Antolin M F, Bosio C F, Cotton J, Sweeney W, Strand M R, Black W C
Department of Biology, Colorado State University, Fort Collins 80523, USA.
Genetics. 1996 Aug;143(4):1727-38. doi: 10.1093/genetics/143.4.1727.
The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito. Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A aygpti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.
利用聚合酶链反应（PCR）的随机扩增多态性DNA（RAPD-PCR）可高效构建饱和连锁图谱。然而，通过琼脂糖凝胶电泳分析时，大多数RAPD-PCR标记作为显性等位基因分离，减少了获得的连锁信息。我们描述了利用RAPD标记的单链构象多态性（SSCP）分析，在单倍二倍体寄生蜂麦蛾茧蜂和二倍体蚊子埃及伊蚊中构建连锁图谱。RAPD-SSCP分析揭示了在那些似乎作为显性（条带存在/条带缺失）标记分离或在琼脂糖凝胶上表现不变的标记处共显性等位基因的分离情况。我们的SSCP方案使用银染法来检测在大型薄聚丙烯酰胺凝胶上分离的DNA，并且比琼脂糖凝胶电泳揭示更多的多态性标记。在麦蛾茧蜂中，六周内用12个RAPD引物定位了79个标记；在埃及伊蚊中，五周内用10个RAPD引物定位了94个标记。在麦蛾茧蜂中45%的标记作为共显性位点分离，而在埃及伊蚊中11%作为共显性位点分离。对RAPD-PCR标记进行SSCP分析为构建许多物种的密集连锁图谱提供了一种快速且廉价的方法。
