Bristol, United Kingdom
University of Bristol A small but significant maize functional genomics facility --Edwards, D, Barker, G, Wilson, I, Grimmer, M and Edwards KJ   High throughput transposon tagging. We have produced a variety of transposon mutagenesis resources based on Robertson's Mutator (Mu) transposable elements. Currently over 700 amplified Mu-tagged insertion sequences are available on a blast searchable database MuDB and seed harbouring these insertion events are available on request (http://www.cerealsdb.uk.net/). In 1998, we developed a classic PCR screen for mutants within known maize genes; however, this has now been superseded by the MuArray. Muarray has been developed for the rapid identification of maize plants harbouring Mutator transposon insertions within specific genes. Mu-flanking sequences have been amplified from over eight thousand individual Mu-active plants (representing over 400,000-insertion events) using an optimised MuAFLP protocol. These transposon-flanking sequences have been arrayed at a high density onto glass slides, with each microarray feature representing an individual Mu-active maize plant. Hybridisation of these arrays with Cy dye labelled genes identifies plants with Mu-insertions within these genes. Knockout plants are being characterised using a range of physiological, biochemical and molecular methods including gene expression microarrays to provide an insight into gene function. This resource is available for screening by the international maize community by contacting [email protected] or via our web site www.cerealsdb.uk.net

EST-based SNP discovery. We have developed a SNP detection program to identify putative polymorphisms between orthologous and parologous sequences from expressed sequence databases. By calculating SNP scores based on redundancy and co segregation of haplotypes, we can differentiate between errors due to sequencing and true genetic polymorphisms. This has advantages in throughput and accuracy over previous methods, which relied on the analysis of original sequence trace files. This program has been applied initially to the >100 000 expressed maize sequences available in the public databases and will be used to identify putative polymorphisms within expressed sequences derived from the Bristol-based IGF program and other public wheat sequence repositories. Please contact Dave Edwards for further details.

Microsatellite-based SNP discovery. By characterising the flanking regions of 52 sequence tagged microsatellites from 11 Zea mays inbred lines we have identified a total of 324 sequence polymorphisms. These polymorphisms consisted of both single nucleotide polymorphisms and indels in a ratio of approximately two to one. Interestingly, the level of sequence variation within the flanking regions of microsatellites linked to expressed sequence tags appears to be lower than microsatellites that were unlinked to expressed sequence tags. Conversion of the SNPs into allele specific oligonucleotides followed by covalent binding to glass slides allowed the sequence polymorphisms to be used in a simple hybridisation-based genotyping procedure. This procedure allowed us to discriminate between different inbred lines and allowed variations within a single inbred to be identified. The sequence information generated and the assay developed could be used as a starting point for the further development of a non-gel based, multi-locus, multi-allele screen for large-scale maize genotyping (Mogg et al. In press).

ZEASTAR: A Functional Blueprint for the Zea mays Endosperm Cell factory. In March 2001 the Bristol lab began to co-ordinate a 10 partner EU Framework V programme designed to generate a set of resources for studying Flint-maize grain development. These resources will include numerous cDNA libraries from key stages in endosperm development; high-density EST insert arrays, a proteomics-based description of the endosperm and numerous transposon tagged lines. It is our hope that by characterising the Flint endosperm to this level we will be able to provide both further research materials for the academic laboratories involved and material for European-based plant breeders and food processors.
 
 

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