The Genetic Diversity Of The Plants From The Seedings Of Large Embryo Buds In Loquat (Eriobotrya Japonica Lindl.)

The plants derived from the seeds of large embryo buds were used for experimental materials to study their genetic diversity. Through the morphological observation in the field measurement, clustering analysis was used to assess the morphological diversity. Root tip squash method was used to observe the chromosome number. A modified CTAB method was developed to extract genome DNA of high quality for RAPD analysis. RAPD reaction system was further optimized and screened for the reaction conditions of the present study, and random primers were selected, cluster analysis and similarity coefficient computing were carried out in the study. The main results were as follows.1. Through the field morphological measurement statistics of120material, it was shown that the material in leaf attitude, leaf shape, leaf length, leaf width, leaf apex shape, leaf margin shape, leaf margin serration depth, leaf color(upper side), leaf veins (under side), leaf blade(shape of upper side), leaf texture and so on all had certain differences.2. Using root tip squash method to count the chromosome numbers of the big embryo bud derived plants, the results showed that polyploidy was not present in these materials, namely the size of the embryo buds had nothing to do with the chromosome ploidy of the plants.3. The modified CTAB method was used to successfully extract genome DNA from fresh leaves. DNA extracted by the resultant method of this study was of high quality, OD260/OD280of DNA was1.85-2.03,so it can be used for RAPD analysis later. DNA extracted by electrophoresis results we can see that the quality is better, more complete.4. Using five factor and five level orthogonal design, an optimized PCR reaction system for RAPD was established for the present study:in a total volume of25μL, the reaction mixtures contained2.5μL10×PCR buffer,2.0mmol/L Mg2+,0.20mmol/L dNTPs,1.2U Taq polymerase,0.3μmol/L primer,50ng template DNA, with sterile water making up25μL. The temperature profile used for RAPD-PCR was94℃for5min, followed by40cycles at94℃for1min,38℃for1min, and72℃for1.5min, and was terminated with a7min DNA extension step at72℃. Amplification products were conserved at4℃.5. Adopting the optimized reaction system and screened primers from100random primers, after repeating three times,11primers were selected which had good polymorphism, amplification replication with more clear bands. Using the11primers to amplify the DNA of both the population from large embryo bud derived plants and the maternal material,119bands were amplified,10.8bands on average for each primer, including104polymorphism bands, accounting for87.4%,9.5polymorphic bands on average for each primer. According to the results of amplification, a UPGMA clustering figure was constructed. The120plants were divided into four major categories.6. Through the analysis by POPGENE1.32, Nei’S gene diversity and Shannon’s information index were obtained for the seedlings from the large embryo buds. A rich genetic diversity and some mutants were found in the individuals from the large embryo buds, which suggested that the seedlings from the large embryo buds could be very useful for breeding in loquat.