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Accueil du site → Master → Afrique du Sud → 1999 → Sequence-tagged sites of cDNA clones in Solanum tuberosum and their evaluation as molecular markers in solanaceae species

Stellenbosch University (1999)

Sequence-tagged sites of cDNA clones in Solanum tuberosum and their evaluation as molecular markers in solanaceae species

Liebenberg J

Titre : Sequence-tagged sites of cDNA clones in Solanum tuberosum and their evaluation as molecular markers in solanaceae species

Auteur : Liebenberg J

Université de soutenance : Stellenbosch University

Grade : Master of Science MSc (Genetika) 1999

Résumé
Potato is an important food crop that is constantly threatened by pests and diseases. To cope with the food shortages in the water-poor subcontinent, Africa will have to increase its yield through the use of disease- and pest-resistant varieties, and increase the area planted by developing drought-tolerant varieties. In order to introduce resistant or stress-tolerant genes, either by classical breeding or genetic engineering, genome analyses with molecular markers are needed to identify and track such genes. Partially sequenced cDNA clones or expressed sequenced tags (ESTs), play an important role in genome analysis. ESTs can be used to tag genes and are a common source for the development of sequence-tagged site (STS) markers. In this study cDNA-based STS marker technology is combined with singlestranded conformation polymorphism (SSCP) analysis to detect polymorphisms. An oligo(dT)-primed cDNA library was constructed from young potato leaves. About 3000 clones were produced, using a plasmid vector. The quality of the library was determined by means of PCR and sequencing. Forty-eight ESTs were produced and based upon similarities to characterised sequences in electronic databases, 37 cDNA sequences could be assigned putative identities. STS oligonucleotide primers were developed from the ESTs, and were subsequently used to screen a panel of 10 potato cultivars. Twelve of the 13 primer pairs could detect polymorphisms in SSCP analyses and between two and six unique profiles were amplified per assay. Five of the polymorphic STSs were characterised by sequencing. It was found that single nucleotide polymorphisms gave rise to the variation for three markers and the variations for the remaining two markers were due to insertions and deletions. Since the STS primers were derived from partially sequenced cDNA clones, they have the advantage of providing not only a marker, but also a tag for a gene. In addition it was shown that most of these markers could also be used in other Solanaceae species. Nine markers could detect variations between six different species by standard agarose gel electrophoresis. In summary, useful PCR-based markers, which are quick and easy to analyse, have been developed for potato and can find application in the genome analysis of potato as well as other Solanaceae species.

Présentation (NRF)

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