Abstract The disease caused by Riemerella anatipestifer ( R. anatipestifer ) causes large economic losses to the global duck industry every year. Serotype-related genomic variation (such as in O-antigen and capsular polysaccharide gene clusters) has been widely used for the serotyping in many gram-negative bacteria. To date, there have been few studies focused on genetic basis of serotypes in R. anatipestifer . Here, we used pan-genome-wide association studies (Pan-GWAS) to identify the serotype-specific genetic loci of 38 R. anatipestifers strain. Analyses of the loci of 11 serotypes showed that the loci could be well mapped with the serotypes of the corresponding strains. We constructed the knockout strain for the wzy gene at the locus, and the results showed that the mutant lost the agglutination characteristics to positive antisera. Based on the of Pan-GWAS results, we developed a multiple PCR method to identify serotypes 1, 2, and 11 of R. anatipestifer . Our study provides a precedent for systematically analysing the genetic basis of the R anatipestifer serotypes and establishing a complete serotyping system in the future. Highlights R. anatipestifer serotype-specific locus was identified by Pan-GWAS for the first time. Molecular serotyping multiplex PCR was developed based on O-antigen biosynthesis gene clusters

Genetic loci of theR. anatipestiferserotype discovered by Pan-GWAS and its application for the development of a multiplex PCR serotyping method