Abstract:

Ramie [Boehmeria nivea (L.) Gaud.] is an important bast fiber crop. To study genetic background of this species, the microsatellite markers of ramie were isolated and characterized. Microsatellites were captured by using ramie cultivar Luzhuqing digested with MseⅠand ligated with adaptors and then linked to biotinylated oligoprobe (CT)₁₅ attached to streptavidin-coated magnetic beads (Dynabeads M-280). Double-stranded targeted fragments were obtained by PCR amplification using 21-mer adaptor oligonucleotide as primer, 300–1 000 bp DNA fragments were recovered from 1.5% agarose gel electrophoresis and then religated into pMD18-T vector and transformed into competent DH5α cell. Thus a genomic library containing inserts (GA)_n repeats fragments was constructed and then screened by PCR using anchored simple sequence repeats as primers for the presence of (GA/CT)_n repeats. A total of 22 clones were identified as positives after randomly picked 36 clones, all the 22 clones contained microsatellites and enrichment efficiency was 61.1%, among the existed microsatellites, the expected type of GA/CT is the most popular which accounted for 83.3% and is compatible to the (CT)₁₅ probe, however some other types including （TG/AC）n and more complicated simple sequence repeats with 3 and 6 bases, such as (GAC)_n, (ACG)n, (TCT)n, (TCG)n, (GAA)n, (CCGACG)n, (GAGAAA)_n could also be detected in the microsatellite-enriched library. About 18 microsatellite loci were found after sequencing. Finally, 18 primer pairs were designed on the basis of DNA sequences flanking the repeat motif. The length of GA/CT repeat units varied between 7 and 40 with a mean value of 19.2, which suggests that they would be polymorphic and powerful genetic markers in ramie. These SSR markers are useful for ramie genotype identification, gene and QTL analysis, marker-assisted selection in breeding and pedigree analysis.

Key words: Ramie [Boehmeria nivea (L.) Gaud.], Microsatellites, Magnetic beads, Genomic library, (GA)n repeats.