RAPD And ISSR Analysis Of Main Pinus Massoniana Genetic Resources In 12 Porvinces Of China

In this experiment 35 accessions of Pinus massoniana Lamb. genetic resources in 12Provinces of China were used as materials. The method of tender tip DNA extraction wasstudied; and then the RAPD and ISSR reaction system was established and optimized; and thegenetic relationship and genetic diversity of Pinus massoniana Lamb were discussed by RAPDand ISSR markers and clustering analysis. The obtained results were as follows:1．The available method of DNA extraction from tender tip of Pinus massoniana. Pinusmassoniana was rich in the secondary metabolites such as polysaccharides, polyphenols andproteins, which makes it rather difficult to obtain high quality genomic DNA from their tissues.The improved CTAB method was suitable to extract pure and high-quality genomic DNA fromtender tip of Pinus massoniana for RAPD and ISSR amplification. But some attentions shouldbe paid in this method. First of all, in order to overcome the intervention of the polysaccharides,the detergent must be used before cell nucleus was split. Secondly, 2% PVP was added in theextraction buffer to overcome the intervention of the polyphenols. Thirdly, beta-sulfhydryl groupalcohol as reducer was used twice to restrain the oxidization of the polyphenols and others.2．Optimization of RAPD reaction system and ISSR reaction system in Pinusmassoniana. Based on the adjusting experiments, the RAPD reaction system in our researchfollowed as: the RAPD amplification system was in a 25μL reaction mixture containing 25 ngDNA, 2．5 mmol/L MgCl, 0．8μmol/L primer, 0．16 mmol/LdNTP, 1．0 U TaqDNA polymerse and1×PCR buffer; and the RAPD PCR program was 94℃for 5 min; 45cycles at 94℃for 1 min,37℃for 2 min, 72℃1min; 72℃for 7 min. The best reaction system for ISSR in our researchfollowed as: the ISSR amplification system was in a 25μL reaction mixture containing 50 ngDNA, 2．5 mmol/L MgCl, 0．5μmol/L primer, 0．2 mmol/LdNTPs, 1．0 U TaqDNA polymerse and1×PCR buffer; and the ISSR PCR program was 94℃for 5 min; 35cycles at 94℃for 30 s,specific temperature for 45 s, 72℃for 2 min; 72℃for 7 min.3．The analysis of RAPD and ISSR polymorphic degree in Pinus massoniana. 18primers were selected from 200 random 10-meroligo-mucleotide primers. They were used to analyze the 35 accessions with RAPD, and 153 bands were generated, among which the numberof polymorphic bands was 137 and the percentage of polymorphism was equaled to 89．5%. Theaverage number of bands directed by each primer was 8．5; 16 primers, which could amplifiedbands clearly, were selected from 60 ISSR primers. As a result, 153 bands were generated,among which polymorphic bands number was 136, and the percentage of polymorphism was88．9%. The average number of bands directed by each primer was 9．6．Through relevant analysis,the results gained through RAPD and ISSR molecular markers were significantly correlated. Theconclusion indicated that the two methods can be used to study the genetic relationship andgenetic diversity in Pinus massoniana and the ISSR was more effective than the RAPD.4．Clustering analysis of RAPD and ISSR in Pinus massoniana. Regardless of RAPD orISSR, the genetic distances among 35 accessions all were between 0 and 1 .The correlations was0．771 between RAPD and ISSR markers, and they were significantly correlated in 0．01 level,which indicated that the two methods were consistent again and they can be used to study thegenetic relationship and genetic diversity in Pinus massoniana.