The Transcriptome Sequencing And Gene Expression Analysis Of Migrant And Resident Mythimna Separata (Walker)

The oriental armyworm Mythimna separata (Walker) is not only a typical long-distance migratory pest, but also a key agricultural insect pest that threatens the three staple food crops, including rice, wheat and maize in China. Migration is the main behavioral strategy for insect species to adapt to the complex variation of environment, maintaining breeding populations and the major reasons for the frequent outbreaks of migratory pests. A migrant population usually consists of parts of migrant and resident.In order to select migratory key target genes and obtain gene expression profiles of migrant and resident and different gender, which will reveal the molecular regulatory mechanism of migratory behavior and population migration hazards of M.separata.48h,96h and 144h after emergence of the resident (1larvae/850 mL) and migrant (10 larvae/850 mL) female moths and 96h after emergence of the migrant male moths were sequenced using Illumina HiSeq2500 and subjected to relevant informatics analysis. q-PCR was used to validate the differentially expressed genes of transcriptome sequencing data. At the same time, we discovered high-throughput SSR markers of M.separata from the transcriptome data. The main results are as follows:1.108 Gb raw data,105.42Gb clean data and 7.53Gb clean data per sample were acquired from the transcriptome sequencing.The Q20 of all samples were higher than 95%, the GC content was between 45.15% and 47.16%.174385 transcripts and 108494 unigenes were obtained. After the annotation of gene function, in the GO classification, there were 21955 unigenes with the specific functional definition and they were classsficated into 3 categories:molecular functions, cellular components and biological processes and 55 branches; in the KEGG classification, there were 8718 unigenes with specific definition, acquired 10611 KEGG function annotation; in the KOG classification, there were 11661 unigenes with specific proteins functional definition, acquired 12889 KOG functional annotation, involving 26 KOG functional categories.2. Differential expression analysis of two conditions was performed using the softwore DESeq. There were 97,724,250 differentially expressed genes between migrant and resident after 48h,96h and 144h emergence, which revealed that the same period migrant and resident had different biological behavior from the gene level. There were 8626 differentially expressed genes between different gender, 4725 genes were up regulated and 3901 genes were down regulated.3. The GO and KEGG enrichment analysis of differentially expressed genes results showed that the migrant had strong flight ability, stress adaptation ability and immune ability.4.15 randomly selected differentially expressed genes were quantified by real-time quantitative PCR. The results showed that the gene expression data based on transcriptome sequencing and qPCR were consistent, which indicated that the gene expression data obtained from RNA-seq was reliable.5. Three kinds of genes related to migration were screened, such as 8 circadian clock genes,4 genes involved in the regulation of JH levels, three genes related to reproduction. Chemosensory genes were screened 26 genes involved in 6 major categories.We screened the key enzyme genes related to metabolism, such as JH carboxylesterase, fatty acid binding protein and acylcarnitine translocase.. In addition, neurotransmitter receptor genes, such as octopamine receptor,5-hydroxytryptamine receptor and dopamine receptor were also screened.Screening of these genes will provide gene resources for future studies on the molecular regulatory mechanism of migratory behavior of M.separata.6. The sofeware MISA was used to filter 108494 unigenes.12483 SSRs were obtained, accounting for 11.51% of all unigenes,which were distributed in 10685 unigenes. The frequency of occurrences for SSRs was 9.85%. In average, there was one SSR site in every 6126 bps. Mononucleotide and trinucleotide repeats were the main types and the number of 10 repeats SSR was the most. There were 42 kinds of repeat motifs existing in the M.separata transcriptome and mononucleotide repeat motifs A/T was the most frequent. Establishing massive SSR molceluar markers will help explore the migratory insect source exchange and population genetic structure and genetic diversity of M.separata in the future.