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Genetic variability of the avirulence gene AvrLm4-7 among Leptosphaeria maculans isolates by high resolution melting analysis.

Felipe Cevallos: Oklahoma State University

<div>The fungal pathogen, <em>Leptosphaeria maculans,</em> is an ascomycete that causes black leg disease of canola (<em>Brassica napus</em>) in Oklahoma and worldwide. Major-gene resistance involves the interaction between avirulence effectors in the pathogen and their corresponding resistance genes in the plant. <em>AvrLm4-7 </em>is an important avirulence effector that is recognized by two resistance proteins encoded by the genes <em>Rlm4</em> and <em>Rlm7</em>. A rapid detection, and discrimination tool for these effectors is needed for characterization of pathogen races. Previously reported internal primers AvrLm7Up1 and AvrLm7LOW2 were used for PCR and high resolution melting analysis. Melting temperatures profiles were determined for <em>L. maculans</em> isolates with different avirulence allele profiles. Some of the melting curves obtained using the internal primers were different from those previously reported. To analyze the entire <em>AvrLm4-7 </em>region, its reference nucleotide sequence was retrieved from NCBI-Genbank and aligned using MEGA 6. Two sets of external primers were designed using the software Primer3 (AvrLm47A and AvrLm47B). The melting curves obtained from the external region using AvrLm47A primers were similar among isolates with a functional <em>AvrLm4-7</em> gene and some isolates with a non-functional <em>AvrLm4-7</em> gene. Minor variations of the melting curves were detected in the external region using AvrLm47B primers. Most isolates with a non-functional <em>AvrLm4-7 </em>gene were not amplified by any set of primers. The obtained melting profiles allows the examination of the allelic variability among <em>L. maculans</em> isolates.</div>