Establishment Of AFLP System For Kidney Bean Reasearch And Varieties Selection For Low-elevation Area Of Chongqing

Two Netherlandish scientists(Zabeau and Vos)developed AFLP(Amplified fragment length polymorphism)marker system in 1993.The advantage of RFLP and RAPD were combined in AFLP.This technique need only less DNA sample and need no DNA hybridization procedure and sequence information compared with RFLP.AFLP offeres more sTab amplified products than that of RAPD.Thus it has beem used widely in fingerprints map,the construction of genetic linkage map,and the genetic diversity research.In some expriments with less genetic diversity,for example,in near isogenic line(NIL)and the BSA analysis,AFLP analysis could produce abundant genetic polymorphism information and has been widely applied to the high-density genetic map and fine genetic map.The AFLP technology has been used in wheat,paddy rice,corn,soybean and cotton et al.,Moreover,AFLP has been used widely in vegetabs(potato,tomato,chick-pea and so on),forest plants(hangs a birch,Monterey pine)and Arabidopsis.Until now,most of the research results mentioned above indicated that AFLP produces more abundant genetic diversity than that of RFLP,RAPD and so on.AFLP analysis has a lot of steps and the whole procedure need a long time to complete,which need people who do this work should be skillful and familiar with this methed.Therefore,it need us to grasp the key technical and potimizing the experiment condition so as to achieve sTab results with high effciency.In this paper,we have worked on AFLP key technique development and optimizing for kidneybean so that wo could use this technique to study the diversity of cytoplamis male sterility(CMS)kidney bean.The main results were as follows:1.Via repeat experiments the method is fished out for the purification of kidney bean mitochondrial DNA which is so pure that can be used for the analysis of AFLP.2.By the research of enzyme dosage,digestion time,ligation times,we set up the suiTab silver staining AFLP analysis system of kidney bean,In 20μL digestion system,it contained purified DNA250ng,T₄Ligase1.5U,T₄Buffer3.5μL,EcoR I 3U,Mse I 3U,EcoR I adapter 5pmol,Mse I adapter 50pmol,add ddH₂O to 25μL,digestion under 37℃for 4h;It took more than 8h(or overnight)for adapter ligation under 37℃.Ligation products should be diluted by 5 times before pre-amplification;Then selective amplification products should be subjected to denaturring after adding 5μL loading buffer under 95℃for 10 minutes,then cooled on ice immediately for 5 minutes;then the denatured products were separated in 6%denaturing poly-acrylamide gel electrophoresis(PAGE)analysis and detected by silver staining.3.In my experimentâ… compared the purposes of different combinations of endoenzyme(Mseâ… ,EcoRâ… ,Mseâ… -EcoRâ… )and find that Mseâ… -EcoRâ… easily finds the polymorphism by primers combinations suiTab for AFLP.And by means of this there are many polymorphism strips.So the double endoenzyme(Mseâ… -EcoRâ… )of AFLP was exercised for the analysis of kidney bean.In the cource of my expriment,16 primer pairs of Mseâ… and EcoRâ… were exerted to analyse the DNA of kindney bean.4.Through the primary filtration better primer pairs were abtained which have many polymorphisms between the sterilities and holdings in DNA of kidney bean,;5.By statistic the photograghâ… get 1774 strips and at the mean time there are 475 polymorphism strips which is account of the high polymorhpism of AFLP.The experiment will give much help for the research of molecular marker for kidney bean.three varieties were selected by yeild test for two years,as Xiaohei kidney bean,LS-141-1,LRK333.Their high yeild and good economic traits are all fit for Chongqing to develop.