salmonis was the leading cause of mortality in farmed Atlantic salmon and rainbow trout ( Sernapesca, 2020b). In Chile, the bacterium is widely distributed and costs the salmon farming industry up to US$700 million per year ( Maisey et al., 2017). Piscirickettsia salmonis is a facultative intracellular Gram-negative bacterium ( Fryer and Hedrick, 2003 Mauel et al., 2008) and the etiological agent of Piscirickettsiosis, a systemic infection that affects all farmed salmon species ( Mauel et al., 2008). Finally, the genome-based multiplex PCR protocol presented in this study is a rapid and affordable alternative to classical sequencing of PCR products and analyzing the length of restriction fragment polymorphisms. This method also identified the pathogen in field samples infected with LF-89 or EM-90 strains, which is not possible with other methods currently available. The Multiplex-1 prototype was chosen for reliable genotyping because of differences in annealing temperatures and respective reaction efficiencies. salmonis (LF-89 and EM-90), with 253 and 291 unique sequences for LF-89 and EM-90 genogroups, respectively. A total of 1,801 clusters of coding sequences were shared among all tested genomes of P. The results revealed 2,322 sequences shared between 35 LF-89 genomes, 2,280 sequences in the core-genome of 38 EM-90 genomes, and 331 and 534 accessory coding sequences each genogroup, respectively. The present study reports the development of a multiplex PCR for genotyping LF-89 and EM-90 genogroups based on comparative genomics of 73 fully sequenced P. salmonis genogroups in field samples is vital for veterinary diagnoses and understanding the population structure of this pathogen. Therefore, the development of methods for quick genotyping of P. The monophyletic genogroup LF-89 and a divergent genogroup EM-90 are responsible for the most severe Piscirickettsiosis outbreaks in Chile. It provokes Piscirickettsiosis outbreaks in the marine phase of salmonid farming, resulting in economic losses. Piscirickettsia salmonis is a bacterial pathogen that severely impact the aquaculture in several countries as Canada, Scotland, Ireland, Norway, and Chile. 10Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.9Beagle Bioinformatics, Santiago, Chile.8Instituto Vandique, João Pessoa, Brazil.7Laboratorio de Ecopatología y Nanobiomateriales, Departamento de Biología, Facultad de Ciencias Naturales y Exactas, Universidad de Playa Ancha, Valparaiso, Chile.6Laboratorio de Biología de Redes, Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile.5Programa de Doctorado en Genómica Integrativa, Vicerrectoría de Investigación, Universidad Mayor, Santiago, Chile.4Centro de Modelamiento Molecular, Biofísica y Bioinformática – CM2B2, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile.3Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad Santo Tomás, Santiago, Chile.2Interdisciplinary Center for Aquaculture Research (INCAR), University of Concepcion, Concepción, Chile.1Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile.Levipan 7 Denise Haussmann 3 Jaime Figueroa 1,2 Maria Cecilia Rauch 1 Vinicius Maracaja-Coutinho 4,8,9* Alejandro Yañez 2,10* Eduardo Martinez-Hernandez 4,5,6† Héctor A.
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