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

Abstract

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.

Morphological variation associated with trophic niche expansion within a lake population of a benthic fish

“…The sampling sites included almost all types of bottom environment where P. esocinus inhabits (Fig 1, Table 1). We screened 48 microsatellite loci developed by Takeshima et al (2016) [33] using specimens from the mtDNA group A and B and developed 14 microsatellite primer pairs available for the both groups (S2 Table).…”

Interploidy gene flow involving the sexual-asexual cycle facilitates the diversification of gynogenetic triploid Carassius fish

“…To characterize the genetic composition of gynogenetic triploid Carassius , we conducted target resequencing on available nuclear markers used in Cyprinidae phylogeny 31 and previous studies on Carassius 27 , 32 , 33 . The PC1 axis of principal component analysis (PCA) based on 1,100 informative single nucleotide polymorphisms (SNPs) from 30 nuclear markers of 15 sexual diploid and 19 gynogenetic triploid Carassius (Fig.…”

“…Ethanol-fixed tissues were used for total genomic DNA extraction using the Wizard Genomic DNA Purification Kit (Promega). The ploidy of C. auratus -complex specimens was estimated using the amplified number of alleles from 12 microsatellite loci 23 , 33 . This method had previously achieved > 97% matching with the result of flow cytometry in three geographically distant Japanese populations 23 .…”

“…We first screened the available nuclear markers used in Cyprinidae phylogeny 31 and previous studies on Carassius 27 , 32 , 33 . Target resequencing of 30 well-amplified loci was conducted for 15 diploid and 19 triploid geographically representative C. auratus -complex specimens, in addition to C. cuvieri and Cyprinus carpio (Fig.…”