AFLP Analysis On Mitochondrial DNA Of Cytoplasmic Male Sterilities And Its Holdings

The research of cytoplasmic male sterility in plant is important in that of plant male sterility and of using the heterosis.In 1921 CMS was first found in flax,and so far,it has been found in maize,broomcorn,rice,wheat,rape and sunflower. And the phenomenon is more effective in the using of heterosis.Soybean is sylleptic crop of protein and fat.And the yield is always the restrict factor in the production of soybean.The hybrid soybean can evidently increase the yield (13-20%,and so much as higher).So heterosis is a main approach for the productionof our soybean. At present,what is much promising applied in hybrid soybean production is however the three series of male sterility.Davis(1985,American) got the cytoplasmic-nuclear male sterile and holding lines by the cross of cultivated soybeans,but there has been nothing more. Sun huan et al.(1997) raised the cultivated sterility and and soybean the three lines was developed completely and successfully.And they has carried through accurately research of heteditary and cytogenetics. The research of the sterility interplay between cytoplasm and nucleolus in soybean has come into the phase of heterosis use and insect pollination for seeds.At the same time ,there is an urgent research of the soybean male sterility,but up to now the molecular biology research related to the mechanism of cytoplasmic-nuclear male sterile has no report in the world.AFLP is a new method to examine the polymorphism of DNA developed by the Netherlandish scientists named ZABEAU and VOS. It needs less DNA and needn't the cross of Southern,and it needn't know the sequence information of the target genes,such as SSR and ISSR.And the results of the method is steady and credibility,which is much better in the aspect of repeat and accord to the rule of Meng De Er.In one reaction of AFLP there can be measured 50-100 locations,and the polymorphism is so strong that it is very fit for the researchs of the analysis of polymorphism of genetics,the identification of germ plasm, orientation of genes,rapid designing the atlas of heredity,which will be widely applied.As a result of the reasons aboved, AFLP was used recurring to the unique materials(JLCMS1-1A and JLCMS1-1B; JLCMS1-2 A and JLCMS1-2B; JLCMS1-3A and JLCMS1-3B) rooted in Zhao li mei who is working in the center of National Engeering Research for Soybean to analyse the tag of these materials in order to find the differential segments from the mitochondrial DNA between soybean cytoplasmic male sterility and itsholding.AH these will give much help for the orientation of the soybean cytoplasmic male sterility gene and for the guidance of breeding and production.The main results are as follows:1 Via repeat experiments the method are fished out for the purification of soybean mitochondrial DNA which is so pure that can be used for the analysis of AFLP.2 Compare the sequence related to NADH between JLCMS1-2BNADH and other species to get the tree-fig of relativity among many species.3 In my experiment I compare the purposes of different combinations of endoenzyme(Mse I -Hindlll ,Mse I -Pst I ,Mse I -EcoR I) and find that Mse I -Hindlll easily finds the polymorphism by primers combinations suitable for AFLP analysis.And by means of this there are many polymorphism strips. So the double endoenzyme (Mse I -Hindlll) of AFLP was exercised for the analysis of soybean cytoplasmic male sterility.In the cource of my experiment, 64 primer pairs of Mse I and Hindlll were exerted to analyse the twe mtDNA pools which are made of sterilities and holdings and the polymorphisms of sterilities and holdings.4 Through the primary filtration 10 primer pairs were abtained which have many polymorphisms between the sterilities and holdings in mtDNA of soybean,the ratio of polymorphism appreance is nearly 30%.5 By statistic the photogragh I get 2127 strips and at the mean time there are 607 polymorphism strips which is on account of the high polymorphism of AFLP. And getting 7 strips most related to soybean cytoplasmic male sterility which appear in the sterilities but not in their holdings which will give much help for the orientation of the soybean cytoplasmic male sterility gene and for the guidance of breeding and production.6 The system of analysis in the soybean mitochondrial DNA was established using the method of AFLP which includes the preparation of template(mitochondrial DNA),the enzyme-cut and joint of AFLP,preliminary extend,selective extend and the optimized process of silver staining-inspcetion which have formed a working flat roof for other crops using for reference to the analysis related to mitochondrial DNA.At last, the clear and discernable strips obtained in the experiment have provide much beneficial information for the fiirthern research and prove the feasibility of the method of AFLP.