| The citrus red mite, Panonychus citri (McGregor), is a worldwide citrus pest leading to a huge fruit yield loss every year. It is also a common sensitizing allergen of asthma and rhinitis. It has the short life cycle and high reproductive rates, infesting over 80 species of plants, such as citrus, rose, almond, pear, castor bean, and several broadleaf evergreen ornamentals. At present, it has developed strong resistance to many registered acaricides. However, the available genomic sequence of P. citri is very limited and has severely hampered the study of resistance mechanisms. In order to gain insight into molecular mechanisms of P. citri resistance, we used next generation sequencing technology (Illumina HiSeqTM 2000) to investigate the global transcriptomes between resistant strains (RS) and susceptible strains (SS), and further compared gene expression profiles of P. citri between different strains using a digital gene expression system.1. P. citri hexyzhiazox resistance selectionA colony of P. citri was collected from citrange next to citrus orchard fences (Citrus Research Institute, Chinese Academy of Agricultural Sciences), which had never been exposed to acaricides for many years. These mites were reared in citrange for 3 years under acaricide-free conditions and considered as a susceptible strain(LC5o=0.056μg/ml). The susceptible strain was treated with hexyzhiazox (about 70% population mortality rate in every spraying) and continuously screened for 20 generations-this resultant strain was regarded as the resistant strain, continuously selected with hexyzhiazox for 20 generations, a resistant strain(LC5o=197.799μg/ml) was obtained with resistance ratio of 3532.12 compared with the susceptible strain.2. De novo assembly of P. citri transcriptomeBy transcriptome sequencing,25 and 26 million of 90 bp clean reads were obtained from RS and SS respectively after filtering the dirty raw reads. First, we use SOAPdenovo software to produce 215,641 contigs with the mean length of 178bp in RS, and 282,291 contigs with the average length of 151bp in SS. And then with the paired-end reads joining and gap-filling, the above contigs were further assembled into 38,178 scaffolds with mean size of 453bp in RS and 44,017 scaffolds of mean size 406bp in SS, where the number of the scaffold size more than 1000bp is 4,643 in RS and 4,270 in SS. The scaffolds with further assembly, there were 30,466 RS-unigenes with the mean length of 536 bp and 34,159 SS-unigenes with the average length of 489 bp. With the same process,32,217 all-unigenes were assembled by using a combination reads from RS and SS. There are total 17,581 annotated unigenes from SS&RS reads by BLAST searching databases of nr, the Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) with an E-value≤le-5, in which 7,075 unigenes were annotated in the COG database,12,712 unigenes were found in the KEGG database,3,812 unigenes were assigned to Gene ontology (GO) and 17,457 unigenes had a nr hit.3. Difference analysis of unigenes related to general pesticide detoxification and targetsWe identified 200 metabolic genes of general pesticide which included121,30,43,2 and 4 unigenes related to cytochrome P450 monoxygenases (CYPs), Glutathione S-transferase (GSTs), Carboxylesterase, NADH dehydrogenase and Superoxide dismutase (SODs), respectively from transcript of P. citri. Only 46 of the P450s were functionally annotated according to their degree of sequence matching (E-value, le-5), owing to the low sequence similarity of other P450s. We found 17 genes including 9 of P450s,4 GSTs,3 carboxylesterases and 1 esterase that were greater than value of 2 based on a log2ratio formula. Particularly,4 of the P450s belonging to the CYP3 clade (which includes CYP3, CYP6 and CYP9 members) were annotated to the xenobiotic metabolism pathway (ko00982 and ko00980). One up-regulated GST was annotated to the xenobiotic metabolism pathway (ko00980).4. Analysis of genes related to the growth and reproduction process 105 and 194 genes related to growth and reproduction respectively were identified, based on the mode of action of Hexythiazox. By comparison nucleotide sequences of sodium channel, GABA receptor and reproduction related genes between susceptible and resistant citrus red mite, we obtained 24,12 and 23 SNPs respectively. Among these SNPs, G-A,T-C and C-T were the main type of SNPs.5. Difference analysis of gene expression profile between resistant and susceptible strainsBy further analysis of RS and SS’s digital gene expression profiling(DGE), we obtained 148 unigenes which Log2Ratio values were greater than 2 and 246 unigenes having a Log2Ratio value more than 1. Enrichment analyses demonstrated that there were 65 unigenes and 22 unigenes involved in GO enrichment and Pathway enrichment respectively. Among those notable differential expressed genes, the three most up-regulated unknown transcripts, Unigene21272_All, Unigene23131_All, Unigene25542_All, were founded and up to 15.47,14.50,13.78 times respectively.To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in phytophagous mite resistance. This study identified differential unigenes related to general pesticide resistance and organism growth and reproduction in P. citri. Furthermore, the 3 top up-regulated unigenes in resistance-hexyzhiazox strains of P. citri were obtained by analysis DGE data. The assembled, annotated transcriptomes provide a valuable genomic resource for further understanding the molecular basis of resistance mechanisms. |
PDF Full Text Request