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First published online November 20, 2002; 10.1105/tpc.004630

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The Plant Cell, Vol. 14, 2985-2994, December 2002, Copyright © 2002,
American Society of Plant Biologists

GENOMICS ARTICLE

A High-Throughput Arabidopsis Reverse Genetics System

Allen Sessions1,a, Ellen Burkea, Gernot Prestinga, George Auxb, John McElverb, David Pattonb, Bob Dietrichb, Patrick Hoa, Johana Bacwadena, Cynthia Koa, Joseph D. Clarkea, David Cottona, David Bullisa, Jennifer Snella, Trini Miguela, Don Hutchisona, Bill Kimmerly2,a, Theresa Mitzelc, Fumiaki Katagiria, Jane Glazebrooka, Marc Lawb and Stephen A. Goffa

a Torrey Mesa Research Institute, Syngenta, 3115 Merryfield Row, San Diego, California 92121
b Syngenta Biotechnology Incorporated, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709
c Syngenta Seeds Incorporated, 7240 Holsclaw Road, Gilroy, California 95020

1 To whom correspondence should be addressed. E-mail allen.sessions{at}syngenta.com; fax 858-812-1337

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ~100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.




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