2005). According to guidelines formulated in the literature (van Katwijk et al. 2009 and references therein), knowledge of the genetic properties of eelgrass populations is one of the most important factors determining the strategy for its restoration and the selection of an appropriate donor population. In this paper, we present the results of genetic analyses selleckchem of the eelgrass population from Puck Bay and two other populations – from Cudema Bay and Greifswalder Bodden, which are potential sources of planting material for the restoration of the Puck Bay underwater meadows. We developed two multiplex PCR assays
for screening 12 highly polymorphic microsatellites (msDNA) arranged in two sets and loaded on two sequencing panels. The genetic polymorphism indices of the three populations that we studied were compared with those obtained by other authors (Olsen et al., 2004 and Diekmann and Serrao, 2012). Eelgrass specimens (floating shoot fragments) were collected in Puck Bay (PB), Poland (N = 23), Cudema Bay (CB), Sarema Island, Estonia, (N = 24)
and Greifswalder Bodden (GB), Rügen Island, Germany (N = 23) ( Figure 1). At each location shoots Mitomycin C order were collected at 1 m intervals at least. Care was taken to collect samples from various parts of each of the three bays. After collection, shoot fragments were cryopreserved in liquid nitrogen and stored at − 70°C for further analysis. DNA was extracted using the modified phenol:chloroform protocol (Sambrook & Russell these 2006). Shoot fragments were homogenised in Fast Prep-24 Instrument (MP Biomedicals) in 1 ml of extraction buffer (0.2 M TRIS, 1% SDS, 1 mM EDTA, pH = 8) using lysing matrix tubes A (MP Biomedicals); for better precipitation of the DNA, 100 μl of 3 M NaAC (pH = 5), 100 μl of LINEAR ACRYLAMIDE (Invitrogen) and 6 μl of PINK (EMD Millipore)
were added. To prevent DNA degradation all manipulations were performed on ice. DNA was resuspended in 100 μl of water (Sigma-Aldrich). 12 msDNA loci (Table 1) developed for eelgrass by Reusch et al. (1999) and Reusch (2000a) were assigned to the two multiplexes according to the published allele length. Each multiplex was checked in silico using FASTPCR v.3.8.41 software ( Kalendar et al. 2011) and each primer pair was tested for potential primer dimerisation. PCR reactions for microsatellite amplification were performed with forward primers labelled with either 6-FAM, HEX, TAMRA or ROX fluorescent dyes (Applied Biosystems). The optimised reaction mixture (10 μl) contained approximately 100 ng DNA, 1 x MasterMix (Qiagen) and primers at the concentrations given in Table 1. The reactions included an initial denaturation step (15 min at 95°C) followed by 35 cycles of denaturation (30 s at 94°C), annealing (90 s at 60.