Rapid Development of Microsatellite Markers with 454 Pyrosequencing in a Vulnerable Fish, the Mottled Skate, Raja pulchra

Abstract

The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1–10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy–Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni’s correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.

Genetic characterization of Mytilus coruscus and M. galloprovincialis using microsatellite markers

“…Kang et al design, facilitating the rapid and cost-efficient parallel processing of millions of templates (Abdelkrim et al, 2009;Santana et al, 2009;Zalapa et al, 2012). Recently, NGS has been effectively used to define the MS markers of marine organisms (Saarinen and Austin, 2010;Greenley et al, 2012;Kang et al, 2012;Wang et al, 2012). Another advantage of using NGS for MS markers is that a large number of candidate markers may be identified by pyrosequencing; thus, facilitating the use of identified MS markers in higher cross-species transfer analyses of closely related species (Guichoux et al, 2011;Wang et al, 2012).…”

Development of microsatellite markers for a hard-shelled mussel, Mytilus coruscus, and cross-species transfer

“…The large number of MS candidates identified by pyrosequencing can be especially useful in cross-specific marker transfer, as has been shown for several species (Kang et al, 2012;Wang et al, 2012). The taxonomic range and taxonomic group are the primary factors affecting marker transferability.…”

“…NGS techniques have been applied successfully for the development of MS markers in several marine organisms (Kang et al, 2012;Greenley et al, 2012;Wang et al, 2012). One advantage of NGS for MS marker development is that a large number of candidate sequences can be selected even with stringent criteria.…”

“…Individual reads with NGS can capture individual MSs and their flanking sequences for polymerase chain reaction (PCR) primer design. This new approach has proven to be effective for the development of MS markers from several aquatic organisms (Kang et al, 2012;Greenley et al, 2012;Wang et al, 2012).…”

Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species

“…The large numbers of random sequence reads that contain many thousands of microsatellite markers eliminate the enrichment process used in traditional methods (Hamilton et al, 1999;Zane et al, 2002;Kircher and Kelso, 2010). The other advantage of NGS methods is the high crossspecies transferability of the developed makers, which is due to the large number of candidates that can be tested in related species and has been proven to be effective in several marine species (Saarinen and Austin, 2010;Greenley et al, 2011;Kang et al, 2012).…”