Identification of insertion and point mutations in all Arabidopsis genes is a major goal of the Multinational Coordinated Arabidopsis 2010 project on functional genomics. Currently, the genome project uses three different mutational approaches that are based on the identification of chemically induced mutations with the TILLING technology (Colbert et al., 2001; McCallum et al., 2000a; McCallum et al., 2000b) and the isolation of insertion mutations by either transposons or the T-DNA of Agrobacterium. Transposon-tagged mutant populations carry insertions of the autonomous maize transposon En (ZIGIA En/Spm lines: Baumann et al., 1998; Wisman et al., 1998a; Wisman et al., 1998b), and stabilized inserts of the Suppressor–mutator (SLAT-lines, Tissier et al., 1999); En-I (ITS-lines, Aarts et al., 1995; Speulman et al., 1999; Speulman et al., 2000); and Ac/Ds (Bancroft and Dean, 1993; Bancroft et al., 1992; Fedoroff and Smith, 1993; Long et al., 1993; Long et al., 1997) two-element transposon tagging systems. Mutations are also induced by the tobacco retrotransposon Tto1 (Okamoto and Hirochika, 2000) and several other transposon constructs exploiting the enhancer-trap and Cre/Lox recombination technologies (Martienssen, 1998; Osborne et al., 1995; Smith et al., 1996; Sundaresan et al., 1995). In addition to the first widely distributed T-DNA insertion mutant collections (Azpiroz-Leehan and Feldmann, 1997; Feldmann, 1991; Koncz et al., 1992), new mutant populations are also available (with gene and promoter trap inserts) that drive specific expression of β-glucuronidase (GUS) and green fluorescent reporter proteins (GFP) (Devic et al., 1995; Goddijn et al., 1993; Kiegle et al., 2000; Topping et al., 1991); and with activator T-DNA tags that facilitate screening for dominant mutations (Weigel et al., 2000). Saturation T-DNA mutagenesis is now performed using in planta transformation (Bechtold et al., 1993; Clough and Bent, 1998) and exploited to identify gene mutations by direct sequencing of transposon and T-DNA insert junctions (Balzergue et al., 2001; Liu et al., 1995; Mathur et al., 1998; Okamoto and Hirochika, 2000; Parinov et al., 1999; Samson et al., 2002; Speulman et al., 1999; Tissier et al., 1999; Yephremov and Saedler, 2000; http://flagdb-genoplante-info.infobiogen.fr/projects/fst;http://genetrap.cshl.org;http://signal.salk.educgi-bintdnaexpress; http://www.jic.bbsrc.ac.uksainsbury-labjonathan-jonesSINS-databasesins.htm; http://www.nadii.com/pages/collaborations/garlic_files/GarlicAnalysis.html). The availability of the complete Arabidopsis genome sequence (Arabidopsis Genome Initiative, 2000) also supports the wide ranging application of PCR-based reverse genetic approaches. Several T-DNA and transposon insertion mutant collections have been used to prepare arrays of pooled PCR DNA templates to screen for mutations in known genes and gene families (Bouché and Bouchez, 2001; Galbiati et al., 2000; Krysan et al., 1996; Krysan et al., 1999; Mahalingam and Fedoroff, 2001; McKinney et al., 1995; Meissner et al., 1999; Parinov and Sundaresan, 2001; Thorneycroft et al., 2001; Winkler et al., 1998; Young et al., 2001). However, the need to identify the insertions by Southern hybridization still limits the efficiency of PCR-based mutant screening techniques.
To contribute to the identification of gene knockouts, we describe here an improved PCR technique that allows simple detection of amplified T-DNA insert junctions by agarose gel electrophoresis, and subsequent isolation and sequencing of the PCR products. To exploit this method we have generated a new collection of 90 000 T-DNA tagged Arabidopsis lines by in planta transformation with the Agrobacterium gene fusion vector pPCV6NFHyg (Koncz et al., 1989). Estimation of the average number of insertion loci and T-DNA copies indicates that the collection contains at least 116 100 independent insertion loci, over 50% of which carry concatenated T-DNA copies. To estimate the mutation frequency, 39 700 lines have been screened for T-DNA tags in 154 genes, which resulted in 87 confirmed knockouts in 73 genes. Based on this result, the technique is adjusted such that the entire population of 90 000 lines can be screened with 170 PCR reactions, leading to the identification of a sequenced T-DNA tag in a segregating M2 mutant family within 4 weeks.