ABSTRACT
As one of the top 10 plant viruses, the severity of losses to crop productivity caused by the tomato spotted wilt virus (TSWV) has resulted in an urgent need to develop a more sensitive and rapid method of detection. In this study, we developed a CRISPR/Cas13a-based detection system to diagnose TSWV in tomato and western flower thrips (Frankliniella occidentalis). The detection system relies on recombinase polymerase amplification and Cas13a-mediated collateral cleavage activity. Positive results can be distinguished after 20 min by a significantly enhanced fluorescence signal. We tested the sensitivity of CRISPR/Cas13a-based detection system and found that the detection system that we developed has limits of detection that reaches 2.26 × 102 copies/µl and a 10-fold increase compared with the sensitivity of using RT-PCR to detect the virus. Furthermore, the CRISPR/Cas13a-based detection system has a high selectivity for the TSWV without interference from other viruses. The CRISPR/Cas13a-based detection system was utilized to detect the TSWV in samples of tomato leaves and the transmission vector F. occidentalis that were fully consistent with the results when RT-PCR was used to detect the virus.
ABSTRACT
Aphidius gifuensis is a parasitoid wasp that has been commercially bred and released in large scale as a biocontrol agent for the management of aphid pests. As a highly efficient endoparasitoid, it is also an important model for exploring mechanisms of parasitism. Currently, artificially bred populations of this wasp are facing rapid decline with undetermined cause, and mechanisms underlying its parasitoid strategy remain poorly understood. Exploring the mechanism behind its population decline and the host-parasitoid relationship is impeded partly due to the lack of a comprehensive genome data for this species. In this study, we constructed a high-quality reference genome of A. gifuensis using Oxford Nanopore sequencing and Hi-C (proximity ligation chromatin conformation capture) technology. The final genomic assembly was 156.9 Mb, with a contig N50 length of 3.93 Mb, the longest contig length of 10.4 Mb and 28.89% repetitive sequences. 99.8% of genome sequences were anchored onto six linkage groups. A total of 11,535 genes were predicted, of which 90.53% were functionally annotated. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed the completeness of assembled genome is 98.3%. We found significantly expanded gene families involved in metabolic processes, transmembrane transport, cell signal communication and oxidoreductase activity, in particular ATP-binding cassette (ABC) transporter, Cytochrome P450 and venom proteins. The olfactory receptors (ORs) showed significant contraction, which may be associated with the decrease in host recognition. Our study provides a solid foundation for future studies on the molecular mechanisms of population decline as well as host-parasitoid relationship for parasitoid wasps.
