Annua Liu Microsatellite Enriched Library Construction And Screening Marker,

Salix L., belonged to Salicaceae, had an abundance of species, about 500 species of arbor and frutex totally. It was important forestry colony, ecologically and commercially imported. Especially, it had high heterozygosity and easy hybridization, some species generation was short, yearly blossoming and seeding. For all its characteristic, it had a high potential for use in forest genetical study.Microsatellites (SSR), short stretches of tandemly repeated 1-6 nucleotide sequences, were ubiquitously present in eukaryotic genomes. Microsatellites markers were co-dominantly inherited, high degree of polymorphism, conserved markers. Therefore, it had widely been applied for studing population, genetic linkage mapping, QTL location, assistiant selection as a marker system seemed the best candidate.However, The actual use of microsatellites markers for analysis of Salix L. had been very scarce, which limited its application in Salix L.The objectives of this work were to seek afer some strategies for developing new microsatellite markers for Salix L., its emphasization on construction enriched microsatellites libraries of S.caprea developed a rapid and efficient identification microsatellite procedure in Salix L.: with biotinylated(CT)₁₅ and (GT)₁₅ oligoprobes to cross-hybidize to Sau3A I-digested microsatellites fragments in S.caprea, according to affinity between biotinylated oligoprobes and Streptavidin Magnesphere Paramagnetic Particles, 1056 clones were gained. Then, 384 clones were secondly screened by DIG-(CT)₈ and (GT)₈. At last, 360 clones and 24 positive clones after second selected were sequenced. It was suggested that SSR-containing clones is 183(162 from 360 clones(45%), 21 from24 positive clones after second selected(89%)).Of the 44 SSR-containing clones identified in non-homology plaques, 53 SSR loci were gained. Through analysis of repeats types, the proportion of (TC/AG)n, (GA/CT)n and (CA/GT)n repeats isolated occurred relatively high, approximately 74% totally. There existed other motifs including mononucleotide repeats (A/T)n, dinucleotide repeats (AT/TA)n and trinucleotide repeats (GGT)n, (ACC)n, (CTC)n, (GCT)n, (CCT)n, (GGC)n.In addition, analysising the sequenes by Repeatmasker searching the database of Salix in Genebank, 78 microsatellites-containing sequences were identified from 480(16.25%). The di-motif (AT)n, (CT)n, (TG)n types were the most abundant microsatellites. 8 SSR sequences, obtained from Salix viminalis Lambda TriplEx cDNA, were used for designing as primer pairs, which presented data for in-depth analysis, as well as providing ready access to mine data resources availed.19 SSRs, out of 53, were designed as primers and then tested for crossamplification on pooled panels comprising individuals from 5 species of Salix L.. As a result, 5 pairs preliminary identificated were successfully cross-amplifiedted in the most cases. Moreover, 2 of the 5 microsatellites(5c5l and Scs3) revealed polymorphism within 16 individuals of S.suchowensis, 10 individuals of S.integra, respectively.6 microsatellites primers of P. trichocarpa Hook., out of 23, gave amplification on S.suchowensis and S.integra tested, one microsatellite revealed polymorphism when being further tested within 23 individuals of S.integra. The fact experimentally confirmed good transportability across Populus L.and Salix L.