The genome of the eastern spruce budworm, Choristoneura fumiferana Clem., Lepidoptera: Tortricidae is ~500Mb (n=30). A parsimonious approach to spruce budworm genome study is to study genome-wide expression of genes as expressed sequence tags (ESTs). ESTs are generated through cDNA libraries made from expressed mRNA which represent the genes that are expressed in the tissues from which the mRNA was isolated. The EST approach has been widely used for novel gene identification, monitoring genome-wide expression of genes, physical map construction and annotation of genomic sequences.

We have initiated a study on the construction of EST collections from different tissues of the spruce budworm. We will construct collections of ESTs from three key tissues, selected on the basis of their physiological importance and virus interaction, namely the midgut, epidermis and fat body. The midgut is involved in digestion, absorption, defence against pathogens and site of entry of oral pathogens; the epidermis is involved in molting, metamorphosis, and protection; and the fat body is involved in secretory protein synthesis, metabolism and detoxification. The spruce budworm has six larval stages and basically each instar is a repeat of the previous stage, except for the last instar where there is a metamorphic change to the pupal stage. Establishment of ESTs for the three tissues described above will set up a reservoir for the discovery of novel genes for insect pest control.

We constructed cDNA libraries using mRNA isolated from different tissues and the cDNA clones are randomly selected and the inserts are sequenced in single-pass reads from 5’ or 3’ ends. These raw sequences are trimmed automatically and manually to generate high-quality sequences. The sequences are assembled, using the Phrap program, into unique contigs. Annotation of the contigs is performed against Swiss-Prot/TrEMBL protein knowledgebase (http://us.expasy.org). Classification of molecular functional, cellular component and biological process of the unique sequences is conducted using the Gene Ontology classification system.

The spruce budworm EST database is generated and can be searched by BLAST, Keyword, and Sequence ID. The database currently (June 30, 2003) contains 9000 high-quality unique sequences from different libraries.

ESTs are identified and selected for further sequencing and comparative and functional analysis. The ESTs will be used for analysis of the global expression of genes that may be involved in the interaction between the insect and the viruses and other pathogens using DNA microarrays.