Quantitative detection of Cucumber vein yellowing virus in susceptible and partially resistant plants using real-time PCR.
A method for the detection of Cucumber vein yellowing virus (CVYV) that combines reverse transcription with real-time PCR (SYBR((R)) Green chemistry) was developed using specific primers designed from a nucleotide sequence of the RNA polymerase gene (NIb) conserved among all the available CVYV strains. This method provided a linear assay over five to six orders of magnitude and reproducibly detected titres as low as 10(3) molecules of the target CVYV cDNA. Real-time PCR gave reproducible results for the quantification of CVYV in young leaves of susceptible and resistant cucumber landraces after mechanical inoculation. Significant differences in the starting amount of target cDNA were found between the analyzed genotypes, indicating differences in viral accumulation that correlated to their different levels of resistance. Real-time PCR results validated our previous findings using slot-blot hybridization, the dominance of the strong resistance to CVYV displayed by C.sat 10, and provided improved reliability and sensitivity of detection. This method has great potential in resistance breeding for germplasm screening, characterization of resistance mechanisms and genetic studies.
Picó
Belén
B
Center for the Conservation and Breeding of Agricultural Biodiversity (COMAV), Camino de Vera 14, Valencia 46022, Spain.
Sifres
Alicia
A
Nuez
Fernando
F
D052160:Benzothiazoles; D018553:Cucumis sativus / Q000821:virology*; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism; D007113:Immunity, Innate / Q000235:genetics*; D009930:Organic Chemicals / Q000378:metabolism; D010935:Plant Diseases / Q000235:genetics / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019176:Potyviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D018076:DNA, Complementary; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I