Subtropical Horticulture Research Station, USDA-ARS, Miami, FL 33158, USA.
Mars/USDA Cocoa Laboratory, Miami, FL 33158, USA.
Int J Mol Sci. 2019 Nov 26;20(23):5944. doi: 10.3390/ijms20235944.
Routine identification of bark and ambrosia beetles is done using morphology. For people lacking the necessary taxonomic knowledge, proper identification of a novel specimen can be challenging and time consuming. This study compares the usefulness of four genetic markers (28S, EF-1a, ITS2, and COI) and five primer pairs (D2F1/D3R2, eflafor1/eflarev1, ets149/efa754, ITS2F/ITS2R, and LCO1490/HCO2198) to identify Scolytinae beetles, and outlines a molecular identification strategy, with results possible in two days. Markers COI and EF-1a were selected based on the ability of the respective primers to amplify DNA from multiple genera (, , , , and ) and the ability of the resulting sequences to provide accurate and unambiguous matches in GenBank. BLASTn analysis of EF-1a sequences (both primer pairs) correctly identified four out of the five genera and COI sequences identified at least one sample of every genus tested and was the only primer pair to correctly identify specimens. Further, 28S sequences successfully identified , and but not or . The low number of EF-1a (1), 28S (7), and ITS2 (0) sequences from individuals present in GenBank compared with COI (137) is likely the reason that only the latter marker was capable of identifying members of this genus. ITS2 sequences were insufficient to identify any of the samples tested. This study also determined the minimum quantity of DNA that could be used for molecular identification. Primers D2F1 and D3R2, which had the highest rate of amplification in all genera tested, could yield an informative sequence with as little as 0.00048 ng of DNA, however, at least 0.0024 ng was needed for reliable amplification.
常规的树皮甲虫和粉蠹甲虫的鉴定是通过形态学完成的。对于缺乏必要分类学知识的人来说，正确鉴定一个新的标本可能具有挑战性且耗时。本研究比较了四种遗传标记（28S、EF-1a、ITS2 和 COI）和五种引物对（D2F1/D3R2、eflafor1/eflarev1、ets149/efa754、ITS2F/ITS2R 和 LCO1490/HCO2198）在鉴定小蠹科甲虫方面的有用性，并概述了一种分子鉴定策略，结果可在两天内得出。选择 COI 和 EF-1a 标记是基于以下原因：相应引物能够扩增来自多个属（,,, 和 ）的 DNA，以及所得序列在 GenBank 中能够提供准确且明确的匹配。EF-1a 序列（两个引物对）的 BLASTn 分析正确识别了五个属中的四个，而 COI 序列则识别了测试的每个属的至少一个样本，并且是唯一能够正确识别 的引物对。此外，28S 序列成功鉴定了 、 和 ，但不能鉴定 或 。与 COI（137）相比，GenBank 中 个体的 EF-1a（1）、28S（7）和 ITS2（0）序列数量较少，这可能是后者标记能够识别该属成员的原因。ITS2 序列不足以鉴定测试的任何样本。本研究还确定了可用于分子鉴定的最小 DNA 量。在所有测试的属中，扩增率最高的引物 D2F1 和 D3R2 可以产生具有信息量的序列，最低只需 0.00048 ng 的 DNA，但至少需要 0.0024 ng 才能可靠扩增。
