Construction of a ‘unigene’ cDNA clone set by oligonucleotide fingerprinting allows access to 25 000 potential sugar beet genes

Abstract

SummaryAccess to the complete gene inventory of an organism is crucial to understanding physiological processes like development, differentiation, pathogenesis, or adaptation to the environment. Transcripts from many active genes are present at low copy numbers. Therefore, procedures that rely on random EST sequencing or on normalisation and subtraction methods have to produce massively redundant data to get access to low-abundance genes. Here, we present an improved oligonucleotide fingerprinting (ofp) approach to the genome of sugar beet (Beta vulgaris), a plant for which practically no molecular information has been available. To identify distinct genes and to provide a representative 'unigene' cDNA set for sugar beet, 159 936 cDNA clones were processed utilizing large-scale, high-throughput data generation and analysis methods. Data analysis yielded 30 444 ofp clusters reflecting the number of different genes in the original cDNA sample. A sample of 10 961 cDNA clones, each representing a different cluster, were selected for sequencing. Standard sequence analysis confirmed that 89% of these EST sequences did represent different genes. These results indicate that the full set of 30 444 ofp clusters represent up to 25 000 genes. We conclude that the ofp analysis pipeline is an accurate and effective way to construct large representative 'unigene' sets for any plant of interest with no requirement for prior molecular sequence data.

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

“…ESTs (21 321) representing 13 618 genes have been cloned from sugar beet (3 February 2005 release, http://compbio. dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=beet; Herwig et al, 2002;Bellin et al, 2007). There are 26 870 ESTs deposited in the GenBank (accessed on 13 November 2007), of which about 20% are estimated to represent defence, signal transduction and secondary product synthetic genes.…”

LNA-Modified Oligodeoxynucleotide Hybridization with DNA Microarrays Printed on Nanoporous Membrane Slides

“…In a classical protocol of oligofingerprinting, PCR products are spotted on the nylon membrane and hybridized with radioactively labeled oligoprobes [18]. The method has been further modified by using fluorescence detection and application of PNA-modified oligoprobes [19].…”

“…It has been shown that reduction of the probe length results in a reduced number of hybridizations necessary for production of the unique fingerprint. For example, only 50 7-mer probes are theoretically required to deliver the same sequence information on particular DNA clones, whereas application of the 8-mer probes requires using 220 oligodeoxynucleotides [18]. Reduction of the number of hybridizations results, in turn, in a drastic reduction of costs in terms of reagents and time consumption.…”

Generation, Annotation, Evolutionary Analysis, and Database Integration of 20,000 Unique Sea Urchin EST Clusters

“…A total of 107,283 clones were simultaneously analyzed by ONF as described earlier (Meier-Ewert et al 1998;Herwig et al 1999Herwig et al , 2002Poustka et al 1999;Clark et al 2001). To prevent overclustering by ONF, the clustering stringency was trained on the real representation of the cDNA clones representing 30 different genes, whose representation in the libraries was determined by backhybridization to the macroarrays of the used libraries.…”