Identification Of The Varieties And Analysis On Genetic Relationships Of Vaccinium Spp. Using RAPD Markers

This paper established a method of rapid extraction of genomic DNA from leaves of Vaccinium spp. and the optimum system of Random Amplified Polymorphic DNA (RAPD) for Vaccinium. Genetic relationships among 17 varieties of Vaccinium, including 2 Chinese wild species and 15 cultivars were evaluated by a RAPD analysis. The results will serve as reference to future identification, reservation, utilization, variety selection and cultivation of Vaccinium.The results showed that DNA obtained from the CTAB method was of higher quantity and quality than that from methods of SDS and High salty Low pH. A new method for rapid isolation of genomic DNA based on the CTAB method was also introduced. The genomic DNA extracted by this rapid method was assessed by ultraviolet photometer with value of OD_260nm /OD_{280 nm} from 1.7 to 2.0, and the average DNA production was 68μg/g, which meet the requirements for general molecular and biological experiments, such as PCR, and RAPD and so on. The optimum system of RAPD for Vaccinium was: in 20μL reaction volume system, DNA template concentration 0.5-1.5ng/μL, dNTPs 0.25mmol/L, ten base random primer 1 pmol/μL, Taq Enzyme 1U, and annealing temperature 37℃.15 random primers were screened out with good repetitive property and high polymorphism from 70 random primers. Using these 15 random primers, 115 markers were generated, of which 98.26% were polymorphic. The genetic similarity was ranged from 0.4870 to 0.9130. Cultivar Coville had the greatest genetic distance with the two wild species, V.uliginosum L. bore the greatest genetic similarity with Blomidon, and V.vitis-idaea L. had the greatest genetic similarity with Bluechip. The results also showed that RAPD method could be used for identification of Vaccinium, using one primer to identify two varieties with the same pedigree, and a group of primers to identify all of the experimental materials.The dendrogram of 17 Vaccinium samples was established using UPGMA. The unweighted pair-group results showed that 17 samples could be classified into three major groups: two groups of the each wild species, and one group of 15 cultivars, indicating that the cluster results had some relativity with the pedigree of cultivars.