Establishment And Application Of STab AFLP System For Soybean Research

Great success has been achieved in the development of hybrid varieties in several major crops,including rice and maize. Due to lacking of genetic male sterile resources or mutants, soybean is oneof a few crop species, in which heterosis has not been exploited until in 1993 Huan Sun et al. foundthe soybean CMS line after about twenty years' work, which promoted the research and applicationin this area, however, there is still no work was reported on molecule mechanism untill now.Two Netherlandish scientists (Zabeau and Vos) developed AFLP (Amplified fragment lengthpolymorphism) marker system in 1993. The advantages of RFLP and RAPD were combined inAFLP. This technique need only less DNA sample and need no DNA hybridization procedure andsequence information compared with RFLP. AFLP offeres more sTab amplified products than that ofRAPD. Thus it has been used widely in fingerprint map, the construction of genetic linkage map, andthe genetic diversity research. In some experiments with less genetic diversity, for example, in nearisogenic line (NIL) and the BSA analysis, AFLP analysis could produce abundant geneticpolymorphism information and has been widely applied to the high-density genetic map and finegenetic map. The AFLP technology has been used in wheat, paddy rice, corn, soybean and cotton etal., Moreover, AFLP has been used widely in vegeTabs (potato, tomato, chick-pea and so on), forestplants (hangs a birch, Monterey pine) and Arabidopsis. Untill now, most of the research resultsmentioned 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,whichneed pepole who do this work should be skillful and familiar with this method. Therefore, it need usto grasp the key technical and optimizing the experiment condition so as to achieve sTab results withhigh efficiency.In this paper, we have worked on AFLP key technique development and optimizing for soybeanso that we could use this technique to study the diversity of cytoplamic male sterility (CMS) soybean.The main results were as follows:1. Via repeat experiments the method is fished out for the purification of soybean mitochondrialDNA which is so pure that can be used for the analysis of AFLP.2. By the research of enzyme dosage, digestion time, ligation time, dilution times, we set up thesuiTab silver staining AFLP analysis system of soybean. In 20μL digestion system, it containedpurified mtDNA of soybean 500 ng, T₄ Ligase (5 u/μL) 0.5μL, T4 Buffer 2.5μL, BSA(10 mg/μL) 0.1μL, Hindâ…¢(Dalian Po biotechnology company)3 U, Mseâ… (NEB company)3 U, Hindâ…¢adapter(50μM) 0.1μL ,Mseâ… adapter (50μM)0.1μL, add ddH₂0 to 20μL, digestion under 37℃for fourhours; It took more than ten hours(or overnight) for adapter ligation under 8℃. Ligation productsshould be diluted by 5 times before pre-amplification and then pre-amplification products should bediluted by 5 times for selective amplification; Then selective amplification products should besubjected to denaturing after adding 5μL loading buffer under 95℃for 10 minutes, then cooled onice immediately for 5 minutes; then the denatured products were separated in 6ï¼…denaturingpoly-acrylamide gel electrophoresis (PAGE) analysis and detected by silver staining.3. In my experimentâ… compare the purposes of different combinations of endoenzyme(Mseâ… -Hindâ…¢, Mseâ… -Pstâ… , Mseâ… -EcoRâ… ) and find that Mseâ… -Hindâ…¢easily finds thepolymorphism by primers combinations suiTab for AFLP analysis.And by means of this there aremany polymorphism strips. So the double endoenzyme (Mseâ… -Hindâ…¢) of AFLP was exercisedfor the analysis of soybean cytoplasmic male sterility.In the cource of my experiment, 64 primerpairs of Mseâ… and Hindâ…¢were exerted to analyse the twe mtDNA pools which are made ofsterilities and holdings and the polymorphisms of sterilities and holdings.4. Through the primary filtration 10 primer pairs were abtained which have manypolymorphisms between the sterilities and holdings in mtDNA of soybean, the ratio of polymorphismappreance is nearly 30ï¼….5. By statistic the photogragh I get 2127 strips and at the mean time there are 607 polymorphismstrips which is account of the high polymorphism of AFLP. And getting 7 strips most related tosoybean cytoplasmic male sterility which appear in the sterilities but not in their holdings which willgive much help for the orientation of the soybean cytoplasmic male sterility gene and for theguidance of breeding and production.