Articles
ISOLATION AND CHARACTERISATION OF RESISTANCE GENE ANALOGS (RGAS) IN GRAPE
Article number
603_53
Pages
419 – 427
Language
English
Abstract
The class of R-genes encoding a Nucleotide Binding Site (NBS) and/or a Leucine Rich Repeat (LRR) region is responsible for a mechanism of resistance to pathogens in many crops.
The use of degenerate primers designed on widely conserved domains has proved to be a fast techinque for the identification of candidate R-genes in different species.
We isolated Resistance Gene Analogs (RGAs) of the NBS-LRR class from grape.
Eleven degenerate primers designed on four domains of plant R-genes were arranged in seven primer combinations and used for amplifying genomic DNA of Vitis amurensis and Vitis riparia. PCR-generated fragments arising from a multi-gene family were cloned into a plasmid vector.
A set of 452 clones were fingerprinted by either RFLP-analysis or SSCP-analysis of the inserts in order to find out unique clones before sequencing.
A total of 87 unique DNA sequences were blasted against GenBank accessions, leading to the identification of 80 grape RGAs characterised by the structural features of NBS-containing R-genes and clustered in 25 groups on the base of sequence similarity.
Further analysis focused on one sequence per each group.
The sequences were probed onto Southern blots of genomic DNA from grapevines either resistant or susceptible to downy mildew (RFLP analysis). One of the probes showed a clear-cut polymorphism between resistant and susceptible genotypes, thus confirming that NBS-sequences may lie in genomic regions conferring disease resistance in grape.
Nested primers were used to generate specific-tagged RGAs per each class.
Specific-RGAs were characterised by conformational polymorphism (SSCP analysis) in a panel of 56 Vitis genotypes carrying different levels of resistance and some of them in two populations segregating for resistance against downy mildew.
The use of degenerate primers designed on widely conserved domains has proved to be a fast techinque for the identification of candidate R-genes in different species.
We isolated Resistance Gene Analogs (RGAs) of the NBS-LRR class from grape.
Eleven degenerate primers designed on four domains of plant R-genes were arranged in seven primer combinations and used for amplifying genomic DNA of Vitis amurensis and Vitis riparia. PCR-generated fragments arising from a multi-gene family were cloned into a plasmid vector.
A set of 452 clones were fingerprinted by either RFLP-analysis or SSCP-analysis of the inserts in order to find out unique clones before sequencing.
A total of 87 unique DNA sequences were blasted against GenBank accessions, leading to the identification of 80 grape RGAs characterised by the structural features of NBS-containing R-genes and clustered in 25 groups on the base of sequence similarity.
Further analysis focused on one sequence per each group.
The sequences were probed onto Southern blots of genomic DNA from grapevines either resistant or susceptible to downy mildew (RFLP analysis). One of the probes showed a clear-cut polymorphism between resistant and susceptible genotypes, thus confirming that NBS-sequences may lie in genomic regions conferring disease resistance in grape.
Nested primers were used to generate specific-tagged RGAs per each class.
Specific-RGAs were characterised by conformational polymorphism (SSCP analysis) in a panel of 56 Vitis genotypes carrying different levels of resistance and some of them in two populations segregating for resistance against downy mildew.
Authors
G. Di Gaspero, G. Cipriani
Keywords
Vitis, genetics, breeding, disease resistance, molecular markers
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