Thesis (MSc)--Stellenbosch University, 2003. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53624 |
| Date | 12 1900 |
| Creators | Mkhize, Thokozani M |
| Contributors | Botha, F. C., Mansvelt, E. L., Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 132 pages : illustrations |
| Rights | Stellenbosch University |
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