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Development of PCR primers for the specific amplification of unique DNA sequences of Peronospora sparsa
A dual culture of Peronosclerospora sorghi and sorghum callus was established as a model system for a dual culture of Peronospora sparsa on rose callus. Though the identity of the fungus could not be verified microscopically, it was verified by DNA amplification with PCR primers specific for P. sorghi, demonstrating that the primers could amplify fungal DNA from infected callus. A dual culture of P. sparsa and rose callus was sought to provide pure DNA for sequencing and primer development but could not be established using similar procedures. Five inoculation techniques including callus inoculation with a conidial suspension, callus inoculation by transfer of conidia, callus inoculation with sections of a sporulating rose leaf, callus inoculation by sporulating leaves in a sporulation chamber, and use of infected rose leaf sections as explants for callus initiation were attempted. Calli were incubated under conditions favorable to fungal growth for two months, but no P. sparsa infection developed. Sufficient DNA from P. sparsa was extracted from conidia on sporulating rose leaves that a dual culture of the fungus was not necessary. The ITS regions of the RDNA were amplified by various combinations of conserved ITS primers. These products were cloned into a pNoTA/T7 vector and transformed into E. coli cc-complementation cells. Inserts from transformants were sequenced by the Gene Technologies Lab, Texas A&M University, and aligned using the Sequencher program to deten-nine the complete sequence of the ITS regions of P. sparsa. This sequence of P. sparsa was compared to fungi with similar sequences as well as common saprophytes of rose, using the BLAST search provided by Genbank, to select putative PCR primers unique to P. sparsa. The primers were tested against DNA from rose leaves infected with P. sparsa, plasmid DNA containing an insert of the ITS region of P. sparsa, rose callus, a water control, and the control fungi Fusarium oxysporum, Epicoccum sp., Alternaria sp., Cladosporium sp., Botrytis sp., and Pestalotia sp. A nested PCR protocol was used in which DNA was first amplified with conserved ITS primers I and 4, diluted 1: I 000 with water, and amplified with specific primers to yield a visible product only for samples containing P. sparsa DNA.
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Includes bibliographical references: p. 43-46.
Issued also on microfiche from Lange Micrographics.
Ross, Sharon (1997). Development of PCR primers for the specific amplification of unique DNA sequences of Peronospora sparsa. Master's thesis, Texas A&M University. Available electronically from
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