Alan Burbidge

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.

Identification of the tomato ABA-deficient mutant sitiens as a member of the ABA-aldehyde oxidase gene family using genetic and genomic analysis

The sitiens (sit) wilty mutant of tomato (Solanum lycopersicum L.) is deficient in functional enzyme activity at the final step in abscisic acid (ABA) biosynthesis. The biochemical lesion is believed to be an impaired aldehyde oxidase (AO). Molecular mapping using various interspecies crosses has previously shown sit to co-map with a cluster of unresolved RFLP markers on the short arm of chromosome 1. Here, the utilisation of bridging lines to produce interspecies mapping populations involving a self-compatible S. peruvianum accession (LA2157) allowed the fine mapping of sit within this cluster. Identification of a novel AO gene, within the region now known to contain the sit locus, was confirmed by analysis of the tomato whole genome shotgun sequence assembly. This novel AO protein shares 76-78% identity at the amino acid level with the previously characterised tomato AO proteins. The DNA sequence of this putative sit gene was characterised in wild type and in two allelic sit mutants (sit and sitw): changes in DNA sequence were identified in these mutant alleles that cause a truncation of exon 2 and the deletion of exon 7, respectively. These results establish the identity of the tomato sit gene and are consistent with its proposed function of encoding the ABA aldehyde oxidase apoenzyme.

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modifiedDs transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsisthaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.

Re-orientation and integration of the classical and interspecific linkage maps of the long arm of tomato chromosome 7

Abstract To obtain reliable classical and integrated interspecies maps of the long arm of chromosome 7 of tomato, detailed mapping work was undertaken and several phenotypic and molecular markers were assigned loci on both maps to provide reliable cross-reference points. To maximise the value of the new maps, pair-wise segregation data for classical genetic markers from the literature were included, based on large segregating populations with readily scorable phenotypes. In addition, to increase confidence in these maps, introgression lines were used to confirm important map locations. The revised classical map is based on two- and three-point test-cross data from a number of F2 and BC1 mapping populations. The integrated interspecies map is based on F2 mapping populations derived from crosses of Lycopersicon esculentum with Lycopersicon pennellii (LA716). The genetic analyses for both maps were performed using the computer package JoinMap. The revised composite classical map indicates that some of the map positions reported in the literature are incorrect. The linear order of the classical markers common to both the revised classical and integrated interspecies maps are in complete agreement. Production of the integrated interspecies map resulted in re-orientation of the existing molecular map.

Strategies for targeted transposon tagging of ABA biosynthetic mutants in tomato.

The ABA biosynthetic pathway has been studied in detail and the steps impaired in some ABA-deficient mutants are known. However, little is known of the molecular control mechanisms regulating ABA production in planta. A direct route for improving our understanding of these mechanisms is to transposon tag and clone the wild-type counterparts of the ABA mutant alleles. On the basis of the observation that maize transposons move preferentially to linked sites in both homologous and heterologous systems and in doing so disrupt gene function, a targeted transposon mutagenesis strategy is being developed towards cloning ABA biosynthetic genes from tomato. The possibility of using marker genes to identify T-DNA insertion sites in selected parts of the genome has been examined and compared with an inverse PCR/RFLP approach to mapping T-DNAs.