CONSTRUCTION AND IDENTIFICATION OF DNA LIBRARIES ENRICHED FOR MICROSATELLITE REPEAT SEQUENCES OF GLYPTOSTERNUM MACULATUM

Microsatellite marker (SSR) has been widely used in population genetics and genetic map construction. In order to determine the genetic diversity of G. maculatum, this study was undertaken to develop and characterize the microsatellite sequence firstly for further to develop the microsatellite markers. Genomic DNA was extracted from muscle tissue using a traditional proteinase K digestion and phenol-chloroform extraction procedure with RNA removed by RNase. Approximately 2 g of total genomic DNA was digested with MboI, then ligated to the adapters (Linker A and Linker B). The treated DNA sample was then pooled and fragments were separated on a 1.5% agarose gel prior to size selection. The resulting fragments (4001000 bp) were extracted from the gel matrix using a column and amplified 20 cycles with Linker B primers. The amplified DNA was hybridized with 5 L of 5'-biotinylated (CA)12 repeat oligos in a total volume of 100 L of 6 SSC and 0.1% SDS. The mixture was incubated at 95℃ for 5min, followed by anneal at 65℃ for 60min and cooled to room temperature. During this hybridization, the 100 L (per treatment) of Streptavidin coated beads was resuspended in 300 L 1 hybridization buffer (6 SSC + 0.1% SDS) and washed three times. The hybridization mixture was added to the washed beads and incubated for 30 min at room temperature. The beads were washed twice with 1 hybridization buffer, twice with high wash solution (0.2 SSC + 0.1% SDS) and twice with high wash solution at 10℃ below the annealing temperature used. The enriched fragments were recovered from the beads by resuspending the beads in 50 L of 1 TE (pH=8.0) and heating to 95℃ for 5min. The enriched solution was removed from the beads immediately using a magnetic stand. DNA containing repeats was amplified with Linker B primers. Cycle conditions were: an initial denaturalization at 94℃ 2min, followed by 20 cycles of 94℃ for 30s, 55℃ for 30s, 72℃ for 60s, with a final extension at 72℃ for 10min. Cleaned products were ligated into a pMD18-T vector (TaKaRa) and transformed into DH5 competent cells. Recombinant clones were detected by PCR amplification using M13 forward and reverse primers as well as (CA)12 probe primer, if the PCR products of some clones contain repeat sequence, the product is two or more bands, or only one band. This experiment obtained 720 clones containing microsatellite sequence, of 720 (89.2%) positive clones, 124 sequences contained repetition that repeated no less than 7 times, according to Weber's classification rules, these sequences were divided into three categories: perfect sequences (64.5% of total), imperfect repeat sequences (32.3%), and compound repeat sequences (3.2%), the repeat time ranged from 7 to 165 with an average of 52. There are 59 sequences can be designed the primer of the 124 sequences and will be used to design primer and test polymorphism in further research. This study provides a base for molecular breeding and assessment of germplasm resources of the Glyptosternum maculatum.