The detection of cherry leaf-roll nepovirus and the use of molecular markers for germplasm identification in walnuts (Juglans regia L.)

Mkhize, Thokozani M (2003-12)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed.

AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om twee algemene opsporingstegnieke te kombineer: serologiese toetsstelle en genetiese vingerafdrukke om cherry leaf-roll nepovirus (CLRV) te eien en om In merkersisteem te ontwikkel wat okkerneut kiemplasma kan karakteriseer. Die opsporing van plant virusse is baie moeilik. As gevolg van kwarantyn vereistes, word daar beperkinge geplaas word op die invoer van plant materiaal vanuit die buiteland. Moderne tegnieke soos hierdie een wat op PKR berus, word benodig om te verseker dat CLRV nie in plantmateriaal teenwoordig is nie. In Stel inleiers is ontwerp wat In 430 bp nie-koderende homoloë area amplifiseer. Hiervoor is konsensus volgordes bestudeer en slegs die volgordes wat 98,5% homologie getoon het, is gekies. In vergelyking met ELISA was die sensitiwiteit van hierdie deteksie metode 100 maal beter. DNA volgordebepaling is op die resulterende fragment gedoen om die PKR produk te verifieer. AFLP tegnologie is gebruik om polimorfiese fraqmente vir 6 okkerneut kultivars en 'n onderstok te identifiseer en SCARs is uit hierdie fragmente ontwikkel. Die AFLP tegniek kon tussen al die okkerneut kultivars en die onderstok onderskei. Die omskakeling van die AFLP fragmente in SCAR merkers om sodoende In eenvoudige kragtige tegniek vir kultivar onderskeiding te ontwikkel, was egter nie suksesvol nie. Met die gebruik van 27 AFLP inleier kombinasies, kon polimorfiese fragmente van so hoog as 47.8% verkry word. Die rede hoekom omskakeling onsuksesvol was lê by die aard van die AFLP tegniek. Die SCAR inleiers is ontwikkel uit volyordes intern tot die AFLP inleiers, maar die spesifisiteit van die merkers het juis in die AFLP inleiers gelê en nie in die interne volgordes nie. In hierdie studie, met die gebruik van AFLP, is gevind dat okkerneut kultivars baie naby verwant is. Die AFLP inleierstelle wat gebruik is, het polimorfiese fragmente gelewer. Uit hierdie fragmente is 7 SCAR merkers ontwikkel. Daar is verwag dat die SCARs wat uit die AFLP merkers ontwikkel is, klein verskille tussen kultivars sou opspoor. Dit was egter net die Paradox SCAR merker wat die kultivars in twee groepe kon verdeel. Restriksie ensiem vertering met Rsalop die Chandler SCAR produkte het dieselfde bandpatrone as die van die Paradox SCAR produkte gelewer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53624
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