磁珠富集法筛选大叶藻zostera marina l.微卫星分子标记-screening of zostera marina l microsatellite molecular markers by magnetic bead enrichment method.docxVIP

磁珠磁珠富集法筛选大叶藻(Zostera marina L.)微卫星分子标记 PAGE PAGE VIMicrosatellite Enrichment by Magnetic Beads inZostera marina L.AbstractZostera marina is the most wide-ranging marine flowering plant in Northern Hemisphere. It helps to physically form the habitat and provides food for a variety of marine organisms. Zostera marina is an important member of the coastal ecosystem. Due to impacts from coastal development, degraded water quality, and climate change, the resource of Z.marina has decreased sharply and seagrass meadows has been one of the most threatened ecosystems for many locations worldwide. For the purpose of scientific conservation and sustainable exploitation of these genetics resources, it is necessary to investigate the genetic background using a set of appropriate microsatellite markers.This study was to isolate microsatellite markers from Z.marina genome by magnetic beads enrichments. Through hybridization of biotin-labeled microsatellite oligonucleotide probes, which were captured by streptavidin-coated magnetic with the adaptor-ligated enzyme-digested genome fragments single-stranded DNA fragments containing microsatellites were obtained. The genomic DNA was cut by the enzyme of Mse I and targeted segments were collected with the size of 100 to 800 base pairs. Then the segments were purified in a low melting agarose gel and ligated with a short linker. This genomic DNA was hybridized with a biotin-labeled microsatellite probe (AC)15. The hybridemixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SRS fragments, the eluted single-stranded DNA contains the selected microsatellite DNA. The selected DNAs were then amplified using primers designed complementary to the linkers. The enriched DNA were ligated into pMD-18T and then transformed into E.coli DH5α competent cells. The method of PCR was used to screen positive clones from the libraries, 360 positive clones were obtained. One hundred and three microsatellites w