Microsatellite Marker Enrichment with Magnetic Beads in Flax

The objective of the study was to set up an efficient protocol for developing microsatellite markers in flax (Linum usitatissimum L.). Fractions of 300–1500 bp flax DNA containing microsatellite sequences were captured by hybridizing the digested genomic DNA fragments with the oligonucleotide probes (CT)15, which were attached to streptavadin-coated magnetic beads (Dynal). The enriched DNA fragments were ligated into pMD18-T vector and then transformed into Escherichia coli Top10 competent cells to form an enriched microsatellite sequence library. PCR screening 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 93.3%. Comparison analysis showed that 51 of the 97 microsatellite sequences were of high similarity. PCR amplification using a pair of primers designed from these sequences successfully identified the clones containing these high similar microsatellite sequences before sequencing. This method is proved to be efficient in screening specific markers in microsatellite libraries.