Apr 122017
 

Once we realised that most of our plate of Schistidium ITS2 amplifications had been successful, it was an easy decision to process them all for DNA sequencing. If a higher proportion had failed, we would have had to “cherry pick”, selecting and transferring the successful reactions into new tubes. Every sequencing reaction has a cost, and so deliberately sending a lot of failed reactions through the process, knowing that they won’t generate DNA sequences, is worth avoiding. However, transferring the reactions into new tubes is a meticulous job. In a plate of reactions, there are 96 wells arranged in 12 columns (1-12) and eight rows (A-H).  The first sample is at 1A, then 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A… all the way to 12H. Imagine if the first failed sample is at 1B and the second at 1G – then we’d transfer 1A to 1A, 1C to 1B, 1D to 1C, 1E to 1D, 1F to 1E, 1H to 1F, 2A to 1G, etc… it’s easy to see that any interruptions can be fatal (this sort of task is why people in the lab sometimes have a “Do Not Disturb” sign stuck on the back of their lab coats). Thus, with only a few failures, we’re better keeping any liquid transfers simple (and manageable using a multichannel pipette).

A 96-well plate on ice

After electrophoresis and visualisation of the Schistidium ITS PCR products under blue, or U.V., light, the next step is to clean up these PCR products. This is where we remove unincorporated primers and dNTPs from the reactions. In our PCRs, we added all the ingredients in excess, so that there was more of everything than the reaction needed. This might seem wasteful, but compared to the costs of the time and plastics that would be required to carefully optimise each reaction, throwing lots in and getting quicker results really isn’t too profligate.

When a PCR is successful, the primer (an oligonucleotide, or short single stranded DNA molecule) binds to the end of the region that is getting copied, and a new strand of DNA is synthesised from the end of the primer, incorporating the primer into the new strand. Thus, primers get used up. The building blocks for DNA synthesis, the nucleotides (As, Ts, Gs and Cs), are the dNPTs (deoxynucleoside triphosphates) that we added to the PCR reaction, again in excess. So even when we’ve built a lot of copies, we still have some primers and dNTPS that weren’t used up.

This didn’t matter when it came to looking at the results on the gel, but does become important in our next reaction, the sequencing PCR. In the sequencing PCR, we add just a single primer, and we add a very precise blend of nucleotides. So we don’t want to carry over primers and dNPTs from the previous reaction. There are several ways of removing these.

The cheapest is through ethanol precipitation of the synthesised DNA, where the unincorporated primers and dNTPs stay in solution and are thrown away. This is less easy to scale up to plates, and the moment where you turn it all upside down and toss the liquid out is rather worrying – the cost of plates of subsequent sequencing failure if the DNA pellets were lost is huge, and the pellets tend not to be visible.

For several years we used a column-based approach, a bit like our DNA extraction method, where the PCR product is bound to a membrane, and the unincorporated primers and dNTPs are flushed off it, before the PCR product is eluted back into solution.

However, the method we now use most regularly involves a combination of two enzymes, Eco1, and Shrimp Alkaline Phosphatase (SAP), which work elegantly in combination, and which involves methodology that is easily scalable for working with plates and multichannel pipettes. The Eco1 enzyme digests single-stranded DNA, so it cuts the primers up into individual nucleotides. The SAP enzyme dephosphorylates the nucleotides (the dNTPs) so that they are no longer functional. Thus after using these enzymes, although nothing has been physically removed from the tubes, the unwanted reagents have been rendered unusable. For convenience, we buy in a commercial combination of the two enzymes called ExoSAP-IT™, and add a small amount of that to our reactions (in this case, around 1 μl ExoSAP-IT to about 15 μl of PCR product).

The enzymes work best at 37ºC, and so the reactions were put on a heating block for 15 minutes. The enzymes are killed at a higher temperature, and so the next step was to heat the reactions to 80ºC for 15 minutes, to make sure that no viable enzymes are left to interfere with the sequencing reaction. After that, the plate of cleaned Schistidium ITS2 amplicons was left in the fridge until it was needed for the sequencing reactions.

 

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Mar 302017
 

The moss Campylopus introflexus, native to the southern hemisphere, is now considered an invasive plant in parts of Europe and North America. While it occurs on some natural sites within Edinburgh, notably on Arthur’s Seat, it is also no stranger to man-made habitats. At the Botanics, the species forms large tactile ball-like clumps between the glass panes of the Research House roof. However, as it can damage the roof, it is one of our less welcome bryological volunteers.

Gunnar Ovstebo holding a tactile moss ball from the Research House roof

 

Wolfgang Hofbauer and Gunnar Ovstebo checking out the bigger and better Campylopus moss-balls that are out of reach further up the roof

 

Furry Campylopus plants growing on the Research house roof

Campylopus introflexus moss ball

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Mar 292017
 

There are very few bryophytes growing in the living collections of the Royal Botanic Garden, Edinburgh. What I mean by this is that there are very few bryophytes that we have carefully selected as wild plants, and have planted and nurtured as part of our curated collection, databased and in possession of accession numbers. On the other hand, there are lots and lots of different bryophytes growing here, having volunteered to become part of our living landscape. Given that part of the work that we are doing with visiting Synthesys researcher Dr Wolfgang Hofbauer is looking at ways to increase and promote the growing of mosses, and to better understand their place in the built environment, horticulturalist Gunnar Ovstebo and I started off Wolfgang’s visit with an excursion into the garden, to see what we already have.

