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

dc.contributor.advisorWarnich, L.en_ZA
dc.contributor.advisorPeeters, A. V.en_ZA
dc.contributor.authorLiebenberg, Junitaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Chemistry & Polymer Science.
dc.date.accessioned2012-08-27T11:34:30Z
dc.date.available2012-08-27T11:34:30Z
dc.date.issued1999-12
dc.identifier.urihttp://hdl.handle.net/10019.1/51422
dc.descriptionThesis (M.Sc.) -- University of Stellenbosch, 1999.
dc.description.abstractENGLISH ABSTRACT: 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 single-stranded 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 polymorph isms 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.
dc.description.abstractAFRIKAANSE OPSOMMING: Die aartappel is 'n belangrike voedselgewas wat gedurig deur peste en siektes bedreig word. Om die voedseltekorte in die droogtegeteisterde subkontinent te oorkom, moet Afrika deur die gebruik van weerstandbiedende varieteite die oes verhoog, asook die area onder verbouing vergroot deur droogtetolerante varieteite te ontwikkel. Om die weerstands- en droogtebestande gene oor te dra, deur klasieke planteteelt of genetiese ingenieurswese, is genoomanalises met merkers nodig om gene te identifiseer en oordrag daarvan te volg. Die volgordes van cDNA klone of ESTs ("expressed sequenced tags"), speel 'n belangrike rol in genoomanalise. ESTs kan gebruik word om gene te merk en hulle dien as bran vir die ontwikkeling van STS ("sequence-tagged site") merkers. In hierdie studie is cDNAgebaseerde STS merkertegnologie met SSCP ("single-strand conformation polymorphism") analise gekombineer om polimorfismes waar te neem 'n cDNA-biblioteek van jong aartappelblare is vervaardig met oligo(dT)-inleiers. Ongeveer 3000 klone is geproduseer deur van 'n plasmied vektor gebruik te maak. Die kwaliteit van die biblioteek is bepaal met behulp van PKR en volgordebepaling. Ag-en-veertig ESTs is geproduseer en volgens ooreenkomste met gekarakteriseerde volgordes in elektroniese databasisse, is 37 cDNA volgordes voorlopige gerdentifiseer. STS inleiers is ontwerp vanaf die ESTs en is gebruik om 'n paneel van 10 aartappelkultivars te amplifiseer. Twaalf van die 13 inleierpare kon polimorfismes waarneem in SSCP analises en tussen twee en ses unieke profiele is per merker geamplifiseer. Vyf van die polimorfiese STS'e is deur volgordebepaling gekarakteriseer. Daar is gevind dat enkel nukleotied polimorfismes verantwoordelik was vir die variasie van drie van die merkers en die verskille van die oorblywende twee merkers was as gevolg van insersies en delesies. Aangesien die merkers van cDNA-kloonvolgordes afgelei is, het dit die voordeel dat dit nie slegs 'n merker nie, maar ook 'n geen vir 'n kaart verskaf. Daar is gevind dat die merkers ook in ander Solanaceae spesies gebruik kan word. Met nege merkers is variasie tussen ses verskillende spesies met agarose jelelektroforese waargeneem. Opsommend kan gese word dat nuttige PKR-gebaseerde merkers, wat maklik en goedkoop ge-analiseer kan word, vir die aartappel ontwikkel is en toegepassing in die genoomanalise van aartappel en ander Solanaceae kan vind.
dc.format.extent88 pages : ill.
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch University
dc.subjectPotatoes -- Disease and pest resistance -- Genetic aspectsen_ZA
dc.subjectGenetic markersen_ZA
dc.subjectSolanaceae -- Geneticsen_ZA
dc.subjectDissertations -- Geneticsen_ZA
dc.titleSequence-tagged sites of cDNA clones in Solanum tuberosum and their evaluation as molecular markers in Solanaceae speciesen_ZA
dc.typeThesisen_ZA
dc.rights.holderStellenbosch University


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