siRNAs Derived from Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus Down-modulated the Expression Levels of Endogenous Genes in Phalaenopsis equestris

Lan, Han-hong; Wang, Cui-mei; Chen, Shuang-shuang; Zheng, Jian-ying

sRNA-seq and analyzing. The RNA extracted from infected and healthy P. equestris (n = 3) was used for library construction and sequencing for three times by Novogene Technology Co. Ltd. (Beijing, China) as described previously (Lan et al., 2015) . To identify siRNAs, using the software Bowtie v.0.12.7 with 0 mismatch, we aligned all the cleaned reads to the viral genome sequences and calculated the average depths and the genome coverages. The average depth is calculated as the total number of nucleotides of the aligned reads divided by the read-covered positions on the reference genome. The genome coverage represents the proportion of read-covered positions against the genome length. Reads obtained above were analyzed using Excel. Single-base resolution maps along with virus genomes were created using Bowtie tools and in-house Perl scripts. sRNAs from each sample were assembled de novo using Velvet. Assembled contigs were used to search the Gen-Bank/EMBL/DDBJ database using BLASTn or BLASTx. Coverage and distribution of virus-specific contigs by siRNAs were determined using the program MAQ under default parameters, and results were exported to Microsoft Excel for further analysis. Primers were designed for amplification and Sanger sequencing based on the identified viral contigs using Vector NTI (Invitrogen). Phylogenetic tree analysis was performed using MEGA 5.0.