基于种特异性COI引物(SS-COI)的桉树枝瘿姬小蜂A、B隐种快速鉴定技术

Species-specific COI(SS-COI)primers for rapid identification of Leptocybe invasa cryptic species A&B

隐种A和隐种B是桉树枝瘿姬小蜂Leptocybe invasa两种重要的全球入侵隐种,对多国林业生产造成了严重危害。由于桉树枝瘿姬小蜂体型微小,且无法从形态上区分隐种类型,给该害虫的防治造成困难。本研究基于线粒体DNA细胞色素C氧化酶亚基I(mtDNA COI)基因序列的种特异性(species-specific COI,SS-COI)PCR方法,研究桉树枝瘿姬小蜂隐种快速分子检测技术。基于隐种A、B的COI序列分别设计特异性SS-COI引物各1对(AF/AR和BF/BR)。使用这两对引物扩增同一桉树枝瘿姬小蜂样品DNA,即可有效进行隐种鉴定,同时两对引物也能互相验证鉴定结果。引物鉴定灵敏性检测结果显示,AF/AR与BF/BR均具有较高的鉴定灵敏性,其对DNA的有效鉴定浓度阈值分别为11.42 pg/μL和28.32 pg/μL。本研究开发的桉树枝瘿姬小蜂隐种A、B的快速鉴定方法解决了桉树枝瘿姬小蜂入侵地区隐种鉴别的难题,极大缩短鉴定时间、降低鉴定费用,为进一步探究桉树枝瘿姬小蜂隐种A、B的生物学差异以及它们对不同抗性品种桉树的适应能力提供技术参考。

Cryptic species A and B are two important globally intrusive cryptic species of Leptocybe invasa,which have caused serious damage to forestry production in many countries.Due to the tiny size and undistinguishable cryptic species in morphology,it makes pest control more difficult.In this study,we researched the rapid molecular identification method of cryptic species of L.invasa,basing on the speciesspecific COI(SS-COI)PCR method of mitochondrial DNA cytochrome C oxidase subunit I(mtDNA COI)gene sequence.According to the COI sequence,two pairs of cryptic species A and B SS-COI primers(AF/AR and BF/BR)were respectively designed.Using these primers to amplify the DNA of the same L.invasa sample,by which could not only effectively identify the cryptic species,but also mutually verify the identification accuracy.The primer identification sensitivity detection showed that both AF/AR and BF/BR had high identification sensitivity,and their effective identification concentration thresholds for DNA were 11.42 pg/μL and 28.32 pg/μL.The rapid identification method developed for the cryptic species A and B of L.invasa in this study can addresse the challenge of discriminating cryptic species in areas invaded by L.invasa.It significantly shortens identification time,reduces identification costs,and provides technical references for further exploration of the biological differences between cryptic species A and B of L.invasa,as well as their adaptability to different resistant varieties of Eucalyptus.