苹果属植物RAPD分析的影响因素及其稳定性研究

Factors Influencing RAPD Analysis in Genus Malus

以富士苹果和山定子为试材对苹果属植物ＲＡＰＤ分析的最佳反应条件及其可重复性等进行了研究，结果表明，ＲＡＰＤ反应的的各个条件因子，包括模板ＤＮＡ浓度，引物浓度ｄＮＴＰ浓度，ＤＮＡ聚合酶的种类和浓度ＰＣＲ扩增循环数以及反应总体等都对ＲＡＰＤ结果有影响，都有其最适用量范围，各因子在低子最适值时会导致扩增带数的减少甚至无带，高于最适值则无出现带的弥散或缺失。

Fuji apple and Malus haccata were employed to study the factors influncing RAPDanalysis in genus Malus. The results be that the RAPD band patterns of genus Maluswere affeced by a variety of factors including the concentrations of template DNA, primer.dNTP,Taq polymrase, as well as the source of Taq polymerase, the number of PCR cyclingand the total volume of reation mixture. The optimized RAPD reaction conditions for genusMalus were 5~20ng template DNA, 20~40ng primer(0. 13~0.27μM), 0. 8~ 1. 6 bot Taqpolymrase,and 100~200μM dNTP in a total of 35 ~50μL reaction mixture. Lower concen-tration caused the decrement or lack of RAPD bands and higher amount resulted in smear ofRAPD bands or failure of amplification. Under optimized reaction condition, RAPD band pat-terns of genus Malus were quite repeatable. The kind (leaf or bark) and growing stage(youngor old) of plant tissue from which template DNA were extracted as well as the DNA isolationmethods had no significant influence on RAPD band pattem. RAPD products could keep stableunder 4℃ for at least 3 months.