Plant Genome IV Conference Plant Genome IV Conference
Our goal is to isolate the Mi1 root-knot nematode-resistance gene first identified in Lycopersicon peruvianum then introgressed into tomato, L. esculentum. After finding recombinants in L. esculentum and in L. peruvianum which localized Mi1 to a 100 kb region, we isolated bacterial artificial chromosome (BAC) subclones of this region to test for functional complementation in transgenic plants. A BAC library was prepared from a 500 kb YAC (Keygene, unpublished) which includes the entire Mi1 region. The library had 2800 BAC clones with average insert size of about 50 kb, or about 6 yeast-genome-equivalents. 5 BACs that span the region to which Mi1 maps were identified by hybridization to two probes which flank Mi1, and to additional interior probes. Selectable markers and T-DNA border sequences were added to our BACs by Cre/Lox mediated recombination to pAL221 (Alan Lloyd, unpublished). Unfortunately, while the resultant plasmids were stable in E. coli, deletion of variable amounts of the BAC inserts occurred upon conjugation into recA Agrobacterium strain AGL1 by 3-way mating. Nonetheless, a BAC contig in Agrobacterium is being constructed to make transgenic tomato tobacco, and Arabidopsis, which will be tested for nematode resistance.