The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

BackgroundThe mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding. Methodology/Principal FindingsThree mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique. Conclusions/SignificanceA COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.研究背景 分类学家已广泛将线粒体细胞色素c氧化酶亚基I（COI）作为标准DNA条形码（DNA barcode）序列，用于众多动物物种的鉴定。然而，COI区域在部分物种的鉴定中应用受限，且并非在所有动物类群中都能通过聚合酶链式反应（PCR）高效扩增。为评估COI作为DNA条形码的实用性并筛选其他候选条形码基因，本研究以长管蚜亚科（Lachninae，半翅目Hemiptera：蚜科Aphididae）为研究对象。我们对比了COI与另外两个线粒体基因——线粒体细胞色素c氧化酶亚基II（COII）与细胞色素b基因（Cytb）的鉴定结果。此外，我们提出了一种新方法，以提升DNA条形码技术在物种鉴定中的效率。 方法与主要结果 我们对3个线粒体基因（COI、COII与Cytb）进行了测序，并将其用于80余种长管蚜亚科物种的鉴定。结果显示，COI与COII的PCR扩增效率优于Cytb。基于COII序列的物种鉴定成功率更高，其中“最佳匹配法（best match）”达96.9%，“最佳近缘匹配法（best close match）”达90.8%；且相较于另外两个分子标记，COII的种内遗传分化更低、种间遗传分化更高。我们提出的“标签条形码（tag barcodes）”新技术，是将物种特异性标签连接至标准DNA条形码的策略。通过该方法，可几乎完全消除“条形码重叠”现象。最终，在使用COI结合“最佳近缘匹配法”技术时，我们将鉴定成功率从83.9%提升至95.2%。 结论与意义 基于COII的鉴定系统在长管蚜亚科物种鉴定中的效果优于COI或Cytb。然而，由于Cytb基因序列多样性较高，其可作为蚜虫种群遗传学研究的有效分子标记。此外，“标签条形码”技术可通过降低或消除种内与种间遗传分化值之间的重叠，提升DNA条形码鉴定的准确性。