Identification and comparison of circular RNAs in Ascosphaera apis mycelium and spore

Abstract:

[Objective] The objective of this study is to clarify the differences of number, type and expression profile of circRNAs between Ascosphaera apis mycelium and spore, and discuss the potential role of common circRNAs, specific circRNAs and differentially expressed circRNAs (DEcircRNAs). [Methods] Based on previously gained high-quality RNA-seq data of A. apis mycelium (AaM) and spore (AaS), circRNAs were predicted using find_circ software. Common circRNAs and specific circRNAs were filtered via Venn analysis. DEcircRNAs of AaM vs AaS were screened following the standard of P ≤ 0.05 and|log₂ fold change|≥ 1. Function and pathway annotation of source genes of circRNAs was done via alignment against GO and KEGG databases. CircRNA-targeted miRNAs and miRNA-targeted mRNAs were predicted using TargetFinder software. Cytoscape software was used to visualize competing endogenous RNA (ceRNA) regulation network. DEcircRNAs were verified by RT-qPCR. [Results] There were 13210156 and 19011000 anchors reads in AaM and AaS, respectively, among them 6124922 and 11392886 ones can be mapped to the reference genome of A. apis. In AaM and AaS groups, 1868 and 2225 circRNA were respectively identified, and the numbers of common circRNAs, specific circRNAs in AaM and specific circRNAs in AaS were 1098, 770 and 1127, respectively. In addition, the length of circRNAs in AaM and AaS was mainly distributed among 1000-2000 nt, and the most abundant circularization type was intergenic circRNA. Further, 456 upregulated circRNAs and 97 downregulated circRNAs were contained in AaM vs AaS comparison group. Source genes of common circRNAs were annotated to 29 functional terms and 14 pathway classifications; source genes of specific circRNAs in AaM were annotated to 31 functional terms and 17 pathway classifications; source genes of specific circRNAs in AaS were annotated to 34 functional terms and 16 pathway classifications; source genes of DEcircRNAs were annotated to 29 functional terms and 40 pathways. ceRNA regulation network analysis showed that 36 common circRNAs can target four miRNAs, further regulating six mRNAs related to endocytosis. Additionally, four (255) specific circRNAs in AaM (AaS) can target two (two) miRNAs, further regulating eight (two) mRNAs related to biosynthesis of secondary metabolites; nine DEcircRNAs can target two DEmiRNAs, further regulating three DEmRNAs associated with MAPK signal pathway. The result of RT-qPCR displayed that the expression trend of 10 DEcircRNAs was consistent with that in the sequencing result, verifying the reliability of our sequencing data. [Conclusion] Common circRNAs, specific circRNAs and DEcircRNAs in A. apis mycelium and spore may regulate material and energy metabolisms, endocytosis, biosynthesis of secondary metabolites, and MAPK signaling pathway through controlling the expression of source genes and serving as ceRNAs, further affecting the mycelium growth, spore germination, and pathogenicity of A. apis.

Key words:chalkbrood;Ascosphaera apis;mycelium;spore;circular RNA;microRNA;competing endogenous RNA