Detection Of Korla Fragrant Pear Virus By Transcriptome And Small RNA Sequencing

Object:The objective of this study was to use high-throughput sequencing to perform transcriptome and small RNA sequencing of Korla fragrant pear flowers,the gene sequence related to plant virus was selected as candidate virus analysis,and the gene sequence of the virus was screened by RT-PCR technology.Explored a new method to analyze the genome sequence information of Korla fragrant Pear virus,and lay a foundation for the research of Korla Pear virus detection,virus control and breeding of virus-free seedlings.Methods:The Korla fragrant pear flowers collected in mid-April 2014,mid-April 2017,and mid-April2018 were sent to Novogene company in the same year,and commissioned them to use the Illumina HisSeq^TM2500 sequencing platform for transcriptome sequencing.The raw reads was filtered to eliminate low-quality data as a clean reads,and Trinity was used to stitch clean reads.The transcript sequence obtained by Trinity splicing,Corset was used to hierarchically cluster the transcripts by comparing the reads and expression patterns of the upper transcripts,and the longest Cluster sequence was obtained after Corset hierarchical clustering for gene function annotation.Plant virus long fragment sequences were used as candidate viruses,and primers for candidate virus sequence designed were verified by RT-PCR.Small RNA sequencing data has been obtained in 2014.The raw reads were filtered by the sequencing data to obtain the clean reads.The reference sequences of various plant viruses were downloaded from GenBank database of NCBI,and the clean sequences were compared with nucleic acid database and protein database by local BLAST program,and the sequences with high similarity to plant virus sequences were screened out,so as to obtain candidate viruses for analysis..The results of the study are as follows:1?Analysis of transcriptome sequencingAccording to the bioinformatics analysis results of the three groups of transcriptome sequencing data,a total of 66 gene sequences,202 gene sequences and 921 gene sequences were selected from the gene sequences annotated as plant viruses and 1 gene sequence analysis annotated as plant viroid.Three kinds of viruses have been reported to infect pears and one kind of viroid,respectively was apple stem pitting virus isolate PA66,apple stem groove virus strain P-209,apple stem groove virus isolate Korean,apple chlorotic leaf spot virus,hop stunt viroid.An unreported brassica yellows virus.According to the designed specific primers,randomly collected Korla fragrant pear branch samples were amplified and verified by RT-PCR technology,and only the apple stem pitting virus and the apple stem groove virus amplified the target fragment.2?Analysis of small RNA sequencingThe BLAST program was used to compare the clean sequences obtained with the NCBI nucleic acid database and protein database,respectively,22 gene sequences annotated as plant viruses and 32 gene sequences annotated as plant viroids were screened and analyzed.Two kinds of reported viruses and one kind of viroid that infect pears were found to be apple stem pitting virus,apple stem groove virus and apple scar skin viroid.