Methylketone Biosynthesis in Tomato F2 Segregant Populations

Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f glabratum, which synthesizes and accumulates insecticidal methylketones (MKs) such as 2-undecanone and 2-tridecanone in glandular trichomes, and Solanum lycopersicum (cultivated tomato), which does not, demonstrated that MK metabolism in the wild species can be attributed to several loci. Comparative trascriptome analysis of the glandular trichomes of F2 segregants bulked into low- and high-MK plants identified several genes whose transcripts were either more or less abundant in the high-MK plants. The total RNA was extracted from isolated trichomes of bulked segregants; high and low methylketones accumulators, from the F2 population (5 plants in each bulk). The samples for Gene expression were labeled using Agilent Low RNA Input Linear amplification Kit (Part number:5184-3523). Five hundred nanograms each of the Control and test samples were incubated with reverse trancription mix at 42°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for aRNA generation. aRNA was generated by in vitro transcription and the dyes Cy3 CTP(Agilent) for control samples and Cy5 CTP(Agilent) for test samples were incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled aRNA was cleaned up and quality assessed for yields and specific activity. The labeled aRNA samples were hybridized on to a Custom Tomato Gene Expression 4x44k (AMADID:19003). 825ng of cy3 labeled samples and 825ng of cy5 labeled samples were pooled, fragmented and hybridized and data from these channels were used for comparison to test hybridizations of respective channels. Fragmentation of labeled aRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327) and scanned using the Agilent Microarray Scanner G Model G2565BA at 5 micron resolution. Four arrays were used and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all four replicates.