Rico C, Rico I, Hewitt G
University of East Anglia, School of Biological Sciences, Norwich, U.K.
Proc Biol Sci. 1996 May 22;263(1370):549-57. doi: 10.1098/rspb.1996.0083.
Primers for 18 microsatellite loci originally isolated from whiting (Merlangius merlangus, n = 6), stickleback (Gasterosteus aculeatus, n = 5) and cod (Gadus morhua, n = 7) were tested across a panel of diverse fish species, representing the three principal superclasses and most principal superorders of fish, to examine conservation of microsatellite regions across distantly related taxa. Three methods were used. First, amplified fragments were analysed by Southern blotting using the relevant microsatellite motif probes. A total of 17 of the tested primer pairs gave a product in the expected size range in at least four of 11 tested species. Second, for two study loci the amplified polymerase chain reaction products were cloned and sequenced in five fish species to reveal a high level of conservation of the flanking and microsatellite sequences. Finally, the 17 loci successfully amplified in non-source species were tested for polymorphism in groups of unrelated individuals from nine species, in several cases revealing extensive polymorphism. Levels of polymorphism were generally high in species from which the loci were derived or among closely related species. The conservation of flanking sequences for particular microsatellite motifs over the span of fish evolution represented in the test species (470 million years) far exceeds that hitherto reported and lends support to the suggestion (derived from studies of whales and marine turtles) that the rate of base substitution in nuclear and mitochondrial sequences is lower in aquatic than terrestrial organisms. A further explanation could be that these sequences, although generally considered neutral, may play an important role in eukaryotic genomes, and may be under strong selective constraints. The study suggests that heterologous primers will be a ready source of polymorphic markers among fish species, but also indicates that caution should be used in cross-species comparisons of variability.
最初从牙鳕（欧洲无须鳕，n = 6）、棘鱼（三刺鱼，n = 5）和鳕鱼（大西洋鳕，n = 7）中分离出的用于18个微卫星位点的引物，在一组多样的鱼类物种中进行了测试，这些鱼类代表了鱼类的三个主要纲和最主要的目，以研究远缘分类群中微卫星区域的保守性。使用了三种方法。首先，使用相关的微卫星基序探针通过Southern印迹分析扩增片段。在11个测试物种中，共有17对测试引物在至少4个物种中产生了预期大小范围内的产物。其次，对于两个研究位点，在5种鱼类中对扩增的聚合酶链反应产物进行了克隆和测序，以揭示侧翼和微卫星序列的高度保守性。最后，对在非源物种中成功扩增的17个位点在来自9个物种的无关个体组中进行了多态性测试，在几个案例中发现了广泛的多态性。在位点来源的物种或密切相关的物种中，多态性水平通常较高。在测试物种所代表的鱼类进化过程（4.7亿年）中，特定微卫星基序的侧翼序列保守性远远超过迄今报道的水平，并支持了（源自对鲸鱼和海龟的研究）水生生物核序列和线粒体序列中碱基替换率低于陆地生物的观点。另一种解释可能是，这些序列虽然通常被认为是中性的，但可能在真核基因组中发挥重要作用，并且可能受到强烈的选择限制。该研究表明，异源引物将是鱼类物种中多态性标记的现成来源，但也表明在跨物种变异性比较中应谨慎使用。
