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Title: Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations

item BIASI, ANTONIO - University Of Reggio Calabria
item Martin, Frank
item SCHENA, LEONARDO - University Of Reggio Calabria

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/2015
Publication Date: 1/17/2015
Citation: Biasi, A., Martin, F.N., Schena, L. 2015. Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations. Journal of Microbiological Methods. 110:61-67.

Interpretive Summary: This manuscript describes the development of molecular markers (simple sequence repeats) for investigating the population biology of Phytophthora nicotianae, a plant pathogen with a wide host range and distribution around the world. This species has been reported to cause disease on more than 255 plant genera in 90 families and is an important economic pathogen on tobacco (tobacco black shank disease), citrus (citrus root rot and gummosis) and ornamentals (root rot). The markers were developed by examining DNA sequence data from genomic sequencing projects and using comparative genomics to identify sequences for development of the technique. These markers will enable large-scale studies on the population biology and genetics of this important pathogen.

Technical Abstract: A large number of SSR loci were screened in the genomic assemblies of 14 different isolates of Phytophthora nicotianae and primers were developed for amplification of 17 markers distributed among different contigs. These loci were highly polymorphic and amplified from genetically distant isolates of the pathogen. Among these, nine were further validated using a multiplexed genotyping assay with differentially labeled primers (FAM or HEX) to allow for duplex PCR amplification. The use of reverse primers with a 5' PIG tail was important to increase the quality and reliability of the analyses. A total of 46 alleles were detected in five tester isolates of P. nicotianae representing the breadth of diversity in the species. Furthermore, a high incidence of heterozygosity was determined with two alleles detected in 67% of the primer/isolate combinations. Three different alleles where detected for a single locus/isolate combination, indicating variation in ploidy. These markers represent a valuable new tool for the characterization of populations of P. nicotianae.