Abstract:Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects shrimp. Although EHP infection does not cause widespread mortality in shrimp, it significantly slows down their growth and makes them more susceptible to other pathogens, resulting in significant economic losses. Thus, it has emerged as a major threat to the shrimp farming industry. The successful decoding of its genome sequence and genetic code has provided a robust foundation for various fundamental research endeavors. These endeavors provide valuable data essential for the identification and exploration of important functional genes in EHP. Here, we aimed to study the basic information regarding microsatellites in the EHP genome and provide theoretical support for molecular marker development, population evolution, and genetic diversity studies of EHP and its related species. We used bioinformatics to analyze the quantity, distribution, and frequency of SSR (simple sequence repeat) sites in the whole EHP genome and compared them with the corresponding properties of five other microsporidian fungal species. A total of 794 SSRs were identified in the EHP genome, with a total length of 21773 bp, accounting for 0.72% of the genome. The average length of SSRs was 27.43 bp, with a relative abundance of 262 loci/Mb and a relative density of 7184.43 bp/Mb. Of the six identified types of SSRs, tetranucleotide SSRs were the most prevalent, with 570 SSRs and a total length of 12152 bp; they accounted for 71.79% of the total SSRs, the average length of SSRs was 21.32 bp, with a relative abundance of 188.08 loci/Mb and a relative density of 4009.79 bp/Mb. Dinucleotide SSRs were the least prevalent, with only 20 SSRs and a total length of 280 bp; they accounted for 2.52% of the total SSRs, the average length of SSRs was 14 bp, with a relative abundance of 6.6 loci/Mb and a relative density of 92.39 bp/Mb. The most frequently occurring SSR in the genome (≥10) included AACT, TTAG, TAGT, ACTA, AGTT, CTAA, AACTA, GTTA, TTAGT, TAAC, and AT. Thirty multi-nucleotide SSRs with sequence lengths≥20 bp were selected for primer design for species identification. The genes containing SSRs were functionally annotated using the Gene Ontology database, and a total of 28 genes (3.53%) were annotated into three categories: molecular function, biological process, and cellular component. Compared to other microsporidian fungi, EHP has a moderate abundance of SSRs, and the quantity of SSRs is independent of genome size. This study provides a theoretical foundation for population genetics and evolutionary research of EHP.