| From 1990 s to today, as one of the world’s most important fruit producing areas and consumer markets, fruit trading between China and ASEAN has been in a good development trends. Especially China-ASEAN free trade area, to further promote the sustainable and stable growth of China and ASEAN fruit import and export trade, was built in January 1, 2010. With the increasing of trade volume, quarantine pests which have been intercepted by inspection and quarantine departments in China from ASEAN entry fruits, is increasing. The mealybug pest, one of the most important pests of fruit, has attracted increasing interest, as its serious damage, difficult treatment ect [1].Mealybug Mealybug is a kind of important pests in the world, and also is the second biggest family in coccoidea [2]. It brings a huge threat to fruit, vegetables and other import and export trade. In general, most mealybug which have been intercepted by quarantine department are nymphs and even debris. At the same time, it has cryptic species, and the differences between them are smaller, which takes great difficulty to identification of mealybug timely and accurately basing on morphological characteristics[3]. Mealybug belongs to Pseudococcidae, commonly known as mealybugs, Latin name is Pseudococcidae, is a small sucking insect. It distributes worldwide, and can adapts to any environment. The female is oval, with very slow action. It is about 1 centimeters in length, covering with a sticky white powder in vitro. Female and larvae accumulate on the back of leaves. There is a pair of developed fore wings in the male’s middle chest, and a developed genital sheath at the end of body[2].Generally, mealybug identification is based on morphological observation of the female adult. However, the slide production process is complicated and requires professionalism to do identification, in addition, the samples of larvae and residue can not be well identified [4]. Many experts began to apply to molecular identification technology in the scale insect research field, including phylogenetic analysis [5-6], species identification [7-14]. In recent years, the reports of using mitochondrial DNA, DNA mt DNA and cytochrome C oxidase subunit I gene as molecular marker, by DNA barcoding technology for species identification, is increasing[15]. There are some studies on scale insects yet. At the same time, some experts have found that 28 S gene sequence variation also provides some information as a supplement to COI bar code identification, which can improve the accuracy of identification. DNA bar code is a special DNA sequence. It can be used to identify species. China inspection and quarantine departments have intercepted a large number of mealybugs from ASEAN entry fruits, many of them are quarantine pests, however mealybug individuals are small, only by morphological observation methods can not be identified timely and precisely [16].DNA barcode is becoming a important molecular identification technology. In the present study, DNA barcode of mealybug based on mealybugs and Ceroplastes rusci, which were intercepted from fruits imported at Pingxiang Port, was carried out. DNA barcode sequences of 77 individuals(11 species of mealybug and C.rusci) and 28SD2 region sequences of 48 individuals(10 species of mealybug and C.rusci) were sequenced. The phylogenetic tree based on COI sequences was constructed by MEGA6.0.6, and the genetic distance was analyzed by Kimura-2-parameter model. The validity of the mitochondrial COI gene sequence for the DNA barcoding of mealybug species identification was verified.The results indicated that, DNA barcode could effectively distinguish all the mealybugs, except for D.lepelleyi, which were clustered into two branches, and P.minor/P.citri, which were clustered into one branch. The topological structure of NJ phylogenetic tree based on 28SD2 sequences was quite similar to the tree based on COI sequences. It suggested that 28 S sequences might be used for multiple genes DNA barcode technology. |
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