RAPD And ISSR Analysis On Superior Clones Of Pinus Massoniana Lamb.

Pinus massoniana Lamb.is one of the important indigenous plantation and tree species for industrial raw material in south of China.In this experiment 60 accessions of Pinus massoniana Lamb.clones were used as materials to carry out the studies on the genetic relationship and genetic diversity of Pinus massoniana Lamb.by RAPD and ISSR markers.The obtained results were as follows:1.Integrating the previous research on the traits of Pinus massoniana Lamb.,the simple, rapid and accurate method about extraction and purification of Pinus massoniana Lamb.genetic DNA was explored.The improved CTAB method was suitable to extract pure and high-quality genomic DNA from non-lignification and tender tips of Pinus massoniana Lamb.All the aboves were fully suitable for genetic diversity analysis on Pinus massoniana Lamb.clones by RAPD and ISSR markers.Besides,a fit of reliable method for extracting DNA from Pinus massoniana Lamb. was explored and a suit of analysis system of RAPD and ISSR was established.The RAPD reaction system in the research was as follows:the whole volume of the reaction is 25μL reaction mixture containing 0.18 mmol/L dNTP,2.5 mmol/L MgCl₂,0.8μmol/L primer,25 ng/25μL DNA, 1.0 U/25μL TaqDNA polymerse and 1×PCR buffer.And the RAPD PCR program was 94℃for 5 min;45 cycles at 94℃for 1 min,38℃for 1 min,72℃2 min;72℃for 7 min,4℃for 30 min. The best reaction system for ISSR in the research was as follows:the ISSR amplification system was in a 25μL reaction mixture containing 0.2 mmol/L dNTPs,2.5 mmol/L MgCl₂,0.6μmol/L primer,50 ng/25μL DNA,1.0 U/25μL TaqDNA polymerse and 1×PCR buffer;and the ISSR PCR program was 94℃for 5 min;35 cycles at 94℃for 30 s,specific temperature for 45 s,72℃.for 2 min;72℃for 7 min,4℃for 30 min.2.18 random primers were selected and used to analyze the 60 accessions with RAPD,and 202 bands were generated,among which the number of polymorphic bands was 201 and the percentage of polymorphism was equaled to 99.5%.The average number of bands directed by each primer was 11.2;16 random primers were selected and used to analyze the 60 accessions with ISSR,as a result,169 bands were generated,among which polymorphic bands number was 162,and the percentage of polymorphism was 95.1%.The average number of bands directed by each primer was 10.6.Besides,both RAPD and ISSR markers amplified separately 26 and 24 specific bands.Meanwhile,the most Clones of Pinus massoniana Lamb.,which were amplified specific bands have very high yield and economic characters.3.There was a high correlation between RAPD and ISSR,and the correlations were 0.826 between RAPD and ISSR markers,which were significantly correlated in 0.01 level,indicating that the two methods were consistent again and could be used to study the genetic relationship and genetic diversity in Pinus massoniana Lamb.But some cultivars still clustered different between the two markers.4.The dendrogram of clustering analysis and similarity coefficient tables were analysized by SPSS11.5 software,showing the genetic similarity coefficients among the 60 tested samples by RAPD and ISSR were all in the range of 0-1,which proved that the genetic background of the clones in Pinus massoniana Lamb.was so complicated that the genetic variation was greatly affected by different environments and characters and therefore possessed high genetic diversity.5.According to the clustering analysis on the results of RAPD and ISSR,a conclusion could be drawn that the genetic diversity of Pinus massoniana Lamb.clones was rich.The results showed that the genetic diversity of Pinus massoniana Lamb.clones was higher,of which differed distinctly from each other,and the variations of their genomes were very obvious among individuals.Through analysis by RAPD and ISSR molecular markers,the 60 Pinus massoniana Lamb.clones could be classified into three genotypes,namely High-yield Resin,Red Duramen, and Fibre Timber Pinus massoniana Lamb.,which were completely consistent with analysis on phenotypic traits.