Proceedings 1st Solanaceae Genome Workshop, 19-21 Sept. 2004
Wageningen, The Netherlands, pp.51.

The Tobacco Genome Initiative: A Gene Discovery Platform

Charles H. Opperman1, Steven A. Lommel1, Mark Burke1, Joanna Carlson1, Carol George1, Sandy Gove1, T. D. Houfek1, Stuart Jefferys1, Sam Kalat1, Ron King Jr. 1, Jennifer Levin1, P. C. Little1, Amy Lumpkin1, T. Ross1, Aimee Salstead1, Betsy Scholl1, Bryon Sosinski1, P. Stephens1, S. H. Zekanis1, J. Ferlinger1, Nate Lakey2, Joey Bidell2, Arief Budiman2, Ferruccio Gadani3, and Alec Hayes3.

1 College of Agriculture and Life Sciences, NC State University, Raleigh, NC 27695
2 Orion Genomics, LLC, St Louis, MO, 63108
3 Philip Morris USA, Research Center, Richmond, VA 23261, USA

Abstract

The Tobacco Genome Initiative (TGI) uses large-scale DNA sequencing and bioinformatics to reveal the genes of Nicotiana tabacum. Ultimately, plant genomics may lead to the genetic modification of tobacco or may speed up conventional breeding with the potential to assist in achieving the goal of reducing the harm associated with cigarette smoking and enhancing the performance of tobacco as a crop in different environments.

Moreover, cultivated tobacco is member of the agriculturally important Solanaceae family, which includes tomato, potato, eggplant and pepper crop plants that may benefit from gene discovery in tobacco. N. tabacum, an amphiploid species (2n=48) likely resulting from an interspecific cross between N. sylvestris (2n=24) and N. tomentosiformis (2n=24), has a very large genome size compared with other cultivated solanaceous plants. At approximately 4.5 billion base pairs, it is 1.5 times the size of the human genome, with the vast majority of the DNA occurring as highly repetitive sequences. We are employing a methyl filtration library approach to identify gene-rich regions in N. tabacum in order to expedite the gene discovery.

To date, we have sequenced 305,280 lanes of a methyl filtered library and observe a nearly 10-fold increase in gene discovery in filtered vs. non-filtered libraries. In addition, we constructed a BAC library (9.7-fold genome coverage) and initiated BAC-end sequencing as part of our physical mapping program. We have fingerprinted 45,331 BAC clones using the four dye protocol and have sequenced 18 BAC clones to ~5X coverage. We also performed EST sequencing from various Nicotiana libraries, and to date, we have sequenced over 60,000 ESTs.