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The Ncm-1 gene for resistance to Cucumber mosaic virus in yellow lupin (Lupinus luteus): molecular studies and marker development

Li, Dora (2012) The Ncm-1 gene for resistance to Cucumber mosaic virus in yellow lupin (Lupinus luteus): molecular studies and marker development. PhD thesis, Murdoch University.

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Cucumber mosaic virus (CMV) is an important virus pathogen of lupins in Australia which causes serious yield losses of up to 60% in epidemic years. In commercially grown lupin (Lupinus angustifolius and L. luteus) crops CMV is spread non-persistently by aphid vectors, but it can also be seed borne and this extends virus infection into successive generations. Resistance to CMV has been identified in L. luteus cv. Wodjil and is the conferred by the Ncm-1 gene. The aims of this research were to study the Ncm-1 gene in order to gain a better understanding of resistance in yellow lupin, and to develop a molecular marker linked to Ncm-1 for use in marker assisted selection.

Previously published data by Jones et al (1996) identified Ncm-1 as being a single dominant resistance gene, however, phenotypic analysis of CMV infection in a segregating L. luteus mapping population in this thesis was consistent with the Ncm-1 gene being a dominant gene modified by at least one other minor gene. The polygenic nature of CMV resistance in this genetic background was further supported by AFLP analysis which identified one major and one minor QTL associated with resistance.

A PCR based approach, using degenerate primers designed on conserved disease resistance protein motifs, was used to identify resistance gene analogues (RGA) in L. luteus. Comparative analysis revealed that RGAs isolated from L. luteus were members of the TIR-NBS-LRR class of R proteins and were similar to the TMV resistance gene N identified in tobacco and the RT4-4 CMV resistance gene from pepper. Extensive comparative analysis using the genomes of model species (including Medicago truncatula, Glycine max, Arabidopsis thaliana and Lotus japonicus) was explored and validated the assignment from L. luteus RGAs to the category of candidate gene for CMV resistance. The RGAs identified in L. luteus were found to be highly conserved in both the CMV resistant and susceptible varieties tested. SNPs which resulted in non-synonymous mutations were identified using cDNA based 5’ RACE and used to develop a single nucleotide primer extension (SNuPE) assays for MALDI-ToF mass spectrophotometric analysis. As SNuPE is based on the allele specific extension of a single nucleotide, genotyping is highly accurate and provides co-dominant information. Two SNuPE assays were developed based on the RGAs isolated and validated on bulked samples from two L.luteus populations segregating for CMV resistance. One assay, SNuPE A267→C was found to associate with CMV resistance. This co-dominant assay is the first of its kind reported for yellow lupin.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Jones, Mike and Appels, Rudi
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