Isolating Genomic DNA From The Leaves Of Litsea Coreana Lèvl.var.Lanuginosa And Establishment Of RAPD Techniques

Using the leaves of Litsea coreana LevL var. lanuginosa as materials, different waysof isolating genomic DNA were tested to find the best one. At the same time, the optimal Random Amplified Polymorphic DNA (RAPD) techniques for Litsea coreana LevL var. lanuginosa (Migo) Yang et P.H.Huang were established and applied to 80 leaves. The results were as follows:1) Using improved SDS method was an economic, easy and fast way to isolate genomic DNA. It could obtain qualified DNA from plants containing high polyphenols. The DNA was qualified for RAPD analysis.2) Using CTAB method and SDS method could isolate only a little amount of DNA, farther more, the DNA contains secondary substances such as pigment and protein. As a result, the amplification was instable or failed.3) The PCR products were stable when the PCR was 45 cycles of predenature at 95 for 1 minute, denature 94 1 minute, annealing 34 1 minute, extension 72 3 minutes, and when the amplification reaction solution (20ul) was consisted of dNTPs 0.4 l, 10 PCR2 l, MgCl22ul, Taq DNA polymerase 0.3 l(0.5u/ l), H2O 11.3 l , template DNA 2ul(40ng), Primer 2 l(0.8Pm/ l).4) The results also showed that Litsea coreana LevL var. lanuginosa possessedmuch higher genetic diversity and the genetic diversity degree was 46.8%. The specifkpresence or absence ef amplified bands of different materials from different primers,which revealed the speciality in sequences of genomic DNA, could be used for identifying variety of Litsea coreana LevL var. lanuginosa.5) Eighty template DNAs were tested with RAPD markers using 16 arbitrary 10-mer primers selected from 38 primers.