HYBRIDIZATION OF MICROSATELLITES TO RAPD - A NEW SOURCE OF POLYMORPHIC MARKERS
HYBRIDIZATION OF MICROSATELLITES TO RAPD - A NEW SOURCE OF POLYMORPHIC MARKERS
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DOI：
10.1093/nar/23.18.3798
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发表时间：
1995-09-25
影响因子：
14.9
通讯作者：
WEISING, K
中科院分区：
文献类型：
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作者：
RICHARDSON, T;CATO, S;WEISING, K
用于分类学、进化和生态学研究的动植物种群和物种的遗传分析极大地受益于各种分子标记技术的发展，这些技术在DNA水平上揭示了多态性。一般来说，基于DNA的标记具有重要的优势，如选择性中性，丰富，高变异性和环境影响的独立性。在植物中最广泛应用的技术是基因组RFLP分析（1）、使用小型或微卫星互补探针的多位点DNA指纹分析（2-3）、单个微卫星位点的PCR扩增（4）和使用扩增基因组匿名区域的任意或半特异性引物的PCR（5-7）。RFLPs和PCR扩增微卫星标记是一种具有位点特异性的共显性标记，在群体遗传学研究和高密度连锁图谱的构建中具有重要的应用价值。然而，这种位点特异性的方法需要分子克隆程序，而多位点指纹和匿名PCR策略不需要。因此，后者的技术是首选的工具，研究物种很少或没有DNA序列信息。本文报道了一种将任意引物或半特异引物PCR与微卫星杂交相结合的新方法，在每个电泳凝胶上产生多个独立的多态性遗传指纹。不需要先前的序列信息。这种新的方法，我们称之为RAMPO（随机扩增微卫星多态性），通常适用于植物，最有可能也适用于动物和人类。基因组DNA首先用单一的任意或微卫星复合物PCR引物扩增。PCR产物经电泳分离和染色后，将凝胶干燥或印迹到尼龙膜上，随后与[32 P]标记的微卫星互补寡核苷酸探针（例如[CA] 8）杂交。放射自显影检测可重复的和多态的指纹图谱不对应于染色模式，并且对于每个探针是完全不同的。在利用微卫星互补寡核苷酸作为PCR引物（MP-PCR）进行基因组指纹分析时，偶然发现了匿名基因组PCR产物中存在的二核苷酸重复序列。我们发现，番茄和猕猴桃模板DNA的Southern印迹MP-PCR或RAPD产物与32 P标记的二核苷酸重复探针[GT] 8或[GA] 8的杂交，在放射自显影后产生强烈而明显的信号（6）。我们怀疑这种带型可能代表了真核生物中多态性标记的另一个来源。在本研究中，我们测试了这一预测与各种物种和品种的属番薯（山药），以及其他一些植物和真菌物种。用不同微卫星引物或随机引物产生的PCR产物进行扩增、印迹，然后与[CA] 8和[GA] g进行杂交。由于已知使用任意引物的PCR对实验条件的变化敏感，我们对每个模板/引物组合进行了四次重复实验以确保再现性。溴化乙锭染色的PCR产物显示不同的带型与任意和微卫星引物。将RAPD和MP-PCR产物与[CA] 8和[GA] 8杂交产生了新的和高度可重复的指纹图谱（数据已提交但未显示）。这些图谱与染色模式完全不同，并且强烈依赖于特异性引物/探针组合。与众不同...
Genetic analyses of plant and animal populations and species for taxonomic, evolutionary and ecological studies tremendously benefited from the development of various molecular marker techniques which reveal polymorphisms at the DNAlevel. In general, DNA-based markers have important advantages such as selective neutrality, abundance, high variability and independence of environmental effects. Among themost widely applied techniques in plants are genomic RFLP analysis (1), multilocus DNA fingerprinting with mini-or microsatellite complementary probes (2-3), PCR amplification of individual microsatellite loci (4) and PCR with arbitrary or semispecific primers that amplify anonymous regions of the genome (5-7). RFLPs and PCR-ampli-fied microsatellites are locus-specific, codominant markers which proved highly useful for population genetic studies and the establishment of high density linkage maps. However, such locus-specific approaches require molecular cloning procedures whereas multilocus fingerprinting and anonymous PCR strategies do not. Therefore, the lattertechniques are preferred tools to study species where little or no DNA sequence information is available. Here we report a new method which combines arbitrarily or semispecifically primed PCR with microsatellite hybridization to produce several independent andpolymorphic genetic fingerprints per electrophoretic gel. No prior sequence information is needed. This novel method which we call RAMPO (random amplified microsatellite polymorphisms) is generally applicable for plants, and most probably for animals and humansas well. Genomic DNA is first amplified with a single arbitrary or microsatellite-comple-mentary PCRprimer. After electrophoretic separationand staining of the PCR products, the gel is either dried or blotted onto a nylon membrane, and subsequently hybridized to a [32P]-labelled, microsatellite-complementary oligonucleotide probe (eg [CA] 8). Autoradiography detects reproducibleand polymorphic fingerprint profiles which do not correspond to the staining patterns, and which are completely different for each probe. The presence of dinucleotide repeats in anonymous genomic PCR products was detected incidentally during the use of microsatellite-complementary oligonucleotides as PCR primers (MP-PCR) for genomic fingerprinting. We found that hybridiza-tion of Southern-blotted MP-PCR or RAPD products of tomato and kiwi fruit template DNA to 32P-labelled dinucleotide repeat probes [GT] 8 or [GA] 8resulted in strong and distinct signals after autoradiography (6). We suspected that such banding patterns might represent an additional source for polymorphic markers in eukaryotes. In the present study, we tested this prediction with a variety of species and cultivars of the genus Dioscorea (yams), as well as some other plant and one fungal species. PCR products generated by different microsatellite or arbitrary primers were electrophoresed, blotted and successively hybridized to [CA] 8 and [GA] g. Since PCR with arbitrary primers is known to be sensitive to variations in experimental conditions we performed four replicate experiments for each template/primer combination to ensure reproducibility. Ethidium bromide staining of electrophoresed PCR products revealed distinct banding patterns both with arbitrary and microsatellite primers. Hybridizing the RAPD and MP-PCR products with [CA] 8 and [GA] 8 yielded novel and highly reproducible fingerprinting profiles (data submitted but not shown). These profiles were completely different from the staining patterns, and strongly depended upon the specific primer/probe combination. Distinctly different …