Relationship Of Crabapple Of Malus By AFLP

Wild crabapple not only have high ornamental value, but also cold resistance, droughtresistance, anti water logging, these wild crabapple has a significant role in the landscapingand rootstock seedlings, cultivation of new varieties, they also could be used in making wineand dried fruit, treating diseases and has a high application value. AFLP is a molecular markertechnology that is widely used, which has some merits of high polymorphism and goodstability, AFLP is often applied to investigation of Malus germplasm resources. We exploredthe genetic relationship of the wild crabapple resources, cultivated species crabapple of Malusand representative species of hawthorn and pear, the cluster analysis of these materials is alsodone, the job we did provide materials for the research of wild crabapple resources andfoundation for their further study.The main research results of this experiment are as followings:1. Improved CTAB method is suitable for the extraction of DNA of Malus, Hawthorn,Pear：Adding3%beta-mercaptoethanol,1%PVP to the3%CTAB extract buffer, the DNAwas deposited with icecold anhydrous ethanol and7.5M ammonium acetate at the same timeand was purified by chloroform/isoamyl alcohol(23:1, v/v) after RNA was digested by RNAenzyme. The DNA was detected by UV spectrophotometer and agarose gel electrophoresis,the results showed that the DNA has good quality, so it could be used in AFLP experiment.2. AFLP silver-staining analysis system suitable for the genomic DNA of tested materialswas determined: the total volume of enzyme digestion is20μL, including10μL ddH2O, DNA5μL(100~500ng),10×Tango buffer4μL, MseI5U, EcoRI5U, it was incubated at37℃for5h;Put5μL connection mixed liquor into the digested solution, then the mixed liquor wasincubated at22℃for12h, after that the pre-amplification reaction was done; Thepre-amplification produce was also diluted10times with ddH2O and used for selectiveamplification; The samples were denatured by adding8μL formamide loading buffer andheated up at95℃for8min,then cooled on ice-bath; Selective expansion product wereanalyzed on6%denaturing polyacrylamide gel and stained by silver.3. Selected4primer pairs that have clear bands and highly polymorphic from64primercombinations and used for further analysis of30tested materials. A total of192bands wereamplified, polymorphic bands were170, polymorphism percentage was88.5%.4. AFLP amplification results of30tested materials were clustered, kinship tree was also established. The results showed that the similarity coefficient of the tested materials rangedfrom0.64to1.00. The tested materials can be divided into4groups according to the SMcoefficient0.73; Two wild species, Hawthorn, Malus, Pyrus each clustered into one group,Malus materials could be assigned to4groups at0.78, the first group and the second groupcan be divided into different subunits according to the similarity coefficient, there is richgenetic diversity between interspecies of crabapple of Malus.5. Separate analysis of M. baccata, M. micromalus, M. spectabilis, M. prunifolia wascarried out. The dendrogram showed that M. micromalus has nearer relationship with one ofits parents，according to the coefficient of genetic distance and dendrogram, comparativeanalysis, we can concluded that another parent of M. micromalus may be M. prunifolia.