Phylogenetic analysis and designing new primers for molecular identification of Drosophila suzukii

. The family Drosophilidae includes over 3750 species worldwide and over 2000 of these are species of Drosophila . Spotted wing drosophila (SWD), Drosophila suzukii is one of the most dangerous species in this family. The insects live on undamaged ripening fruits, using its peculiar serrated ovipositor to break the skin of fresh ripening fruits and lay eggs in it. Drosophila species are very difficult and practically impossible to detect at larval stages. The present investigation was conducted at the All-Russian Plant Quarantine Center and Agrarian and Technological Institute of RUDN University, Moscow, Russia in 2018—2020. The aim of this study was to investigate the method of accurate and rapid identification of D. suzukii , and to design specific primer pairs for pest identification by Real-Time PCR method. The real-time quantitative PCR is a fast, sensitive, repeatable and accurate method for quantifying gene transcript levels. In this study, we designed specific primers (4.Dsuz.FRP) for Real-Time PCR to identify D . suzukii from other relative species. Although D. suzukii is ab-sent in the Russian Federation and has not been reported so far, the project could be a precautionary measure. Acknowledgments. This paper has been supported by the RUDN University Strategic Academic Leadership Program.


Introduction
The spotted wing Drosophila (SWD), Drosophila suzukii, was originally described by Matsumura in Japan in 1931 [1]. Members of the Drosophila genus are not generally considered pests since their larvae are primarily developed on damaged or rotting fruits. Nevertheless, D.suzukii infests healthy ripening fruits while still on the plant. Drosophila suzukii larvae devour the fruit pulp within the fruits rendering them unmarketable and reducing the processed fruit exceptional [2,3]. Furthermore, the wounds created at the infested fruits throughout ovipositing provide access to secondary fungal or bacterial infections leading to additional fruit tissue disintegrate [4][5][6][7][8]. Drosophila suzukii includes exceptional kinds of hosts; for this reason, this type of agricultural pest has come to be a world major pest (Fig. 1) of a large variety of commercial fruit crops [9,10]. The aim of the research was to molecularly become aware of and draw a phylogenetic tree for Drosophila suzukii and distinguish it from other species.  [11]. (https://www.cabi.org/isc/datasheet/109283#toDistributionMaps) The principal purpose of this study was to categorize the molecular species of D. suzukii. Our purpose was to create an accurate and unique identity technique based on primers designed for D. suzukii, this approach is rapid and more efficient than techniques based totally on morphological identity [5,12,13]. The polymerase chain reaction turned into created to discover insects as a reliable and price-effective approach. In addition, in various molecular identification methods, one of the most important parts is the primer design. In this project, we design a specific pair of primers based on Real-time PCR.

Materials and Methods
DNA was extracted from the material under project (insect and larvae, from laboratory collection) by treating the specimens with Proteinase K accompanied with removal of proteins with no extraction with natural solvents and using «DNA Extran-2 Kit», set № NG-511-100 («Synthol», Russian Federation) according to manufacturer's instructions.
PCR-products purification. A 1:1 volume of Binding Buffer was added to the completed PCR mixture (e. g. 100 µL of Binding Buffer per every 100 µL of the reaction mixture) and mixed thoroughly. Then the solution was transferred up to the GeneJET purification column, centrifuged for 30…60 s. The flow-through solution was discarded. 700 µL of Wash Buffer was added to the GeneJET purification column and centrifuged for 30…60 s. The flow-through was discarded. The purification column was placed back into the collection tube. Then the empty GeneJET purification column was centrifuged for 1 min. After that the GeneJET purification column was transferred to a clean 1.5 mL microcentrifuged tube. Then 50 µL of Elution Buffer was added to the center of the GeneJET purification column membrane and centrifuged for 1 min.
Sequencing. DNA extracts were quantified on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., USA). Sequencing was done according to the generally accepted protocol with the use of Genetic Analyzer AB-3500 (Applied Biosystems, USA).
As shown in the phylogenetic tree ( fig. 2), D.suzukii family members, such as D. simulans and D. melanogaster, are very close in genetic code, making it more difficult to identify. Moreover, this further highlights the need to design proprietary primers. Finally, we recommend this primer to other researchers (Table 3).
In this study, we have developed a Real-time PCR assay for the detection and identification of D. suzukii (Fig. 3).
In addition, there is a case of the negative control (sample 3 up to 11) for checking PCR and extraction DNA processes. Two were accurately identified as Drosophila suzukii; two samples (number: 1, from Egypt and number: 2 from Turkey) showed melt peaks in the acceptable range of T: 58 ºC results for and were clearly distinguished from another species. No other melt peaks were reported.   The research has proven that particular host fruits were preferred for ovipositing and development of larvae, at the same time as temperature performed an essential position in D. suzukii development, survival, and fecundity. We have used unique sequences in all stages and the primers were designed for the first time as well.
The primary comparison for the results of the sequence with the GeneBank genetic sequence database was performed by the NCBI BLAST web site (http://www.ncbi. nlm.nih.gov/BLAST). BioEdit v.7.0.5.3 [18] sequence alignment editor was used for sequence checking, alignment, and editing.

Conclusion
Molecular identification for all samples used within the improvement of the technique changed into done by way of PCR amplification. For a long time, polymerase chain reaction (PCR) methods had been used to classify pests, organisms and sicknesses around the sector. The outcomes showed that those primers appropriately diagnosed the gene region in addition to the specific region of D. suzukii. When samples are not sufficient for morphological identification or handiest immature samples are available, real-time PCR molecular detection may be used as a fast and efficient detection approach. For such an economically vital attacker, a one-of-a-kind identification method can also permit extra accurate tracking and detection and save you misidentification. Real-time PCR detection can be used as a fast and efficient detection device. Finally, a phylogenetic tree turned into identified for numerous specimens close to D. suzukii. As an end result, it is far located near the subspecies in the tree.