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Genetics, Vol. 168, 595-608, October 2004, Copyright © 2004
doi:10.1534/genetics.104.034785

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Construction and Evaluation of cDNA Libraries for Large-Scale Expressed Sequence Tag Sequencing in Wheat (Triticum aestivum L.)

D. Zhang*,1, D. W. Choi{dagger},2, S. Wanamaker{dagger}, R. D. Fenton{dagger}, A. Chin{dagger}, M. Malatrasi{dagger}, Y. Turuspekov{dagger}, H. Walia{dagger}, E. D. Akhunov{ddagger}, P. Kianian§, C. Otto§, K. Simons§, K. R. Deal{ddagger}, V. Echenique{ddagger},3, B. Stamova, K. Ross&, G. E. Butler**, L. Strader{dagger}{dagger}, S. D. Verhey{dagger}{dagger},4, R. Johnson{dagger}{dagger},5, S. Altenbach{ddagger}{ddagger}, K. Kothari{ddagger}{ddagger}, C. Tanaka{ddagger}{ddagger}, M. M. Shah§§, D. Laudencia-Chingcuanco{ddagger}{ddagger}, P. Han, R. E. Miller, C. C. Crossman{ddagger}{ddagger}, S. Chao,6, G. R. Lazo{ddagger}{ddagger}, N. Klueva*, J. P. Gustafson&, S. F. Kianian§, J. Dubcovsky{ddagger}, M. K. Walker-Simmons¶¶,7, K. S. Gill{dagger}{dagger}, J. Dvorák{ddagger}, O. D. Anderson{ddagger}{ddagger}, M. E. Sorrells&&, P. E. McGuire, C. O. Qualset, H. T. Nguyen*** and T. J. Close{dagger},8

* Department of Plant and Soil Science, Texas Tech University, Lubbock, Texas 79409
{dagger} Department of Botany and Plant Sciences, University of California, Riverside, California 92521
{ddagger} Department of Agronomy and Range Science, University of California, Davis, California 95616
§ Department of Plant Sciences, North Dakota State University, Fargo, North Dakota 58105
Genetic Resources Conservation Program, University of California, Davis, California 95616
& U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) Plant Genetics Research Unit, Department of Agronomy, University of Missouri, Columbia, Missouri 65211
** Arizona Genomics Institute, Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
{dagger}{dagger} Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164-6420
{ddagger}{ddagger} USDA-ARS Western Regional Research Center, Albany, California 94710-1105
§§ Department of Agronomy, University of Nebraska, Lincoln, Nebraska 68583-0915
¶¶ USDA-ARS, Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164
&& Department of Plant Breeding, Cornell University, Ithaca, New York 14853
*** Department of Agronomy, University of Missouri, Columbia, Missouri 65211

8 Corresponding author: Department of Botany and Plant Sciences, University of California, Riverside, CA 92521.
E-mail: timothy.close{at}ucr.edu

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.




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