Ender A, Schwenk K, Städler T, Streit B, Schierwater B
J.W. Goethe-Universität, Abteilung Okologie & Evolution, Frankfurt, Germany.
Mol Ecol. 1996 Jun;5(3):437-41.
Simple sequence repeats (SSRs, or microsatellites) have been constantly gaining importance as single-locus DNA markers in population genetics and behavioural ecology. We tested a PCR-based strategy for finding microsatellite loci in anonymous genomes, which avoids genomic library construction and screening, and the need for larger amounts of DNA. In the first step, parts of a genome are randomly amplified with arbitrary 10mer primers using RAPD fingerprinting. Labelled SSR-oligonucleotides serve as probes to detect complementary sequences in RAPD products by means of Southern analyses. Subsequently, positive RAPD fragments of suitable size are cloned and sequenced. Using GA and GT probes, we applied this approach to waterfleas (Daphnia) and revealed 37 hybridization signals in 20 RAPD profiles. Thirteen positive RAPD fragments from three Daphnia species and two hybrid 'species' were cloned and sequenced. In all cases simple sequence repeats were detected. We characterized seven perfect repeat loci, which were found to be polymorphic within and between species.
简单序列重复（SSRs，或微卫星）作为群体遗传学和行为生态学中的单基因座DNA标记，其重要性一直在不断增加。我们测试了一种基于PCR的策略，用于在匿名基因组中寻找微卫星位点，该策略避免了基因组文库的构建和筛选，以及对大量DNA的需求。第一步，使用RAPD指纹技术，用任意的10聚体引物对基因组的部分区域进行随机扩增。标记的SSR寡核苷酸用作探针，通过Southern分析检测RAPD产物中的互补序列。随后，将合适大小的阳性RAPD片段进行克隆和测序。使用GA和GT探针，我们将这种方法应用于水蚤（水蚤属），并在20个RAPD图谱中揭示了37个杂交信号。从三种水蚤物种和两种杂交“物种”中克隆并测序了13个阳性RAPD片段。在所有情况下都检测到了简单序列重复。我们鉴定了七个完美重复位点，发现它们在物种内和物种间具有多态性。
