Many sharks and skates are particularly vulnerable to overfishing because of their large size, slow growth, late maturity and low fecundity. Among the 227 species in 25 genera of the family Rajidae, 11 skate species belonging to four genera have been identified in Korea to date. Skates are commercially important in Korea, and the mottled skate, Raja pulchra, is the most favored and valuable species because of its superior meat quality [1]. This species is one of the most expensive fishes sold in South Korea, reaching US $10–30 per kilogram. This species is also valued in China and Japan for their dried wings and meat, but is less expensive (US $5) than in South Korea.

Raja pulchra, a low-boreal subtropical species abundant in the coastal waters of some areas of Korea and Japan, is usually caught at a depth of about 5–100 m [2–4]. This species has been known to inhabit the East China Sea, the Yellow Sea, the East Sea, the Pacific coast of Japan, the southern Kurils, and a recent survey has shown that they are present off the coast of western Sakhalin [5,6].

Catch data from Korea shows a 90% decline over a ten-year period. The average annual catch from 1991–1993 was 2700 metric tons, whereas the catch in 2011–2003 was 220 metric tons [7], reflecting a serious decline in the biomass collected. In China, there has been an overall reduction in all fish biomass due to overexploitation and heavy trawling within the mottled skate’s range, as well as habitat degradation as a result of such fishing activities. The overall decline in numbers of this species has exceeded 30% over the last 15 years (a period of three generations) [8]. As a result, the International Union for Conservation of Nature (IUCN) has designated it as “vulnerable” and in fact, the decline may be severe enough to warrant a status of “endangered” [8]. However, there are no conservation measures in place for this species. Further assessment and monitoring of catches in fisheries and population trends are required.

Microsatellite (MS) or simple sequence repeat (SSR) markers are versatile molecular tools for determining parentage, inferring genetic structure and gene flow patterns and for assessing the origins of introduced populations [9,10]. Traditionally, the isolation of MS DNA loci has relied on the screening of genomic libraries using repetitive probes and sequencing of positive clones to develop locus-specific primers. While effective in many applications, these cloning and sequencing procedures are tedious, labor-intensive and expensive [11,12].

Next generation sequencing (NGS) technologies have revolutionized many life science fields because of its rapid cost-efficient parallel processing of millions of templates [13]. NGS is capable of generating gigabases of sequence data in a single run, with individual reads long enough (>500 bp) to capture individual microsatellites and the flanking sequence for PCR primer design. Additionally, there is no need for enrichment because even a fraction of a run can provide sufficiently large numbers of random sequence reads to contain many thousands of microsatellites markers. Application of this technique to expressed genes would make it possible to develop panels of MS markers for genes underlying phenotypic variation [14].

Based on these advantages, this new technology is expected to replace conventional microsatellite isolation protocols [15], and there are increasing reports employing NGS MS markers in studies of mammals [16], plants [17,18], insects [19], and amphibians [20]. This new technology also has been applied in the development of microsatellite maker in marine organisms and was proven to be effective [21–24].

In this work, we carried out a global analysis of the repetitive fraction of the R. pulchra genome using NGS combined with the bioinformatics approach described above. This work will be useful for future resource management of this economically and ecologically important, but vulnerable, species.

Among 11 skate species identified in Korea so far, R. pulchra is the most economically important species, and it is consumed raw or as fermented fish. It inhabits coastal areas around the Korean Peninsula and was extremely common about 20 years ago in the Yellow Sea, especially around the Daecheong-do and Heuksan-do Islands of Korea. As in other skates, reproduction of R. pulchra is oviparous, and the number of eggs in each spawning is less than 250 [4]. Because there has been a >90% catch decrease over the past ten years, it has been placed on the “vulnerable” species list by the IUCN [8]. Therefore, it is necessary to understand the genetic structure and population diversity for future management and resource restoration.

MS markers have many advantages over other molecular markers for the study of population structure and diversity [25]. However, the cloning and sequencing processes commonly used in MS marker development are time consuming and costly, often with positive clone yields as low as 0.03% [26].

NGS has recently been applied in the development of MS markers in many organisms, greatly reducing the cost, labor and time required [13,15,16,27]. This method has been shown to be more effective when enriched DNA libraries are used for sequencing [28]. Despite the advantages, the development of MS markers using NGS in aquatic organisms has been limited, but has been proven to be effective in recent publications [21–24,29].

