Microsatellite enrichment in Amur sturgeon Acipenser schrencki by magnetic beads

Microsatellite DNAs were prepared from Amur sturgeon Acipenser schrencki genomes by magnetic beads.The genomic DNA was first cut into small pieces by enzyme of Sau 3AI,and the segments with the size of 400-900 bp collected by centrifugation of sucrose density gradient.Then the segments were purified in a low melting agarose gel,connected by a 20 bp linker.Genomic PCR library was created by PCR using the primers designed complementary to the linkers.These genomic DNA fragments were hybridized with a biotin-labeled SRS probe to selected microsatellite DNA.The selected DNAs were amplified,connected by the pGEM-T vector and transformed into competent Escherichia coli DH 5α.In this experiment,the second screening with a radio labeled probe was carried out and partial microsatellite genomic library containing more than 1 400 colonies was constructed.About 300 positive colonies were obtained.Sequencing of 96 positive colonies confirmed that it contained 99 microsatellite loci(number of repeats≥10 times).From these sequences,40 repeat motifs(about 40.4%) were perfect,52 repeat motifs(about 52.5%) were imperfect,and 7 repeat motifs(about 7.1%) were compound.50 pairs of primers were designed with the software Primer 3.0,in which there were 28 pairs amplified special fragments stably and 22 pairs were polymorphism.Meanwhile alleles of the loci were counted in the Amur sturgeon.