| 研究报告 |
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| Isolation of microsatellite DNA from endangered tree species Toona ciliata var. pubescens and optimization of SSR reaction system |
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Abstract T. ciliata var. pubescen is an endangered tree species. The population genetic structure should be essential to conserve it effectively. Genomic DNA was successfully extracted from fresh leaves of Toona ciliata var. pubescens using CTAB method. Microsatellite DNA was isolated from genomic DNA with improved method of streptavitin beads.We got rid of some tedious and inefficient processes, such as gel extraction.The genomic library which enriched with microsatellite was constructed.After sequencing the positive clones,we found eighteen positive clones enriched with microsatellite.Eighteen SSR primer pairs were designed and synthesized.The factors which affected on the SSR reaction of Toona ciliata var. pubescens were studied.The results showed that the optimized concentration of DNA is 30ng ; the concentration of dNTP is 0.3 m mol·L-1;0.3μmol·L-1 is the optimized concentration of primers.Through above PCR system, repetitive and steady SSR reaction system was constructed.This is the base of studying the population genetic structure and genetic variation of Toona ciliata var. pubescens.
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Received: 31 July 2006
Published: 25 December 2006
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