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新菠萝灰粉蚧(Dysmicoccus neobrevipes Beardsley)是我国进境植物检疫性有害生物,与近似种菠萝灰粉蚧(Dysmicoccus brevipes(Cockerell))从形态上很难区分,给口岸快速准确鉴定带来了困难。本研究尝试采用快速、易于操作的DNA条形码技术对两种粉蚧识别鉴定。实验选择两种粉蚧的不同地理种群共65个样本,选用3个分子标记(COI、28S和18S)进行引物筛选、基因扩增、序列分析。结果表明:引物(18S_2880_F/18S_B_R;28S_S3660_F/28S_A335_R;Pco_F/C1-12342_R)能够成功扩增所有测试样品;3个分子标记都存在条形码间隔,COI最大,28S次之,18S最小;结合Gen Bank数据库中的同源序列进行系统发育分析,28S的NJ树图与COI相似,均以高支持率(97%和100%)将所有样本分成2个独立分支,分析结果与形态鉴定结果一致。与28S和COI相比,18S基因序列矩阵构建的聚类树含低支持率分支。本研究扩增的COI及28S基因区段均可作为快速准确鉴定新菠萝灰粉蚧与菠萝灰粉蚧的有效分子标记。
Dysmicoccus neobrevipes Beardsley, a quarantine pest of entry plants in China, is difficult to distinguish from the approximate species Dysmicoccus brevipes (Cockerell), which makes it difficult for the port to be quickly and accurately identified . This study attempted to identify and identify two mealybugs using a fast, easy-to-use DNA barcoding technique. Sixty-five samples from different geographical populations of two mealybugs were selected for the experiment. Three molecular markers (COI, 28S and 18S) were selected for primer selection, gene amplification and sequence analysis. The results showed that all the test samples could be successfully amplified with primers (18S_2880_F / 18S_B_R; 28S_S3660_F / 28S_A335_R; Pco_F / C1-12342_R); all of the three molecular markers had barcode spacing with the largest COI, 28S followed by 18S; The NJ tree map of 28S was similar to that of COI, and all samples were divided into two independent branches with high support rate (97% and 100%). The analysis results were consistent with those of morphological identification. Compared with 28S and COI, the clustering tree constructed by the 18S gene sequence matrix has a low support rate branch. The amplified COI and 28S gene segments can be used as effective molecular markers for the rapid and accurate identification of Neisseria gonorrhoeae and Pincton pineapple.