Isolation And Characterization Of Microsatellites From Ramie(Boehmeria Nivea(L.)Gaud) And Its Application For Analysis Of Genetic Relationships In Sect.Tilocnide

In the research, 31 microsatellites in ramie were isolated by RAPD and Magnet beads enrichment protocols. Of the two enrichment methods, the second one was proved to be more efficient. 31 pair primers were designed by Net Primer and Primer 3 after we analyzed the microsatellite sequnces. The optimization system to SSR reaction of ramie was also obtained. Meanwhile, Those locus-specific SSR markers isolated and characterized by ourselves were used to evaluating the genetic relationship in Sect. Tilocnide.The major results are as follows:1) 250 recombinant positive clones of RAPD microsatellites-enriched library of ramie were rapid screened with PIMA protocol. 13 microsatellites in twelve recombinant positive clones of ramie were obtained after sequencing 26 positive clones, which potentially containing microsatellites. These 13 microsatellites were enrolled in GenBank.2) 36 recombinant positive clones of oligoprobe hybridized microsatellites-enriched library of ramie were screened by PCR protocol for the second method. 18 microsatellites were obtained after sequencing 22 positive clones, which potentially containing microsatellites. Oligoprobe hybridized microsatellites-enriched method was proved to be more suitable for isolating microsatellites of raime because it not only has the advantages of RAPD enrichment protocol, but also has its own advantages: straightforward purpose, high efficiency (61.1%) , long repeat fragments isolated(19.2). It was proved to be a reliable method for isolating abundant microsatellites of ramie.3) The optimization to SSR reaction of ramie was obtained: each PCR reaction contained 50ng of genomic DNA, 1 .5mmol/LMgCl₂, 0.01mmol/L of each dNTP, 0.2μmol/L primer, 1U Taq DNA polymerase.Of these 31 primers, 24 pair primers amplified predicted size fragments and were ploymorpic in germplasms of Sect. Tilocnide.Nine piar primers were chosen to analyze genetic relationship in Sect. Tilocnide.The result showed that B. nivea var viridula was at the lowest position in the dendrogram, and the B. nivea var tenacissima took the second place, B. nivea and B. nivea var nipononivea have the equivalent phylogenetic place. The evolutionary pathway was B. nivea var viridula→ B. nivea var tenacissima→ B. nivea or B. nivea var nipononivea.