Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Genome Sequencing & ESTs
            

THE CONSTRUCTION OF A DURUM WHEAT (TRITICUM TURGIDUM L. SSP DURUM) "TOTIPOTENT" LIBRARY FOR ISOLATION OF EXPRESSED SEQUENCES TAGS (ESTs)

¹ Metapontum Agrobios, S.S. Jonica106, Km 448.2, Metaponto (MT), 75010 Italy

² FIUOTECNICA, S.S. Jonica106, Km 448.2, Metaponto (MT), 75010 Italy

Sequencing of a cDNA library to generate expressed sequence tags (ESTs) is an effective tool for research activities such as gene discovery, generation of molecular markers and characterization of transcription patterns. With this purpose, we developed a method (patent pending) to prepare a "totipotent" cDNA library, from a tetraploid durum wheat cultivar Ofanto. The goal of this work is to generate a cloned set of cDNA fragments containing a representation of most of mRNA sequences from any starting material, regardless of the developmental stage in the original material. Poly(A+) RNA was extracted from etiolated seedlings, and reverse transcribed into sscDNA, which was cloned into the lambda Uni-ZAP XR vector (Stratagene) and a primary library of 2.3x10⁶pfu/µg of vector was obtained. To test the quality of the library 200 random clones were sequenced, analysed, assembled and were assembled into contigs using Sequencher^™ (Gene Codes Corporation). The BLAST similarity searches against NCBI EST sequence database revealed abundant messages for storage proteins (gliadins and glutenins), genes for proteins involved in starch metabolism, root genes, pre-anthesis spike genes, and endosperm ESTs. Approximately 20% of the ESTs showed homology to sequences of unknown function.