RAPD Marker Analysis And Core Collection Construction Of Germplasm Resource In Cold-terra Apple

Germplasm resource of cold-terra apple was studied by RAPD marker in this paper. Theresults showed that germplasm resource of cold-terra apple has great value to fruit tree,further study will help to conserve and utilize scientifically and effectively in the future.RAPD markers were used as the main way of research in the experiment. By researchinggenetic diversity of germplasm resource of cold-terra, it indicate that relative and classifyingstatus of different germplasm and searching special RAPD markers of different resources, itcan offer a proof for resources studying, cultivar identification and hybrid early identification..It will be convenient to manage resources and utilize genetic diversity effectively to constructelementarily core collection of resources of cold-terra of fruit tree. The main researches wereas followed:1. The traditional method of extract DNA has been improved, which was apt to extract DNAfrom the leaves of Malus plant tissues rich in protein, polysaccharides, polyphenolics andsecondary metabolite.2. The suitable reaction system and amplification procedure were found. The reaction mixturecontained 20ng/μl template DNA in a 25μl reaction volume, with 10pM RAPD primer1.0μl,1.0U Taq DNA polymerase, 0.2mmol/L dNTP, and 2.5μl PCR Buffer. Amplification wasperformed in a UNOⅡthermal cycle programmed for 35 cycles (94℃for 45s; 37℃for 45s;72℃for 120s) followed by an extension at 72℃for 5min, and then keep at 4℃.3. The genetic relationships 266 cold-terra apple resources were analysed using randomamplified polymorphic DNA(RAPD).25 primers were selected from 200 random 10 bp merprimers, and a total of 245 DNA bands were amplified, 229 of which (93.47%) werepolymorphic. The average number of DNA band amplified by each primer was 9.8.4. There was more than one cultivar specific markers in 33 cold-terra apples, and cultivaridentification and hybrid early identification might be done by using cultivar specific markers.5. Primary core collection containing 180 apples had been established, and a tree diagram wasconstructed by using ward's method. The main results were as follow:①Compared with predecessor research, the classical taxology phylogenetic relationshipamong M. baccata, M. prunifolia and M. Komarovii were validated. But it is different fromthe phylogenetic relationship of wild species. It needs to further study in the future.②Since there is complexity of the cross genetic relationship and argument on classification attaching, it's a good suggestion that the part of resources on classification was looked as thealone a system.6. Finally, 46 cold-terra apples as core collection were screened from 180 cold-terra appleresources in the first time. The diversity and reliability of core collection was identified.It will lay a foundation for conservation and utilization of the cold-terra apple germplasmresources and for constructing genetic map, marker-assisted selection and map based cloningin cold-terra apple. The results, therefore, will produce important effects on geneticimprovement at the molecular levels.