Microsatellite Molecular Marker Enrichment by Magnetic Beads in Flax

Flax (Linum usitatissimum L.) is one of the important oil and fiber crops in the world and can be used as a model plant for bast fiber genomics. The objectives of this study were to set up an efficient protocol to develop microsatellite markers for flax genetic linkage map construction, gene mapping, and marker-assisted selection (MAS). The 300–1 500 bp flax DNA fractions containing microsatellite sequences were captured by hybridizating the digested genomic DNA fragments with the oligonucleotide probes (CT)15 attached to streptavadin coated magnetic beads (Dynal). The enriched DNA fragments were ligated into pMD18-T vector and then transformed into E. coli Top10 competent cells to form an enriched microsatellite sequence library. PCR screen-ing using adaptor primer and VRV (CT)15 as primers identified 104 microsatellite clones from 422 transformants in the libraries. Sequence analysis of these positive clones confirmed 97 microsatellite sequences, with a high enrichment efficiency of 22.99% and PCR screening efficiency of 93.27%. Comparative analysis of the 97 microsatellite sequences showed that 51 among them were of high similarity for microsatellite sequences. PCR amplification using a pair of primers designed from these 51 sequences could successfully identify clones with these high similar microsatellite sequences before sequencing. This method can be used as an efficient tool to eliminate high copy microsatellite clones in screening microsatellite library.