Barr Norman B
Center for Plant Health Science and Technology, Mission Laboratory, USDA-APHIS, Moore Air Base, Edinburg, TX 78541, USA.
J Econ Entomol. 2009 Feb;102(1):401-11. doi: 10.1603/029.102.0153.
The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method described in a previous publication to document worldwide population structure of Mediterranean fruit flies and analyze probable sources of fly introductions is re-evaluated. Analysis of representatives of the RFLP-based markers using the ND5-tRNA-ND4 mitochondrial locus through DNA sequencing shows that the original markers produced haplotypes that are homoplasic and exclude valuable information. DNA sequences also identified errors in data generated with the protocol because of failure of the XbaI enzyme to efficiently cut PCR product. In all, the PCR-RFLP method is able to document haplotypes present in medfly populations outside of Sub-Saharan Africa and is a cost-effective approach to pathway analysis. New primer pairs for analysis of this locus are reported that reduce the number of PCR steps required to analyze the RFLP marker and the time of each PCR run. For comparison of the PCR-RFLP method, the utility of a DNA sequence-based approach to Mediterranean fruit fly pathway analysis is reported using the ND5-ND4 locus and the proposed "barcode region" of the COI gene. Both gene sequences are more informative than the PCR-RFLP method and document high genetic variation within Sub-Saharan Africa. Application of these methods as tools and their interpretation is discussed.
对先前发表的用于记录地中海实蝇全球种群结构并分析实蝇引入可能来源的聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法进行了重新评估。通过DNA测序对基于RFLP的标记物的代表进行分析，使用ND5-tRNA-ND4线粒体基因座，结果表明原始标记物产生的单倍型是同塑的，并且排除了有价值的信息。DNA序列还识别出由于XbaI酶未能有效切割PCR产物而导致的该方案产生的数据中的错误。总体而言，PCR-RFLP方法能够记录撒哈拉以南非洲以外的地中海实蝇种群中存在的单倍型，并且是一种用于途径分析的经济有效的方法。报告了用于分析该基因座的新引物对，这些引物对减少了分析RFLP标记所需的PCR步骤数量以及每次PCR运行的时间。为了比较PCR-RFLP方法，报告了使用ND5-ND4基因座和提议的COI基因“条形码区域”的基于DNA序列的方法在地中海实蝇途径分析中的效用。这两个基因序列比PCR-RFLP方法提供的信息更多，并记录了撒哈拉以南非洲内部的高遗传变异。讨论了将这些方法用作工具及其解释。
