[Isolation and characterization of nuclear microsatellites from Chamaecypress obtusa Endl]

Using 300 approximately 1 000 bp DNA fragments retrieved from 2% agarose gel electrophoresis on nuclear DNA samples of Chamaecypress obtusa with Nde II restricted digestion, microsatellites were efficiently isolated by using Dynabeads M280 streptavitin beads. These beads could immobilize hybrid products between biotin-labeled (CT) 15 probe, and 300 approximately 1 000 bp DNA fragments previously linkaged to Sau3A I cassette. Two enriched microsatellite libraries with about 2 200 positive white clones were constructed by inserting potential microsatellite fragments into vector pUC118 BamH I /BAP and culturing with host E. coli. After sequence analysis on 480 randomly picked out positive clones, it was revealed that the number of SSR-bearing clones accounts for 65.6%, and 215 clones were identified as non-homology. Furthermore,380 microsatellite locus were found in 215 non-homology clones indicating 1.7 loci per clone. Among the existed microsatellites,the expected type of (GA/CT) is the most popular which accounts for 86.1% and were compatible to the (CT) 15 probe, however some other types including (TG/AC)n and more complicated simple sequence repeats with 3 approximately 4 bases, such as (ATAG)n, (CAGA)n, (TGA)n, (ACG)n, (TCA) n, (TCT)n, could be also detected in these two libraries. Finally, primers for amplifying 128 microsatellite locus from 114 SSR-bearing clones were designed by Oligo 5. 1 (http: // lxg. users4. 50megs. com/ssr. html).