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实蝇类害虫多为国内外检疫对象,其鉴定识别方法主要依据成虫的外部形态特征,而传统的形态学识别法对口岸经常截获的幼体及残缺的虫体,则无能为力。本研究以桔小实蝇Bactrocera dorsalis的幼体(卵、幼虫、蛹)以及成虫残体(足、翅、头部、胸部、腹部)为对象,利用DNA条形码技术,构建实蝇类害虫快速鉴定技术体系,并以其他4种常见实蝇(包括番石榴实蝇B.correcta、瓜实蝇B.cucurbitae、南亚果实蝇B.tau、柑桔大实蝇B.minax)为对象对该技术体系进行应用验证。结果显示,桔小实蝇幼体以及成虫残体的碱基序列与数据库中靶标种COⅠ基因碱基序列的一致性为99.51%～99.84%,其他4种实蝇相应序列与数据库中靶标种COⅠ基因序列的一致性分别为100%,100%,99.81%～99.83%和100%;以邻接法(NJ法)构建系统发育树,靶标种实蝇均与数据库中对应种实蝇聚为一支,且置信度均为100%。以K2-P模型计算种内及种间遗传距离得出,5种实蝇的种间遗传距离为0.0597～0.2363,平均为0.1693;种内遗传距离为0.0000～0.0041,平均为0.0019。这些结果表明,基于DNA条形码的物种识别技术完全可用于口岸截获的实蝇类害虫幼体及残体的准确鉴定。
Fruit flies and pests are mostly quarantine objects at home and abroad, and their identification and identification methods are mainly based on the external morphological characteristics of adults. However, the traditional morphological identification method can not help the larvae and incomplete parasites that are often intercepted at the ports. In this study, the larvae (eggs, larvae, pupae) and adult remnant (foot, wing, head, chest and abdomen) of Bactrocera dorsalis were used as the object to establish rapid identification technique of fruit fly pests by DNA barcoding System, and the other four kinds of common fruit fly (including guava fruit fly B.correcta, melon fruit fly B.cucurbitae, South Asia fruit fly B.tau, citrus fruit fly B.minax) for the technical system Application Verification. The results showed that the nucleotide sequence of the COⅠ gene in the larvae and adult remains of the fruit fly was 99.51% -99.84%, and the COⅠ gene of the target species in the other four fruit fly species The sequence identity was 100%, 100%, 99.81% ~ 99.83% and 100% respectively. The phylogenetic tree was constructed by the neighbor-joining method (NJ method). The target fruit flies were clustered with the corresponding fruit flies in the database, And the confidence is 100%. The K2-P model was used to calculate the intraspecific and interspecific genetic distances. The interspecific genetic distances of the five fruit flies ranged from 0.0597 to 0.2363 with an average of 0.1693. The intraspecific genetic distance ranged from 0.0000 to 0.0041 with an average of 0.0019. These results show that the DNA barcoding based species identification technology can be used for the accurate identification of larvae and debris of fruit fly intercepted by the port.