Forensic DNA Division, National Forensic Service, Wonju 26460, Republic of Korea.
Crime-scene DNA Section, Gwangju Institute, National Forensic Service, Gwangju 57231, Republic of Korea.
Forensic Sci Int Genet. 2021 Nov;55:102581. doi: 10.1016/j.fsigen.2021.102581. Epub 2021 Aug 20.
Opium poppy, a member of the Papaveraceae family, is an ancient herbaceous plant and well-known medical resource in the pharmaceutical industry. However, opium poppies are grown worldwide for producing illicit drugs, significantly increasing the incidence of narcotic drug abuse. Since the narcotic poppy has not yet been genetically investigated, we characterized a novel variable number tandem repeat (VNTR) marker of forensically important poppy species based on the genetic analysis of 164 samples collected from two locations spanning the Jeolla province and Jeju island of South Korea. Comparing analysis of the chloroplast (cp) genome sequences for four representative species of Papaver (Papaver somniferum, Papaver somniferum subs. setigerum, Papaver orientale, and Papaver rhoeas) revealed a unique region with 1-3 repeats for 16 nucleotide motifs in the genome inverted repeat A (IRA, positions 128,651 to 128,698) region. For 16 nucleotide motifs, 3 repeats were found in P. somniferum, and 2 repeats were found in P. somniferum subs. setigerum. Therefore, 10 known and the 133 unknown, seized Papaver species were compared to determine whether the species could be identified via variations in the repeat units. The sizes of a novel VNTR ranged from 181 to 252 bp between the species. Phylogenetic analysis confirmed that a novel VNTR, which we named Pscp1, could clearly distinguish between the narcotic and non-narcotic types of Papaver species based on the patterns of sequence variation. Interestingly, we found that Pscp1 could also distinguish between P. somniferum and P. somniferum subs. setigerum. The regions of eight non-narcotic species displayed similar patterns and also differences were found due to the nucleotide substitution and deletion events. The structural differences of Pscp1 were observed within the two narcotic species or between the narcotic and non-narcotic species, suggesting that these variations may act as a genetic marker. We, therefore, developed a new Pscp1 PCR-capillary electrophoresis (CE) method that can reliably identify the narcotic type of Papaver species. Taken together, our findings suggest that the newly developed Pscp1 can be used as an identification marker of opium poppy, and establish that the Pscp1 genotyping method by PCR-CE is an effective primary screening tool that can also contribute to species discrimination in the field of forensic diagnosis and applications.
罂粟，罂粟科植物，是一种古老的草本植物，也是制药行业广为人知的医疗资源。然而，罂粟在全球范围内被种植用于生产非法药物，这大大增加了麻醉药品滥用的发生率。由于尚未对罂粟进行基因研究，我们基于对韩国全罗南道和济州岛两个地区采集的 164 个样本的遗传分析，从具有法医学重要性的罂粟物种中鉴定出一种新型的可变数目串联重复（VNTR）标记。对四种代表罂粟物种（Papaver somniferum、Papaver somniferum subs. setigerum、Papaver orientale 和 Papaver rhoeas）的叶绿体（cp）基因组序列进行比较分析，发现在基因组反向重复 A（IRA，位置 128651 到 128698）区域存在一个独特的区域，其中有 1-3 个重复的 16 核苷酸基序。在 16 个核苷酸基序中，在 P. somniferum 中发现了 3 个重复，在 P. somniferum subs. setigerum 中发现了 2 个重复。因此，对 10 种已知和 133 种未知的罂粟物种进行了比较，以确定是否可以通过重复单元的变化来识别物种。新型 VNTR 的大小在物种之间为 181 到 252 bp。系统发育分析证实，一种新型 VNTR，我们命名为 Pscp1，根据序列变异模式可以清楚地区分麻醉性和非麻醉性罂粟物种。有趣的是，我们发现 Pscp1 还可以区分 P. somniferum 和 P. somniferum subs. setigerum。八种非麻醉性物种的区域显示出相似的模式，并且由于核苷酸取代和缺失事件也发现了差异。在两种麻醉性物种或麻醉性和非麻醉性物种之间观察到 Pscp1 的结构差异，表明这些变化可能作为遗传标记。因此，我们开发了一种新的 Pscp1 PCR-毛细管电泳（CE）方法，可以可靠地鉴定罂粟物种的麻醉类型。总的来说，我们的研究结果表明，新开发的 Pscp1 可作为罂粟的鉴定标记，并证明通过 PCR-CE 进行 Pscp1 基因分型是一种有效的初步筛选工具，也可有助于法医诊断和应用领域的物种鉴别。
