Chestnut Transcriptome NG Sequencing: a New Tool to Investigate Gall Wasp Response

The Dryocosmus kuriphilus susceptibility in almost all chestnut cultivars and the absence of infestation symptoms in the cultivar ‘Bouche de Bétizac’ led us to investigate in depth plant-pest molecular interaction. For this reason, in 2012, the study of the gall wasp response in chestnut shifted from classical methodologies to an innovative approach potentially able to provide a large amount of information on a species poorly known from a genetic point of view. Next Generation Sequencing platforms are able to realize a massive sequencing of DNA molecules spatially separated in a flow cell. This strategy represents a radical change in comparison with method described by Sanger sequencing, and allows to sequence hundreds of Mbp (million base pairs) to Gbp (billion base pairs) of DNA in a single analytical run. RNA-Seq allows to map and quantify the transcripts present in biological samples. Our approach was to use the Illumina technique. The experimental design was set to compare the genes activated at sprouting in susceptible and resistant chestnut cultivars in the presence and absence of infestation. We collected buds at different stages in order to identify the genes involved in plant-insect interaction at the onset of gall formation (susceptible genotype) or at the time of resistant response causing larva death. The sequencing of the cDNA libraries was carried out by BMR genomics (Padova, Italy). The reference chestnut transcriptome was assembled and included about 1,000,000 contig sequences. Since this is an extremely large amount of data, a cutoff at 500 bp was applied to the library obtaining 40,000 contigs that were used as a reference in the RNASeq analysis. The reads produced by sequencing the 4 treatments were mapped against the reference library. Reference contig identification, currently in progress, is being carried out by the program Blast2GO. This approach yielded an initial transcriptome assembly and will provide transcript sequences to the research community to facilitate further studies.