Rapid and effective isolation of candidate sequences for development of microsatellite markers in 30 fish species by using kit-based target capture and multiplexed parallel sequencing

摘要

Recent advances in next-generation sequencing (NGS) technology have accelerated the development of microsatellite markers for wildlife conservation genetics. Although the discovery of microsatellite-containing sequences based on NGS is more efficient with sequencing of a microsatellite-enriched library than with whole-genome shotgun sequencing, the process of constructing a microsatellite-enriched library is somewhat complicated. Therefore, many researchers prefer to use external services for the microsatellite-enrichment, which requires more time. To facilitate the rapid and effective development of novel microsatellite markers, we attempted to simplify the process of constructing a microsatellite-enriched library for multiplexed parallel sequencing. To capture microsatellite-containing sequences, we applied an easy-to-use commercially available kit for the hybridization and wash steps. After preparing shotgun libraries of 30 fish species for NGS, we captured microsatellite-containing DNA fragments directly from the shotgun libraries by using the commercially available kit. Next, three runs of multiplexed parallel sequencing were conducted on the 454 GS Junior platform. The resulting sequences for each species included high proportions of microsatellite-containing sequences (from 46 to 79%). Thus, sufficient numbers of primer sets, ranging from 1029 to 6606, were effectively designed for each species. Microsatellite capture and sequencing were completed in about a week, so the time required was substantially reduced. To validate the effectiveness of our strategy, we screened 44 potential primer sets designed for ayu (Plecoglossus altivelis). The results of polymorphisms revealed that allelic variability at 23 markers will be useful for studying population structure. These results prove the effectiveness of our improved approach for microsatellite marker development.