Abstract: 【Aim】This study aims to deeply sequence the purified spores of Ascosphaera apis and honeybee larval gut infected with A. apis using RNA seq technology， to de novo assemble a reference transcriptome of A. apis， to further conduct functional and metabolic pathway annotation， and finally to develop SSR markers for A. apis. 【Methods】 A. apis spores purified after differential centrifugation were fed to the 4-， 5- and 6-dayold larvae of Apis mellifera ligustica and A. cerana cerana at a concentration of 1×10⁷ spore/mL. The purified spores of A. apis and the infected honeybee larval guts were sequenced at Illumina HiSeq^TM 2500 platform. After filtration of raw reads， unigenes were de novo assembled with Trinity software and annotated through BLASTX against the NCBI Nr， Swiss-Prot， KOG and KEGG databases. The MISA software was used to search SSRs in all unigenes， and Primer Premier 5 software was used to design specific primers for SSR loci. PCR was carried out to amplify SSR loci in A. apis from different regions in China. 【Results】 In this study， a total of 146 135 308 high-quality reads were produced from RNA seq， and 42 609 unigenes were de novo assembled. BLASTX results indicated that 29 316 unigenes have function or metabolic pathway annotations in the aforementioned public databases. And the most unigenes (6 050) were annotated to Xanthophyllomyces dendrorhous. The results of KEGG pathway annotation revealed that all unigenes were annotated to 117 metabolic pathways， among which ribosome (529 unigenes) was the most enriched one. In total， 7 968 SSRs were predicted from all unigenes， and five SSR markers were developed by PCR amplification. 【Conclusion】 In this study， a reference transcriptome of A. apis was successfully de novo assembled and annotated， providing significant information for further studying A.apis at the molecular level. Five SSRs developed in this study can facilitate the research on A. apis including strain identification， gene map construction and gene localization.

Key words: Ascosphaera apis, reference transcriptome, RNA seq, function and metabolic pathway annotation, SSR