Development of an easy and cost-effective method for non-invasive genotyping of insects
Development of an easy and cost-effective method for non-invasive genotyping of insects
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开发一种简单且经济高效的昆虫非侵入性基因分型方法
DOI：
10.1371/journal.pone.0216998
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发表时间：
2019-06-03
期刊：
影响因子：
3.7
通讯作者：
Zhu, Zhihui
中科院分区：
文献类型：
--
作者：
Ali, Bahar;Zhou, Yicheng;Zhu, Zhihui
非侵入性基因分型方法提供了有关昆虫种群的有价值的信息。然而，糟糕的DNA扩增和耗时的采样程序限制了这些方法，特别是对小昆虫。建立了一种高效、简便的非侵入性、非致死性基因分型方法，通过从昆虫脂肪、蜕皮和食物垃圾中扩增内源和外源、核和线粒体基因，对大型昆虫粘虫和小型昆虫黑腹果蝇进行基因分型。从分离菌的蜕皮DNA中成功检测到几丁质合成基因MsCHSB和COI基因。然而，由于DNA的降解，用聚合酶链式反应不能直接从FRASS中检测到COI片段。为了提高检测效率，首先用phi29 DNA聚合酶进行多重置换扩增，然后用聚合酶链式反应从所有样本中扩增出COI片段。将2龄幼虫单独饲养3d，然后从每个个体的食物垃圾中提取DNA。从大多数样本中成功检测到内源片段Serendipity a(Srya)、外源转基因FC31整合酶和位于Y染色体上的雄性特异标记基因KL-5基因。我们开发了一种简单、非侵入性、非致命性的方法来确定性别，并在幼虫阶段早期识别转基因个体。这种通用的方法适用于大多数昆虫，在昆虫和其他节肢动物的遗传学和生态学研究中具有潜在的应用前景。
Non-invasive genotyping methods provide valuable information on insect populations. However, poor DNA amplification and time-consuming sampling procedures limit these methods, especially for small insects. An efficient and convenient method was developed for non-invasive, non-lethal genotyping of a large insect, Mythimna separata, and a small insect, Drosophila melanogaster, by amplification of endogenous and exogenous, nuclear and mitochondrial genes from insect frass, exuviae, and food waste. For M. separata, the chitin synthesis gene MsCHSB and the COI gene were successfully detected by PCR from exuviae DNA. However, a COI fragment could not be detected directly by PCR from frass, probably due to DNA degradation. To improve the detection rate, DNA from frass was first amplified by Multiple Displacement Amplification with phi29 DNA polymerase, after which the COI fragment was detected from all samples by PCR. For D. melanogaster, second instar larvae were reared individually for three days and then DNA was extracted from food waste of each individual. The endogenous fragment serendipity a (srya), exogenous transgene FC31 integrase, and the kl-5 gene, a Y-chromosome-located male-specific marker gene were successfully detected from most samples. We developed a simple, non-invasive, non-lethal method to determine gender and identify transgenic individuals early in the larval stage. This universal method is applicable to most insects and has potential application in genetic and ecological studies of insects and other arthropods.