Abstract
Here, we report the results of next-generation sequencing on the GS Junior system to identify a large number of microsatellites from the epiphytic moss Orthotrichum speciosum. Using a combination of a total (non-enrichment) genomic library and small-scale 454 pyrosequencing, we determined 5382 contigs whose length ranged from 103 to 5445 bp. In this dataset we identified 92 SSR (simple sequence repeats) motifs in 89 contigs. Forty-six of these had flanking regions suitable for primer design. We tested PCR amplification, reproducibility, and the level of polymorphism of 46 primer pairs for Orthotrichum speciosum using 40 individuals from two populations. As a result, the designed primers revealed 35 polymorphic loci with more than two alleles detected. This method is cost- and time-effective in comparison with traditional approaches involving cloning and sequencing.
Highlights
Due to their high polymorphism and reproducibility, co-dominant SSR markers are widely used in population genetics and phylogeographic studies [1]
This paper describes the development of SSR markers for the epiphytic moss species Orthotrichum speciosum
A single sequencing run of Orthotrichum speciosum DNA library in the GS Junior pyrosequencing system resulted in 139,886 reads with an average read length of 426 bp
Summary
Due to their high polymorphism and reproducibility, co-dominant SSR (simple sequence repeats) markers are widely used in population genetics and phylogeographic studies [1]. SSR markers are applied to determine the taxonomic status of species at the early stages of divergence [2]. Despite their numerous advantages, SSR markers can still be problematic to use. The drawback of highly-specific SSR markers is their laborious development. The traditional method of developing SSR markers is both labor-consuming and expensive, and it often generates a small number of polymorphic loci [3]. Methods in which non-specific markers such as AFLP [4,5], ISSR [6] and RAPD [7] are used for enrichment are commonly employed, which does not exclude the cloning process and clone screening
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