Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization

Abstract

In our previous study, we obtained a large number of differentially expressed genes (DEGs) between second-generation merozoites (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix using RNA sequencing (RNA-seq). Here, we report two subtractive cDNA libraries for MZ₂ (forward library) and MZ₃ (reverse library) that were constructed using suppression subtractive hybridization (SSH). PCR amplification revealed that the MZ₂ and MZ₃ libraries contained approximately 96.7% and 95% recombinant clones, respectively, and the length of the inserted fragments ranged from 0.5 to 1.5 kb. A total of 106 and 111 unique sequences were obtained from the MZ₂ and MZ₃ libraries, respectively, and were assembled into 13 specific consensus sequences (contigs or genes) (5 from MZ₂ and 8 from MZ₃). The qRT-PCR results revealed that 11 out of 13 genes were differentially expressed between MZ-2 and MZ-3. Of 13 genes, 11 genes were found in both SSH and our RNA-seq data and displayed a similar expression trend between SSH and RNA-seq data, and the remaining 2 genes have not been reported in both E. necatrix genome and our RNA-seq data. Among the 11 genes, the expression trends of 8 genes were highly consistent between SSH and our RNA-seq data. These DEGs may provide specialized functions related to the life-cycle transitions of Eimeria species.

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