The AT population was generated at the John Innes Centre in the UK.

The En-I (Spm-dSpm) system for maize (Zea mays) is an efficient means for heterologous transposon tagging in Arabidopsis. Two features of the system make it an attractive tool for the selection of stable transposants: this first is the high frequency of independent transpositions achieved using this system; and the second is its ability to transpose to unlinked sites, rendering selections for segregated transposition efficient

Construct

The maize En-I transposon system is used to construct vectors which were suitable for developing collections of stable I transposon insertions that could act as activation tags in plant genomes. Figure 1 shows an activation tag construct that consists of three main components: the En (Spm) transposase coding sequence under the control of the CaMV 35S promoter and terminator sequences; a mobile, non-autonomous I (dSpm) component housing a tetramer of the CaMV 35S enhancer and BAR gene between the terminal inverted repeats denominated by activating I element (AIE); and the negatively selectable marker SU1, adjacent to the transposon components within the T-DNA.

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Figure 1: Schematic representation of the construct used for plant transformation (click on the construct to enlarge). Relevant EcoRI sites used for the molecular analysis are indicated. LB, Left border; RB, right border; 35SP, 35ST, CaMV 35S promoter and terminator, respectively; EnTPase, En immobile transposase source; ILtir, IRtir, I-element left and right terminal-inverted repeat, respectively; 4 Enh., tetramer of the CaMV 35S enhancer; Pnos, Tnos, promoter and terminator sequences from the nopaline synthase gene, respectively; SSU5’, SSU3’, promoter and transit signal peptide to the chloroplast and terminator of the small subunit of Rubisco gene, respectively. The gene specific probes (BAR and SSU3’) used for blot hybridization are indicated as bars above or below the figure.

The transposon construct was introduced by A. tumefaciens-mediated transformation into the Arabidopsis ecotypes Wassilewskija (Ws) and Landsberg erecta (Ler).

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Selectable Markers

This system makes use of the positive selectable marker BAR (refer to De Block et. al., 1986; Thompson et. al., 1986) which confers resistance to the herbicide Basta. The negative selection marker is the SU1 gene (refer to O’Keefe et. al., 1994) which converts the pro-herbicide R7402 into the herbicide sulfonylurea inhibiting or reducing the growth of plants that contain it. The selectable genes have been chosen to select for stable transpositions because of their ability to screen on soil, thus avoiding the need for sterile media and seeds.

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Isolation of the flanking sequences

To characterize insertions in different mutants, genomic DNA is used to isolate fragments adjacent to the activating I elements (AIEs) by thermal asymmetric interlaced (TAIL)-PCR (refer to Liu et. al., 1995; Liu and Whittier, 1995; Tsugeki et. al., 1996).

The specific nested primers for the I element are:

Int2 (5'-CAGGGTAGCTTACTGATGTGCG-3') for the first TAIL-PCR;

Irj-201 (5'-CATAAGAGTGTCGGTTGCTTGTTG-3') for the second TAIL-PCR;

and DSpm1 (5'-CTTATTTCAGTAAGAGTGTGGGGTTTTGG-3') (refer to Tissier et. al., 1999) for the third thermal asymmetric interlaced-PCR

A fourth primer, the I terminal-inverted repeat 3 (5’-CTTACCTTTTTTCTTGTAGTG-3’) is used to directly sequence the obtained PCR products with high specificity. The sequence of the flanking DNA needs to be compared against the Arabidopsis database using BLASTN This enables the positioning of the insert in the genome and its context with adjacent annotated genes.

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Insert Information	ET Line