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

[摘要] ENGLISH ABSTRACT: Potato is an important food crop that is constantly threatened by pests and diseases. Tocope with the food shortages in the water-poor subcontinent, Africa will have to increase itsyield through the use of disease- and pest-resistant varieties, and increase the area plantedby developing drought-tolerant varieties. In order to introduce resistant or stress-tolerantgenes, either by classical breeding or genetic engineering, genome analyses with molecularmarkers are needed to identify and track such genes. Partially sequenced cDNA clones orexpressed sequenced tags (ESTs), play an important role in genome analysis. ESTs can beused 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 single-strandedconformation polymorphism (SSCP) analysis to detect polymorphisms.An oligo(dT)-primed cDNA library was constructed from young potato leaves. About 3000clones were produced, using a plasmid vector. The quality of the library was determined bymeans of PCR and sequencing. Forty-eight ESTs were produced and based uponsimilarities to characterised sequences in electronic databases, 37 cDNA sequences couldbe 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 13primer pairs could detect polymorphisms in SSCP analyses and between two and six uniqueprofiles were amplified per assay. Five of the polymorphic STSs were characterised bysequencing. It was found that single nucleotide polymorph isms gave rise to the variation forthree markers and the variations for the remaining two markers were due to insertions anddeletions.Since the STS primers were derived from partially sequenced cDNA clones, they have theadvantage of providing not only a marker, but also a tag for a gene. In addition it was shownthat most of these markers could also be used in other Solanaceae species. Nine markerscould detect variations between six different species by standard agarose gelelectrophoresis.In summary, useful PCR-based markers, which are quick and easy to analyse, have beendeveloped for potato and can find application in the genome analysis of potato as well asother Solanaceae species.

[发布机构] Stellenbosch University