PCR-based method for identifying a fusarium wilt-resistant genotype in plants
AssigneeClemson University
InventorsRalph A. Dean, Yi-Hong Wang
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Abstract
The present invention provides a sensitive assay for objectively determining the genotype of cucurbit plants, particularly species of melon, with respect to resistance or susceptibility to Fusarium wilt infection. The assay of the present invention uses a polymerase chain reaction to amplify sample DNA using either an AM or FM oligonucleotide primer pair. The PCR product which results from either primer pair differs in size, depending upon whether the template DNA was obtained from a plant susceptible or resistant to Fusarium wilt, permitting easy and rapid identification of plant genotype.
Claims18
A method for objectively identifying a genotype resistant to Fusarium wilt disease in a whole cucurbit plant, plant extract, or plant seed by using cycles of a polymerase chain reaction, said method comprising the steps of:
- (a) removing a sample of plant tissue from a cucurbit plant to be tested for a Fusarium-resistant genotype;
- (b) extracting genomic DNA from said sample for use as a DNA template;
- (c) replicating a desired region in the DNA template by binding a specific oligonucleotide primer set selected from the primer sets comprising Seq ID:
- 1 and Seq ID:2;
- and Seq. ID:3 and Seq ID:4 to said DNA template in a complimentary fashion, said primer set designed to give rise to a desired PCR product having a size different from the size of a product produced from amplifying undesired genomic DNA;
- (d) amplifying complimentary DNA by conducting the polymerase chain reaction from about 30 cycles to about 40 cycles to produce a PCR product;
- and
- (e) examining said PCR product to determine if said product is amplified from a Fusarium-resistant cucurbit genotype.
A method for objectively identifying a genotype susceptible to Fusarium wilt disease in a whole cucurbit plant, plant extract, or plant seed by using cycles of a polymerase chain reaction, said method comprising the steps of:
- (a) removing a sample of plant tissue from a cucurbit plant to be tested for a Fusarium-susceptible genotype;
- (b) extracting genomic DNA from said sample for use as a DNA template;
- (c) replicating a desired region in the DNA template by binding a specific oligonucleotide primer set selected from the primer sets comprising Seq ID:1 and Seq ID:2;
- and Seq. ID:3 and Seq ID:4 to said DNA template in a complimentary fashion, said primer set designed to give rise to a desired PCR product having a size different from the size of a product produced from amplifying undesired genomic DNA;
- (d) amplifying complimentary DNA by conducting the polymerase chain reaction from about 30 cycles to about 40 cycles to produce a PCR product;
- and
- (e) examining said PCR product to determine if said product is amplified from a Fusarium-susceptible cucurbit genotype.
Dependencies are inferred from the claim text.