A RAPD Study On Genetic Diversity Of 17 Species (33 Materials) In Rubus L. From Southwest Of China

The southwest of China was the center of diversity of Rubus L. and its gerrnplasm resource was abundant. Their resistance to various enviroments, survival rate and fruit flavour were better than the introduced cultivars from abroad, which were a very important characteristic for Rubus breeding. The genetic diversity of 33 materials belonging to 10 subsetion of Sect. Idaeobatus and Sect. Malachobatus collected from southwest of China were studied by using RAPD analyses in order to utilizate the wild germplasm resource better and introduce their special gene to varieties and create new superior cultivars. The results were as follows:(1) Compared DNA extraction of CTAB method, SDS method and nuclear DNA method. The results were shown that nuclear DNA method was a better DNA extraction method of Rubus L., the purity and quality of DNA was high.(2) The 85 random primers screened, 25 primers exhibited sufficient polymorphic band patterns. All the primers can discriminate the materials. Total 589 bands were produced, in which 580 bands were polymorphic. The numbers of DNA bands amplified by each primer were between 19 and 33. The average numbers of DNA bands amplified by each primer were 23.56, the percentage of polymorphic bands was 98.47%, in which the percentage of polymorphic bands of 16 primers was 100%. The materials showed high polymorphism.(3) The optimum reaction system of RAPD-PCR and program for Rubus L. were established. DNA was amplified in 25μL using the reaction mixtures containing 1×PCR buffer, 2.0mmol/L Mg²⁺, 0.24mmol/L of dNTPs, 1.5U of Taq Polymerase, 0.6μmol/L of primer, 20ng of template DNA. The PCR reactions were performed in a thermal cycler (eppendorf) programmed for 1 cycle of 4 min at 94℃followed by 45 cycles of 1 min at 94℃, 50 sec at 36℃and 2 min at 72℃for denaturing, primer annealing and extension, respectively. The last cycle was followed by incubation for 10 rain at 72℃. Amplification products were conserved at 4℃. Amplification RAPD products were analysed by gel electrophoresis run in 1.5% agarose.(4) The result of PCR-RAPD showed the genetic diversity of Rubus L. was abundant in the southwest of China, and it supported the view that the southwest of China was the center of the origin of Rubus L. at molecule level. Abundant germplasm resource could provide more breeding materials and enlarge breeding extent.(5) The genetic diversity of materials was studied and obtained evolution seriatim of subsetion. The results of RAPD analyses were coincident with morphological markers, pollen morphological markers and cytological markers. It demonstrated molecule markers could supply the molecular biology evidence for studying phylogeny of Rubus L..(6) The 33 materials were clustered into 9 groups. The results showed that the genetic relationship and diversity of wild Rubus L. were very complex. The genetic distance among 33 materials were between 0.1957～0.9088. The genetic distance between R. niveus from ya'an laoban mountain and zhangjia mountain was the nearest (0.1957), while R. corchorifolius from ya'an and R. piluliferus from xichong was the farthest (0.9088). The RAPD results were roughly resemble with the materials' morphology.(7) RAPD analyses were useful for identification, classification and genetic diversity analyses of germplasm resource of Rubus L..