LSU rDNA‐BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE) 1
LSU rDNA‐BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE) 1
复制标题
LSU 基于 rDNA 的 RFLP 测定法用于区分亚历山大藻（甲藻科）的物种和菌株 1
DOI：
--
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
D. Anderson
中科院分区：
文献类型：
--
作者：
C. Scholin;D. Anderson
在之前的一项研究中，对来自海洋甲藻 Alexandrium tamarense (Lebour) Balech、A. catenella (Whedon et Kofoid) Balech、A.fundyense Balech、A. affine (Fukuyo et Inoue) Balech、A. minutum Halim、A. lusitanicum Balech 和 A. andersoni Balech 的大亚基核糖体 RNA 基因 (LSU rDNA) 序列进行了比较，以评估种间和种内关系。该研究中比较的许多培养物都含有不止一类 LSU rDNA。对来自单一培养物的 rDNA 汇集克隆进行测序揭示了长度异质性和序列模糊性。这种序列比较变得复杂，因为来自单一培养物的多个 rDNA 克隆必须单独测序，以记录该培养物中存在的不同类别的分子。另一个复杂之处在于观察到的培养内序列变异是否是可靠的遗传标记，或者是否是所采用的聚合酶链式反应（PCR）扩增、克隆和/或测序方法的假象。本研究的目的是使用限制性片段长度多态性 (RFLP) 分析来测试 Alexandrium LSU rDNA 序列的准确性，并设计基于 RFLP 的检测方法来区分该组的代表。对序列的计算机辅助检查使我们能够识别出一组限制性内切酶，预计这些内切酶可以揭示物种、菌株和培养物中 LSU rDNA 的异质性。通过对 PCR 扩增材料进行 RFLP 分析，独立且重复地揭示了通过测序鉴定的所有组。通过限制性消化证实了五个模糊性和一个长度异质性，每个都归因于一组独特的亚历山大藻物种或菌株。观察到的培养物内 LSU rDNA 异质性不是克隆和测序的假象，而是 PCR 反应期间扩增的分子谱的良好代表。因此，培养物中的 LSU rDNA 异质性可作为特定亚历山大藻菌株的独特遗传标记，特别是塔马亚历山大藻、链状亚历山大藻和芬迪亚历山大藻的菌株。然而，其中一些“特征异质性”所代表的 PCR 产物的部分比给定获得的序列所预期的要小。与预测的 RFLP 模式的其他偏差包括不完全消化和出现虚假产物。这些观察结果表明，PCR 产物库中序列的多样性大于通过克隆和测序观察到的序列多样性。这里描述的 RFLP 测试是表征 Alexandrium LSU rDNA 的有用工具，以定义培养物的进化谱系，并且适用于测序所需的时间、成本和劳动力的一小部分。
In a previous study large‐subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter‐ and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment‐length polymorphism (RFLP) analysis and to devise RFLP‐based assays for discriminating among representatives of that group. Computer‐assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR‐amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.