Genetic analysesThe Zostera project in addition to in vitro method has also an interesting part such as genetic research. This research is very important to testify a theory that the Polish coastal zone is populated by clones of one individual Zostera marina. Through genetic analyses we will try to determine whether this theory is true or false.
To perform this type of analyses these indispensable information is needed mostly on how to collect the material for genetic test. Thorsten Reusch has provided the information about a methodology for the collection compiled by Olsen et al. 2004, Reusch et al. 2000, Reusch 2002, Ferber et al. 2004. This protocol has been used for ~60 populations of Zostera marina worldwide.
Collection of eelgrass material for genetic analysis
- Sampling area: 40 m (parallel to shore) x 20 m (perpendicular to shore) within the depth horizon with the densest meadow, typically 2-3 m. Deviations from this scheme are necessary if (i) beds are very patchy (ii) slope is steep and eelgrass grows in narrower band alongshore.
- Nsamples (ramets = "plants") = 50
- Desirable: true random allocation according to random x-y-coordinates (takes 3-4 man hours by experienced divers). The 2nd best option is a haphazard collection to both sides of a 40 m transect line, or if this is not available, in a random walk fashion. Always keep minimal distance of >= 1 m in order to avoid sampling individuals belonging to the same clone. Samples can be best collected into ziplock bags. If mapped locations, take already numbered ziplock bags under water. If non-destructive sampling is desired, a piece of leaf (2-3 cm) can also directly be bagged underwater into numbered ziplock bags (see below).
- We have found that the best method for underwater (SCUBA) or intertidal collections are sandwich size (preferably freezer weight) zip-locks (~2-4 L). These are numbered and taped together at the bottom edge with duct tape (10 at a time is convenient) with the zip-lock edges open. The finished product will look like a book with "fanable" pages. The bags are easy to handle and can be reused many times. If you are working with a partner, it is easiest if one person picks the leaves and the other holds the bags. The process is a bit trickier underwater, but still doable.
- Note: If time is severely constraint, picking 50 plants in bulk collection is ok, processing of single samples can then occur on shore /boat, this will save a lot of time. Here you need to make absolutely sure that each picked leaf bundle represents one defined location 1m away, i.e. that you do not preserve several leaf pieces from the same rhizome cluster, as this will result in inflated clonality estimates.
- Sample preservation by drying: prepare 2 mL Eppendorf tubes with half silica gel. From the larger plant, take 2-3 cm of green leaf (typically 1st to 3rd leaf), blot dry with paper towel (important!), stuff in tube – ready.
- Samples can also be stored in small ziplock bags filled with 3-5 g silica gel. Lab grad silica often comes with a moisture indicator that changes color as water is absorbed (blue is dry to pink when wet, or orange to white). The crystals can be dried any time in any type of oven to 50-60°C (or follow directions on package). The blue indicator (cobalt derivative) has been shown to be toxic, so most brands now use some other indicator (orange, etc.).
- If silica is unavailable, any drying between paper sheets, analogous to prepare a herbarium, is ok. Just make sure that plants dry fast enough, and that samples are later unambiguously marked on the sheet.
- Note down site and individual identifier with water proof felt pen on bag /tube. Please provide info on water depth /GPS coordinates /extent of bed and any other useful information.
- No problem if epiphytes are not completely removed (PCR primers are very specific).
- Once completely dry, samples can be sent by air mail.
Thorsten Reusch, Leibniz-Institute of Marine Sciences IFM-GEOMAR, Düsternbrooker Weg 20, 24105 Kiel, Germany
Ferber S, Stam WT, Olsen JL (2008) Genetic diversity and connectivity remain high in eelgrass Zostera marina populations in the Wadden Sea, despite major impacts. Mar Ecol Prog Ser, 372, 86-97.
Olsen JL, Stam WT, Coyer JA, et al. (2004) Population differentiation and post-ice age recolonization of the North Atlantic by the seagrass Zostera marina L. Mol. Ecol., 13, 1923-1941.
Reusch TBH (2002) Microsatellites reveal high population connectivity in eelgrass (Zostera marina) in two contrasting coastal areas. Limnol. Oceanogr., 47, 78-85.
Reusch TBH, Stam WT, Olsen JL (2000) A microsatellite-based estimation of clonal diversity and population subdivision in Zostera marina, a marine flowering plant. Mol. Ecol., 9, 127-140.