3/) and were tested on cDNA for amplification before qRT-PCR. PCR assays were carried out in a final volume of 25 μl containing 2.5 μl of 10X buffer, 1 μl of 2.5 mM dNTPs, 0.4 μl of each primer 10 μM, 1 μl of Red Taq polymerase (Sigma), and 1.5 μl of a total DNA diluted 1:10. The PCR cycling programme consisted of: 95°C for 5 min, 40 cycles of 94°C for 45 sec, 65°C for 45 sec and 72°C for 45 sec. The primer names and corresponding sequences are listed in additional file . […]

Pipeline specifications

Software tools Statistica, ImaGene, limma, Blast2GO, Primer3
Databases TFGD
Applications Miscellaneous, qPCR
Organisms Solanum lycopersicum
Chemicals Nitrogen