Rapid Identification Techniques For Important Invasive Mealybug Species And Their Close Relatives

The mealybugs(Hemiptera,Pseudococcidae)are herbivorous insects,some of which are phytophagous quarantine species in China.Due to the similar morphological characteristics,complicated interspecific relationship,damaged levels of intercepted specimens,and uneven identification capabilities,it is extremely difficult to identify mealybugs by general methods.Therefore,developing identification methods based on molecular biology is important.In this study,nine quarantine mealybug species in six genera were taken as research object.These mealybug species have large numbers in interceptions and also have a significant impact on China’s agricultural ecology and economic benefits.Using second-generation sequencing technology,five species were sequenced and assembled.The interspecies specific sequences and specific SSR(Simple Sequence Repeats)sites of mealybug insects were predicted by bioinformatics methods.The specific primers that can be used for molecular identification are screened and validated,applied to the rapid identification of quarantine mealybugs,and kits are developed to maximize the speed and quality of detecting quarantine mealybugs,providing an important technical support for national gate quarantine.The main findings are as follows:1.Genome sequencing and analysis of five mealybug species in three generaFive quarantine mealybug species include the genus Planococcus: Planococcus minor,Planococcus lilacinus;Dysmicoccus: Dysmicoccus neobrevipes,Dysmicoccus brevipes;Phenacoccus: Phenacoccus solani.The genomes of five species were sequenced by second-generation sequencing technology.The genome sizes of five mealybugs are 597 Mb,452 Mb,284 Mb,432 Mb and 270 Mb,respectively.These five assemblies’ quality meet the requirements for subsequent analysis.At the same time,Assemble and analyze four mealybug species(Phenacoccus solenopsis,Paracoccus marginatus,Ferrisia virgate,Maconellicoccus hirsutus)genome downloaded from the National Center for Biotechnology Information(NCBI).This paper is based on the analysis of the genome data of the above 9 quarantine mealybugs.2.Identification of quarantine mealybugs based on genome-specific sequencesPerl scripts and Bowtie software were used to analyze and screen the interspecies specific sequences of these nine quarantine mealybugs.Subsequently,375 pairs of primers were designed for the selected specific sequences.Other two mealybugs,Pseudococcus comstocki and Planococcus citri were selected as secondary controls.Among them,the PCR results of 16 pairs of primers showed strong species specificity,which can effectively distinguish eight mealybugs from 11 mealybug species,which is suitable for the rapid molecular identification of mealybugs in entry.3.Identification of quarantine mealybugs based on genome-specific SSR sequencesUsing MISA software on the genomes of the 9 mealybugs mentioned above,the SSR sequence of each species was excavated,and the species-specific SSR sequence was selected.156 pairs of primers were designed based on where the SSR sequence is located,and the 11 mealybug species including P.comstocki and P.citri were amplified by PCR in the electrophoresis experiment.Seven pairs of specific primers can effectively distinguished four of the 11 mealybug species,which proved that SSR has a certain practicability in the identification of mealybugs.This study took a full advantage of the specific sequence of the mealybug genome to develop the molecular identification technology,and effectively classified and detected nine species in six genera of the mealybug family.Established a more reliable,faster,and low-cost molecular detection method that can be used for the identification of quarantine mealybugs,providing more efficient identification technology for the rapid customs clearance of imported fruits at ports,and enriching the taxonomic research of mealybugs provides an important reference.