Wolfgang and Gunnar in the Arabian Research House

We began behind the scenes in the Arabian Research House, where a light moss cover is used to help the establishment of some arid-land ferns.

A mossy bed for young ferns growing in the Arabian Research House

It’s bryophytes galore in the Gesneriaceae Research House, even over the plastic under-bench tanking

We also visited the Gesneriaceae and fern research collections, warm moist glasshouses that are incidentally filled with bryophytes. Some of these might be native to Scotland and have come in from the surrounding area; others could have come from around the world as we have made additions to our living collection.

Gunnar and Wolfgang check out one of the crevices in the Arid House

Moving into one of our public display houses, some cool shaded crevices between the rocks of the Arid House provide habitats for algae, mosses and liverworts, which in turn provide germination sites for some of the larger plants like ferns and begonias. The Arid House is also home to three accessioned species of the complex thalloid genus Plagiochasma, which have established really well between some of the rocks.

The tufa wall in the new Alpine House

The third place we visited is the new Alpine house, which has a tufa rock wall dotted with little rosette-forming plants. Mosses are also growing on the wall, but are not particularly welcome!

Mosses spread along damp mortar-work at the back of the new Alpine House

Venturing round the back of the new building, patches of moss are happily spreading in patches along damp cement-work between the bricks.

Behind-the-scenes again, in the Alpine Nursery, although the tunnels are remarkably bryophyte-free, hundreds of pots fill a series of cold frames, and in and amongst these, many moss volunteers thrive.

The Alpine Nursery – the only glasshouse where we didn’t see any bryophytes

Mosses volunteering in the cold-frames of the Alpine Nursery

Having obtained an idea of what we currently have here, over the next few weeks we will be starting up some moss transplant and cultivation experiments, because although our volunteers are very much part of our garden landscape, as scientists and horticulturalists we also want to be able to grow and display particular selected species and accessions; these may then form part of our research collection or our education programme.

 

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Mar 292017
 

Some Schistidium collections from the RBGE Herbarium

Monday 27th March was the start of a month-long visit to RBGE by the Fraunhofer Institute for Building Physics‘s Dr Wolfgang Hofbauer, funded by the EU Synthesys Access programme. This funding enables researchers from other institutes to get their hands on the natural history collections that they need to see and understand, but it is equally vital for collections-based institutes like ourselves, as it promotes the use and curation of some of the material that we conserve.

DNA barcoding publication resulting from Wolfgang’s previous visit to RBGE

Wolfgang first visited us at RBGE as part of an earlier Synthesys programme, in 2014, which initiated a very useful collaborative project looking at the growth of species from the moss genus Schistidium on the built environment. We used DNA sequence data to try to identify some of these mosses, because the harsh environment in which they grow means that the plants are often malformed or underdeveloped, and difficult to identify using morphology.

Wolfgang has come back to RBGE in order to continue this work, in part by adding to our “Reference Library” of DNA sequences from different Schistidium species, but also to look at ways of developing our ability to grow some of these mosses where and how we want them.

We hope that by the end of this visit, we will be closer to answering the five following questions:

a) What is the taxonomy of Schistidium diversity on modern building surfaces?
b) Can we show geographic patterns of morphological and genetic variation in Schistidium on modern buildings in different European regions?
c) Are certain Schistidium taxa confined to special ecological situations (material, exposure, etc.) and can we use this for management of moss growth on buildings?
d) Which taxa of Schistidium are the best candidates for moss gardening, and is there potential for specially developed masonry/techniques to facilitate their growth?
e) Does improving the taxonomy of Schistidium on building surfaces allow us to find specific natural antagonists that could be used for its biocontrol?

Wolfgang hard at work in the Cryptogam Workroom at RBGE

The outcomes that we would like to see from this work are:

1) A complete baseline DNA-barcode library of Schistidium species as a tool for identification of sterile or morphologically atypical material.
2) Increased insight into the ecology and taxonomy of Schistidium species that grow on modern building structures.
3) A common project with the theme of deliberate growth of suitable cryptogams on building surfaces, as a collaboration between Science and Horticulture Divisions here at RGBE, and the Fraunhofer Institute for Building Physics, where Wolfgang is based.
4) Preliminary information on potentially specific biocontrol of unwanted growth on building surfaces, by identification of the moss lineages involved.
5) Development of an accessioned living collection of Schistidium species that have been identified using DNA barcoding and cultivated at RBGE, to be used for moss cultivation experiments and for public display.
6) Working with RBGE’s bryologists and horticulturalists, the development of a living display of moss colonisation (a kind of “living poster”) that can be used for outreach activities.
7) Published research, both on the DNA barcoding of Schistidium and the diversity of the genus on building surfaces, and also on bryophyte cultivation methods.

 

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE: