Microarray hybridisation and data analysis
Four biological replicates per genotype were used for RNA extraction using the method described above. RNA (200 ng) was used for cDNA synthesis and Cy3-labelling using the Low Input Quick Amp Labelling Kit for One-Colour Microarray-Based Gene Expression Agilent analysis (Agilent, Santa Clara, CA, USA). Linearly amplified and labelled cDNA (1.65 µg) was hybridised for 17 h at 65ºC on 4 X 180 k format 60-mer oligonucleotide probes designed against the S. lycopersicum cv. Heinz 1706 build SL2.40 (annotation 2.3) genome (Agilent design ID = 069672; see Gene Expression Omnibus (GEO) record GPL21602). Each array contained ~5 probes for 34,619 transcripts. Arrays were imaged using an MS200 microarray scanner using only the 480 nm laser using the autogain feature of the NimbleScan software (Roche NimbleGen, Madison, WI, USA). Image (tiff) files were imported into the Agilent Feature Extraction software for quality control assessment, grid alignment and expression value extraction at the probe and transcript level with the RMA algorithm (Irizarry et al. 2003) used to carry out background subtraction, quantile normalisation and summarisation via median polish, and output log2 normalised gene expression levels (GEO record GSE79307)(Ferrández-Ayela et al. 2016). Linear Models for Microarray Data (package LIMMA in R) was then used to fit linear models to pairs of samples, identifying genes that contrasted the most between the experimental pairs (Smyth 2004). Transcripts were deemed to be differentially expressed if they showed a Benjamini-Hochberg adjustedP ≤ 0.05 when comparing rootstocks genotypes.