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【目的】粉蚧是一类重要的世界性检疫性害虫,对果蔬产业以及水果的进出口贸易造成了巨大威胁。通常,口岸截获的粉蚧多为若虫或残体,加之隐存种的存在和较小的近缘种间差异,严重影响了基于形态学特征的粉蚧类害虫识别鉴定的准确性和及时性。本研究旨在明确DNA条形码技术对重大潜在入侵害虫大洋臀纹粉蚧Planococcus minor(Maskell)的鉴定有效性。【方法】以旅检截获的36头大洋臀纹粉蚧为对象、其近缘种柑橘臀纹粉蚧Pl.citri(Risso)为参照,以线粒体COI基因5'端和3'端序列以及核糖体28S r DNA D2-D3区段序列为分子标记进行比对分析,以K-2-P模型计算种内种间遗传距离,以最大似然法(maximum likelihood,ML)构建进化树并进行系统发育分析,同时利用Species Identifier物种识别软件评价3种基因片段对大洋臀纹粉蚧的鉴定效果。【结果】当分别以COI基因5'端和3'端序列为分子标记时,截获大洋臀纹粉蚧的碱基序列与NCBI中大洋臀纹粉蚧的序列一致性分别为100%和99%~100%,而与近缘种柑橘臀纹粉蚧COI基因5'端和3'端的核苷酸序列一致性分别为97%~98%和96%~98%;且大洋臀纹粉蚧和柑橘臀纹粉蚧分别存在5个和11个稳定的物种特异性识别位点;系统发育分析显示,截获的大洋臀纹粉蚧均与数据库中的大洋臀纹粉蚧聚为一支。当以28S r DNA D2-D3区段序列为分子标记时,臀纹粉蚧属各物种间高度保守,无法区分大洋臀纹粉蚧与其近缘种柑橘臀纹粉蚧;种间遗传距离仅为0.004。此外,物种识别软件评价结果显示,基于COI基因5'端和3'端序列的鉴定结果完全正确,而基于28S r DNA D2-D3区段序列的鉴定结果却存在45.2%~61.9%的模糊鉴定。【结论】基于COI基因5'端和3'端的DNA条形码技术完全可用于大洋臀纹粉蚧的快速准确鉴定及检测,对有效阻截其入侵和进一步扩散蔓延意义重大。
【Objective】 Mealybugs are a kind of important quarantine pests worldwide, posing a huge threat to the import and export trade of fruits and vegetables and fruits. Normally, the mealybugs intercepted by the port are mostly nymphs or debris, and the existence of the hidden species and the differences of the minor relatives severely affect the accuracy and timeliness of identification and identification of the mealybug based on the morphological characteristics . The purpose of this study was to determine the validity of the DNA barcoding technique for Planococcus minor (Maskell), a potentially significant pest infestation pest. 【Method】 Thirty-six occipital mealybugs crossed with triploids were used as reference. Their relatives, P. citri (Risso), were used as reference. The mitochondrial COI gene 5 'and 3' end sequences and ribose The sequence of 28S rDNA D2-D3 segments was molecular marker. The K-2-P model was used to calculate the intraspecific genetic distance and the phylogenetic tree was constructed by maximum likelihood (ML) Meanwhile, Species Identifier species identification software was used to evaluate the identification effect of three gene fragments on the mealybugs. 【Result】 The results showed that the sequence identities of mealybugs isolated from Melastomataceae were 100% and 99%, respectively, when they were labeled with 5 'and 3' end of COI gene respectively. ~ 100%, while the nucleotide sequence identities of COI gene from the proximal species citrus mealybugs were 97% ~ 98% and 96% ~ 98%, respectively. There were 5 and 11 stable species-specific recognition sites for citrus glume mealybugs respectively. Phylogenetic analysis showed that the intercepted mealybugs crossed with the mealybugs in the database. When the 28S rDNA D2-D3 segment sequence was used as a molecular marker, the genus Glumetida was highly conserved among all species and could not be distinguished from the mealybugs, 0.004. In addition, the evaluation results of species identification software showed that the identification results based on the 5 'end and the 3' end of the COI gene were completely correct, whereas the identification results based on the 28S rDNA D2-D3 segment sequence were obscure from 45.2% to 61.9% . 【Conclusion】 The DNA barcoding technique based on the 5 'end and the 3' end of the COI gene can be used for the rapid and accurate identification and detection of mealybugs. It is of great significance to effectively intercept and further spread the spread of it.