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. 2002 Dec;14(12):2985-94.
doi: 10.1105/tpc.004630.

A high-throughput Arabidopsis reverse genetics system

Allen Sessions  1 , Ellen Burke, Gernot Presting, George Aux, John McElver, David Patton, Bob Dietrich, Patrick Ho, Johana Bacwaden, Cynthia Ko, Joseph D Clarke, David Cotton, David Bullis, Jennifer Snell, Trini Miguel, Don Hutchison, Bill Kimmerly, Theresa Mitzel, Fumiaki Katagiri, Jane Glazebrook, Marc Law, Stephen A Goff
Affiliations

A high-throughput Arabidopsis reverse genetics system

Allen Sessions et al. Plant Cell. 2002 Dec.

Abstract

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 asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 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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Figures

Figure 1.
Figure 1.
Agarose Gel Analysis of Secondary mTAIL-PCR Products from 48 Lines. Size standards are shown at left, with band sizes indicated in kb. Note that for individual samples, the lower molecular mass bands generally are present in relatively greater amounts than larger products.
Figure 2.
Figure 2.
Sequence Analysis of Left Border mTAIL-PCR Products from One Insertion Line. (A) Scheme of BLAST hits generated for sequence from the plant on plate 1154, well E08, using the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/blast/Blast). The three top hits are color coded for alignment scores, as shown at top. Positions 32 to 68 align with left border T-DNA sequences (green), positions 110 to 315 align with an Arabidopsis chromosome 5 sequence, and positions 338 to 651 align with a chromosome 1 sequence. (B) Regions of the electropherogram of the sequence in (A), with regions denoted by colored bars as in (A). Note that 5′ sequence has an overall higher signal intensity than 3′ sequence and includes lower amplitude signals from the sequence of an additional mTAIL-PCR fragment. This second sequence is obscured by the higher amplitude sequence until base 340.
Figure 3.
Figure 3.
Chromosomal Distribution of Predicted Insertions. Transcription unit insertions are shown in maroon, promoter insertions are shown in blue, and intergenic insertions are shown in green. The number of insertions per 50-kb interval is plotted as peaks off of the sides of each chromosome. The scale is indicated by dashed lines that demark intervals of 100 hits, with a maximum of 250. Numbers at peaks greater than 250 indicate value.
Figure 4.
Figure 4.
Insert Confirmation Using PCR with T-DNA Left Border and Gene-Specific Forward and Reverse Primers. The gene-specific primers were 5′-TTTTCACGATTCTTCTAGAC-3′ (Gf; forward) and 5′-AATAACCTGTTTGGCAAGAG-3′ (Gr; reverse). LB3, left border. (A) Scheme of At4g39030 (EDS5/SID1) and the predicted insertion in line 1255_E09, which is predicted to lie at position 841 in the first intron. (B) PCR analysis of the predicted insertion in wild-type (wt) Columbia (negative control) and 10 progeny plants of line 1255_E09. Products of PCR using the primer sets Gf/Gr and Gr/LB3 were run in adjacent lanes for each sample. Plants 1, 3, 6, 8, and 10 are hemizygous for the insertion, whereas plants 4, 5, and 7 are homozygous for the insertion.
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