New microsatellite loci for the green sea urchin Strongylocentrotus droebachiensis using universal M13 labelled markers

The green sea urchin Strongylocentrotus droebachiensis is one of the dominant grazing species in temperate marine ecosystems. Catastrophic overgrazing events have been recorded in the Pacific [1] and the Atlantic [2] including the northern Norwegian and Russian coast, where approximately 2000 km² of kelp forest were grazed to barren grounds in the 1970s [3, 4]. Because of the persistence of the sea urchin dominance and the large loss of biodiversity and production, it is important to develop new genetic tools for studying and improving our understanding of this species. Microsatellite loci have been previously developed using North American individuals [5]. Further analysis have shown that the Northeast Atlantic population, based on one sampling location in Iceland and one in Norway, is differentiated compared from Northwest Atlantic and Pacific populations [6]. Moreover, when tested on the two Northeast Atlantic locations, locus Sd76 was reported failing to amplify any of the individuals and the remaining 3 loci showed fewer alleles compared with North American locations. These results have motivated our goal to develop the present microsatellite loci using a S. droebachiensis individual from a Norwegian population (Drøbak) in an effort to increase genetic information produced for the study, primarily, of the Northeast Atlantic populations.

A simple optimised DNA extraction was performed using QuickExtract (Epicentre Technologies Corporation, Madison, USA). Briefly, 10 to 20 mg gonad material from one individual preserved in Ethanol 96% was washed in distilled water prior to adding 100 μL QuickExtract buffer. Samples were incubated at 65°C for 10 min followed by 98°C inactivation for 5 min. The lysates were further diluted 10^-2 in Tris EDTA buffer (Fluka Chemie GmbH, Buchs, Switzerland) prior to performing PCR. A 3-primer PCR approach using a M13 tail (5’-TGTAAAACGACGGCCAGT) for the forward primer was used for microsatellite loci amplification at concentrations as described previously [8]. Four different dyes were used for the universal M13 forward primer to enable fragment analysis multiplexing [9]. Simplex PCR amplifications, targeting one locus at a time, were performed using a CFX96 thermocycler (Bio-Rad, Hercules, CA, USA) in 10 μL reaction volume containing 5 μL iProof Master Mix (Bio-Rad), 0.04 μM of the forward primer with M13 5’-tail and 0.16 μM of each reverse and forward tagged M13 primers (Eurofins MWG, Ebersberg, Germany) and 2.5 μL sample. Reaction volume was completed with sterile deionised water. PCR amplifications were optimized and carried out under the following conditions: a denaturing step for 1 min at 98°C, followed by 30 cycles of 98°C for 5 s, 62°C for 10 s and 72°C for 15 s followed by 8 cycles of 98°C for 5 s, 57°C for 10 s and 72°C for 15 s. Up to 4 different simplex PCR plates, each with a different dye (Table 2), were mixed and diluted by transferring 5 μL each to a plate prefilled with 100 μL deionized water per well. From this dilution plate 1.2 μL per sample was transferred to the run plate prefilled with 10 μL Hi-Di Formamide (Applied Biosystems, Foster City, CA, USA) and 40% strength orange standard (MCLAB, San Francisco, CA, USA). PCR product sizes were determined using a 3730XL DNA analyser (Applied Biosystems) and scored using GeneMapper software version 4.0 (Applied Biosystems). GenAlEx software version 6.5 was used to report overall observed (H_o) and expected (H_e) heterozygosity [10]. Linkage disequilibrium and Hardy-Weinberg equilibria (HWE) were tested in Arlequin software version 3.5.1.3 [11].

We observed linkage (Likelihood ratio test, p < 0.05) among loci in all four populations, but no pair of loci was linked in all of them. Five loci (Strdro-1051, Strdro-1356, Strdro-4147, Strdro-5563, and Strdro-5590) showed heterozygote deficiencies and HWE deviations (Exact test, p < 0.05) in all populations, possibly suggesting inbreeding and substructuring or the presence of null-alleles. The fact that simplex PCR was performed for all loci strongly reduces the risk for weak amplification that may be observed in a multiplex assay and hence reduces possible false negative loci amplification results.

The loci presented in this study show higher allelic diversity than that reported previously for Northeast Atlantic populations using loci developed on Northwest Atlantic individuals [6]. Interestingly, heterozygote deficiencies and significant HWE deviation were also found with 3 of the 4 microsatellites used in this prior study [6], hence suggesting that S. droebachiensis may naturally deviate from HWE as it has been reported to be the case for many other marine invertebrates [12]. We believe that these new loci will be useful for the study of S. droebachiensis, in particular for the Northeast populations, for better monitoring the observed ongoing population distribution and densities fluctuations along the Norwegian coastline.