Genetic Diversity Of Pinus Massoniana Lamb.Excelent Families Using ISSR And EST-SSR Markers

Pinus massoniana Lamb is native tree species of Southern China and industrial raw materials, which possessing the characteristics of widely distributing, adaptable, and widely useing. The paper research72individualplants from25excellent family through methods of ISSR and EST molecular markers. And the main results are as follows:1.The paper uses masson pine DNA, discusses the massoniana of EST-SSR PCR reaction system in the template DNA, the concentration of Taq DNA polymerase, dNTPs and Mg2+, primer and primer annealing temperature.The stable and reliable optimal system for massoniana EST-SSR PCR like this:2μL10buffer (Mg2+Free),30ng template DNA,0.187mmol/L, dNTPs,20μL system containing,3.75mmol/L of Mg2+8pmol of primer,1.0U of Taq DNA polymerase, the annealing temperature is56℃.The reaction procedure as follows:94℃pre-denature for5min; denature at94℃for30s, annealing at56℃for30s, and extension at72℃for30s,35cycles; extension at72℃for7min; preservation at4℃. The paper probes the annealing temperature in the gradient of55℃～62℃using PCR instrument functional gradient.2. The paper selects9stable and polymorphic pimers from100ISSR primers, with which amplifing25excellent pinus massoniana pamilies, the results of analyse of amplification as follows:the observed number of alleles was1.9820, the effective number of alleles was1.4714, Polymorphic loci percentage (P) is98.2%, the total shannon information index (I) and a total of Nei's gene diversity (h) respectively is0.4538and0.2927, coeffieient of gene differentiation of massoniana (Gst) is0.7429, gene flow (Nm) was0.8730<1, indicating gene flow between populations, are not sufficient to prevent genetic differentiation caused by genetic drift between populations, all these indicate the massoniana excellent family retains high genetic diversity and complexity since be selected repeatedly. Cluster analysis showed that the most individual from the same family cluster together, and shows a closer genetic relationship, but there exists individual variation. The overall results, massoniana excellent show high genetic polymorphisms dispite being seleted several times,so the method of building seed orchard is desirable.3. The paper selects6pairs primers from51pairs EST-SSR primers, and analyse pinus massoniana excellent families using these primers, the tesult shows that the number of polymorphic locus is27, polymorphic loci percentage (P) is99%, Observe Numbers of alleles is0.3347, the total shannon information index (I) is0.4927. It can be found that trees with good traits cluster together because they may have closer affinities.4. The methods that used in paper have own characteristic.ISSR marker has more polymorphic locus, and its band is stable,repeatable.Otherwise, EST-SSR markers are from express sequences, so EST-SSR marker is related to characteristics, this method providing the conditions for molecular breeding. From the research point of view, two molecules can get more information than a single molecule.