In addition to speed and cost-effectiveness, NGS offers high flexibility in primer design because a large number of reads and sequences containing SSRs and loci with the proper repeat units can be selected. In our study, 17,033 (5.5%) from a total of 312,290 contigs contained dinucleotide SSRs, and 33 loci containing a minimum of nine repeat units were used for primer design, which is only 0.2% of the identified SSRs. In the analysis of MS markers reported in plant, only 1% of polymorphic loci identified using NGS were selected by stringent criteria for primer design, compared with 20% of SSRs discovered using a traditional cloning and sequencing process were selected for primer design [30].

To select optimal primers from a large number of candidates, high-quality standards can be applied in loci selection, which can increase the success rate and reduce the time, labor and cost of marker development. For instance, to minimize the risk of null allele amplification, 454 sequence alignments can be used to design primers using loci with a reasonably high depth of coverage at both primer-annealing sites, and that show no evidence of SNPs within the regions [31]. In our study, 20 (62%) primer sets among 32 loci containing a minimum of nine repeat units produced amplified PCR products, and 14 (70%) of them were polymorphic.

After sequential Bonferroni correction for multiple tests, significant deviations from HWE in the direction of heterozygote deficiency were detected at only two (Rp3-nfrdi and Rp43-nfrdi) of the 14 tested loci in the Heuksan-do population. Generally, heterozygote deficiencies increase due to factors such as inbreeding, substructuring of the population sample, or the presence of null alleles. Indeed, our Microchecker analysis revealed the presence of null alleles at four loci, including Rp3-nfrdi and Rp43-nfrdi. The level of genetic diversity (mean Ho = 0.57, He = 0.61; mean allelic number = 4.7) in this study was slightly higher than that of the common skate, Dipturus batis (mean Ho = 0.35, He = 0.36; mean allelic number = 3.5) [32]. No genetic differentiation between populations by F_ST may suggest that the two populations can be regarded as one population. However, differentiation of genetically related skate population by distance has been observed in northeast Atlantic continental shelf [32]. To reduce the time and cost of MS marker development, pre-existing markers from related species have been applied to the study of many animal, fungus, plant and fish species [33,34]. Moreover, cross-species markers can be used for the identification of invading species and the identification of parental origin in hybrid fish [35]. Although the cross-species transferability of MS markers is unevenly distributed among taxa, over 40% and 25% of polymorphic marker transfers have been observed in fish of different genera within the same family, and of families within same order, respectively [33].

With large numbers of SSR candidates identified by pyrosequencing, the development of cross-species markers has been attempted in diverse organisms [36]. The markers developed in this study were tested for cross-species amplification and possibility of finding universal markers for cartilaginous fish with available fish samples. Among the ten markers polymorphic in R. pulchra, four (40%) were polymorphic in other species within same family, which is comparable to previous data [33]. However, none of them showed any polymorphic amplification of targets in specimens belonging to different families within the same species. One interesting finding was that loci Rp16 and Rp35, which do not amplify the target sequences in the family Dasyatididae, produced monomorphic or polymorphic PCR products in Carcharhiniformes. Although the numbers of genera and samples for each genus are limited, these results suggest the possibility that cross-species polymorphic markers can be developed in fish species using the pyrosequencing technique with large number of SSR candidates for marker development.

The rapid population decline of R. pulchra around the Korean Peninsula, especially in the Yellow Sea, is due to many factors such as overfishing and destruction of their natural habitat. However, there have been no conservation measures in place for this species. Therefore, the molecular markers developed in this study will be useful for future assessment and monitoring of population trends of this species. In addition, this study suggests that pyrosequencing methods will be useful for SSR development in aquatic organisms, and may be preferable to the traditional labor- and time-intensive cloning methods.

The pyrosequencing method was applied in the development of MS markers for R. pulchra, which is listed as a “vulnerable” species by the IUCN. A comparably high primer-to-marker conversion ratio (62%) was achieved by this method. In the cross-species amplification, over 90% of the markers were amplified in the fishes belonging to the same family but the success ratio was much less in the different family. The molecular markers developed in this study can be used for future management of this economically and ecologically important species.