Single Pass cDNA Sequencing – A Powerful Tool to Analyse Gene Expression in Preparasitic Juveniles of the Southern Root Knot Nematode Meloidogyne Incognita

Abstract

Expressed sequence tags (EST) have been widely used to assist in gene discovery in various organisms (e.g., Arabidopsis thaliana, Caenorhabditis elegans, Mus musculus, and Homo sapiens). In this paper we describe an EST project, which aims to investigate gene expression in Meloidogyne incognita at the onset of parasitism. Approximately 1 000 5’-end sequence tags were produced from a cDNA library made of freshly hatched preparasitic second stage juveniles (J2). The EST were identified in the primary transformants of the cDNA library, and clustered into nine functional groups including (candidate) parasitism genes. A large fraction of the EST (45%) did not have a significant homologue in public databases and could not be clustered in a functional group. Sixty five percent of the EST that could be clustered into a functional group had homologues in other nematode species. EST were for virtually all parasitism related genes that have been cloned from M. incognita to date. In addition, several novel genes were tagged, including a xylanase and a chitinase gene. The efficiency of EST projects, which produce sequence data for thousands of genes in months time without any difficult pre-selections of mRNA pools, makes random sequencing of good quality cDNA libraries a superior method to identify candidates for parasitism related genes in plant-parasitic nematodes. The sequences in this paper are retrievable from Genbank with the accession numbers BE191640 to BE191741, BE217592 to BE217720, BE225324 to BE225598, BE238852 to BE239221, and BE240829 to BE240865.