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

[摘要] 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 forquarantine purposes on the importation of plant material from foreigncountries. Modern techniques such as a PCR based screening method forCLRV are required to ensure material do not harbour viruses. A primer pairwas designed to amplify a 430 bp non-coding homologous region. For thechoice of primers, consensus sequences were considered and areas wherethe sequence data shared 98.5% homology, were chosen. The sensitivity ofthis detection method was 100-fold higher when compared to the ELISA. ThePCR fragment was verified by nucleotide sequencing.AFLP technology was used to identify polymorphic fragments for 6 walnutcultivars and a rootstock, and SCARs were developed from AFLP specificbands. The AFLP technique distinguished all the walnut cultivars and therootstock. However, conversion of AFLP fragments to SCAR markers for thedevelopment of a simple robust technique for cultivar discrimination, was notsuccessful. Using 27 AFLP primer combinations, polymorphic fragments ashigh as 47.8% were scored. The reason for the lack of efficient conversionwas as the result of the AFLP technique. The SCAR primers were generatedfrom sequences internal to the AFLP primers but the specificity of the markerswas 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 thesefragments, 7 SCAR markers were developed. It was expected that theseSCARs derived from the AFLP markers would detect slight differencesbetween cultivars. The Paradox SCAR marker was the only one that coulddivide the cultivars into two groups. When Chandler SCAR products weredigested with the restriction enzyme Rsal, the same banding pattern as that ofParadox SCAR products was observed.

[发布机构] Stellenbosch University