Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; South China Sea Environmental Monitoring Center, State Oceanic Administration, Guangzhou 510300, China.
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
Harmful Algae. 2017 Jun;66:20-28. doi: 10.1016/j.hal.2017.04.009. Epub 2017 May 18.
The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.
冈比亚比目鱼藻属是一组底栖甲藻，通常与雪卡鱼中毒（CFP）有关，这种中毒通常发生在世界各地的热带或亚热带地区。属内形态相似的物种毒性可能不同；然而，使用光学显微镜进行物种鉴定可能很困难，有时甚至不可能。因此，核糖体 RNA 基因（rDNA）的 DNA 测序通常用于鉴定和描述冈比亚比目鱼藻属和核糖体型，但对于常规培养筛选和/或大规模监测计划，其费用和时间可能过高。本研究描述了一种基于大亚基 rDNA 分析的限制性片段长度多态性（RFLP）分型方法，该方法可成功鉴定至少 9 种已描述的冈比亚比目鱼藻属物种和 2 种福氏藻属物种。使用 DNAMAN 6.0 和限制酶选择器软件程序来识别一组能够区分大多数具有可用 DNA 序列的已知冈比亚比目鱼藻属物种的限制酶（SpeI、HpyCH4IV 和 TaqαI）。该测定通过计算机模拟分析和培养分离物进行了测试，并且通过 RFLP 分型鉴定的分离物的物种鉴定通过 DNA 测序得到了确认。为了验证该测定并评估 RFLP 图谱中的种内异质性，使用 RFLP 分型对包含 10 种冈比亚比目鱼藻属物种、1 种核糖体型和 2 种福氏藻属物种的 63 个冈比亚比目鱼藻属分离物的鉴定进行了验证，随后该方法用于对从加勒比海收集的分离物进行常规鉴定。本研究提出的 RFLP 测定法通过扫描电子显微镜和/或 DNA 测序减少了与形态鉴定相关的时间和成本，并且为常规冈比亚比目鱼藻属物种分配提供了一种具有系统发育敏感性的方法。
