The SM Lines were generated by Alain Tissier and his colleagues at the John Innes Centre.

Plasmid Construct and Selectable Marker

The constructs that is introduced into Arabidopsis ecotype Columbia via Agrobacterium vacuum infiltration are shown in Figure 1 (see below). The defective Spm (dSpm) element carries a phophilnothricin (PPr) resistance (BAR) gene which selects for T-DNA integration and for transposition reinsertion. A terminator region of the nopaline synthase (nos) gene at the 3’ end of theBAR gene occurs in the opposite orientation to the termination region of the Spm element. Thus, insertions in an intron of a mutagenized gene abolish the gene function.

Figure 1: T-DNA construct used for SM Lines. LB and RB, left and right borders, respectively; P, promoter driving the expression of the transposase (Spm, 35S, or AtDMC1 promoters); Spec, spectinomycin resistance gene for selection in bacteria; SU1, counterselectable marker.

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Excision Marker

The dSpm lies between the cauliflower mosaic virus 35S promoter and the ATG codon of a beta-glucuronidase (GUS) gene, which acts as an excision marker to monitor the activity of the element. The transposase gene fragment, coding for both the TnpA and TnpD proteins, was cloned under the control of a 35S promoter, the Spm promoter, or a meiosis-specific promoter from the AtDMC1 gene (constructs 8313, 8337, and 8353, respectively – refer to Methods in Tissier et. al., 1999 AND see also Klimyuk and Jones, 1997).

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Counterselectable Marker

The SU1 gene from Streptomyces griseolus encodes a cytochrome P450 that confers sensitivity to R7402, which is a sulfonylurea proherbicide from Du Pont (refer to O’Keefe et. al., 1994). The counterselection cassette with the SU1 gene was cloned between the left border and the transposase gene in the same orientation. The selectable genes were chosen for their ability to be screened on soil, thus avoiding the need to prepare sterile media and seeds.

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Primer Sequences

Spm31 (5'-GCTTGTTGAACCGACACTTTTAACATA AG-3')

Inv6-2 (5'-GCTAAGCACATACGTCAGAAACCATTATT-3')

Spm32 (5'-TACGAATAAGAGCGTCCATTTTAGAGTGA-3')

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Mapping of T-DNA Insertion Sites

Fragments flanking the T-DNA inserts can be amplified using thermal asymmetric interlaced (TAIL) PCR (as described by Liu et. al., 1995) with three rounds of amplification with the following nested primers:

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SLAT Line	SAIL Line