Microsatellite enrichment by magnetic beads in chrysanthemum

Chrysanthemum (Chrysanthemum morifolium), originating from China, is an important ornamental, edible, tea and medicine plant, but now there is still a lack of knowledge about its genetic and molecular backgrounds. The objectives of this study were to set up an efficient protocol to isolate microsatellite and screen SSR (Simple sequence repeat) primers for chrysanthemum DNA fingerprint development. Genomic DNAs of chrysanthemum were cleaved with Mse I, fragments of 250-1500 bp DNA were recovered, then added with adapters, and amplified by PCR later. The 300-1500 bp DNA fractions containing microsatellite sequences were captured by hybridizing the digested genomic DNA fragments with the oligo nucleotide probes (CT)15 or (TGA)8 attached to streptavidin-coated magnetic beads (Dynal) respectively. The enriched DNA fragments were ligated into pGEM-T Easy vector and then transformed into Escherichia coli Top10 competent cells to form two enriched microsatellite sequence libraries. 144 and 118 microsatellite clones were obtained from the two libraries respectively with PCR identification of microsatellite arrays (PIMA), further 91 and 92 microsatellite sequences were obtained respectively by sequencing analysis. Eighty-eight microsatellite sequences can be used to design primers. According to the 88 microsatellite sequences 142 primer pairs were designed. 23 SSR primer pairs with high polymorphism were obtained by primary screening.