Tag Archives: DNA

Kia ora from northern Germany

Moin!

That is how you say Kia ora or Hello in Oldenburg, which is where my family and I have been living since August 2013. As I near the half-way point in my 18-month fellowship, I thought I would show you where I am living, update you on what I have been up to in the lab, and introduce you to my lovely colleagues here.

Oldenburg is located in the state of Lower Saxony in northern Germany and has about 160,000 inhabitants, most of whom get around by bicycle, ourselves included.

Getting on our bicycles at Pferdemarkt, Oldenburg, Germany, Sept 2012. Photo by Mauricio López.

Getting on our bicycles at Pferdemarkt, Oldenburg, Germany, Sept 2012. Photo by Mauricio López.

Each day I cycle to the University of Oldenburg, where I am curently based. The best part of my 15-minute daily commute  is cylcing down the last kilometre along Drögen-Hasen-Weg.

My trusty bicycle at the start of Drögen-Hasen-Weg, about 1 km from the University of Oldenburg, May 2013. Note also the beautiful spring flowers! Photo by Heidi Meudt.

My trusty bicycle at the start of Drögen-Hasen-Weg, about 1 km from the University of Oldenburg, May 2013. Note also the beautiful spring flowers! Photo by Heidi Meudt.

My colleagues tell me that "Drögen-Hasen-Weg" means "Dry Feet Way" in Plattdüütsch, the local dialect of German still spoken in this area. In earlier times, this road was a way for the locals to get around without having to cross any waterways. Photo by Heidi Meudt.

My colleagues tell me that “Drögen-Hasen-Weg” means “Dry Feet Way” in Plattdüütsch, the local dialect of German still spoken in this area. In earlier times, this road was a way for the locals to get around without having to cross any waterways. Photo by Heidi Meudt.

The beautiful native trees (“Traubeneiche”, or sessile oak, Quercus petrea) that line this “Eichenallee” (literally, “oak avenue”) are now a protected natural monument.

Sessile oak trees (Quercus petraea) along the Drögen-Hasen-Weg Eichenallee, Oldenburg, Germany. Here they are just beginning to show their new green spring leaves, May 2013. Photo by Heidi Meudt.

Sessile oak trees (Quercus petraea) along the Drögen-Hasen-Weg Eichenallee, Oldenburg, Germany. Here they are just beginning to show their new green spring leaves, May 2013. Photo by Heidi Meudt.

A fellow commuter along the Drögen-Hasen-Weg Eichenallee, Oldenburg, Germany, May 2013. Photo by Heidi Meudt.

A fellow commuter along the Drögen-Hasen-Weg Eichenallee, Oldenburg, Germany, May 2013. Photo by Heidi Meudt.

After cycling through the beautiful tunnel of oak trees (did I mention I get to do this every day?), I arrive at the science campus of the University of Oldenburg, and turn right at this sign to get to my office. Photo by Heidi Meudt.

After cycling through the beautiful tunnel of oak trees (did I mention I get to do this every day?), I arrive at the science campus of the University of Oldenburg, and turn right at this sign to get to my office. Photo by Heidi Meudt.

I have come to Oldenburg to work on a research project regarding polyploidy in New Zealand and European Veronica with Dirk Albach, and in the process learn some new techniques. Polyploidy means whole genome doubling, and it occurs in Veronica species from both areas. We will compare the genes that are expressed in European and New Zealand polyploid species with their closest diploid relatives to determine when these genome doubling events occurred, confirm that the polyploid species likely evolved following hybridisation of diploid ancestors, and compare patterns of evolution of duplicated genes

One of the first things I learned was how to extract RNA, or ribonucleic acid, from leaf tissue.

Here I am with Eike Mayland-Quellhorst, grinding up some Veronica leaf tissue in liquid nitrogen for RNA extractions in the lab, May 2013. Photo by Silvia Kempen.

Here I am with Eike Mayland-Quellhorst, grinding up some Veronica leaf tissue in liquid nitrogen for RNA extractions in the lab, May 2013. Photo by Silvia Kempen.

Once the tissue has been ground to a fine powder, it is poured with the liquid nitrogen into a small tube. Photo by Silvia Kempen.

Once the tissue has been ground to a fine powder, it is poured with the liquid nitrogen into a small tube. Photo by Silvia Kempen.

Here I am extracting the RNA of the ground leaf tissue at the clean bench in the lab. Photo by Simon Pfanzelt.

Here I am extracting the RNA of the ground leaf tissue at the clean bench in the lab. Photo by Simon Pfanzelt.

Once the RNA is extracted and cleaned up, it is checked to determine whether it is of sufficient quality and quantity for sequencing. The next step will be to send the samples to a sequencing facility, and hopefully soon I will get some new data to analyse!

Another part of the project involves determining genome size, that is, measuring how much DNA (deoxyribonucleic acid) a particular plant contains in the nuclei of its cells. By working with Silvia Kempen, one of the technicians in the lab, I have learned how to use a flow cytometer and have measured the genome size of several Veronica species.

Getting everything ready in the flow cytometry lab to measure the genome size of three samples, May 2013. Photo by Silvia Kempen.

Getting everything ready in the flow cytometry lab to measure the genome size of three samples, May 2013. Photo by Silvia Kempen.

Chopping up the leaves from the first sample with a razor blade in preparation for flow cytometry, May 2013. Photo by Silvia Kempen.

Chopping up the leaves from the first sample with a razor blade in preparation for flow cytometry, May 2013. Photo by Silvia Kempen.

Watching Silvia calibrate the flow cytometer, which is the small machine to the right of the computer. Once the machine is calibrated, it is ready to measure the genome size of our prepared samples, May 2013. Photo by Eike Mayland-Quellhorst.

Watching Silvia calibrate the flow cytometer, which is the small machine to the right of the computer. Once the machine is calibrated, it is ready to measure the genome size of our prepared samples, May 2013. Photo by Eike Mayland-Quellhorst.

I must admit, the lab work has had its ups and downs, and it has taken me longer to get to this point than I had planned. One logistical problem we had, was that the plant material collected prior to my arrival did not result in good RNA extractions. That meant we needed to collect fresh plant material and retry the extractions, so I did my part by heading to Mallorca, Spain, on a collecting trip.

Collecting Veronica plants in Mallorca, Spain, with local botanist and PhD student Jaume Seguí Colomar. Photo by Mauricio López.

Collecting Veronica plants in Mallorca, Spain, with local botanist and PhD student Jaume Seguí Colomar. Photo by Mauricio López.

But perhaps delays, hiccups and changes are to be expected when one is learning new techniques, in a new lab, in a new country, and in a new language, no less! Although I speak quite a bit of English at the university, I am taking an evening language course, and I seek out daily opportunities to practice German with my colleagues. Speaking of which, here they are!

Photo of Dirk Albach's working group, outside our office and lab space at the Universtiy of Oldenburg, May 2013. Standing, left to right, Heidi Meudt, Eike Mayland-Quellhorst, Vera Mageney, Niklas Buhk, Katarzyna Palinska, Thomas Schmidt, Jane Looschen, Jennifer Nolzen, Lillian-Lee Müller, Imke Notholt, Simon Pfanzelt, Ute Friedrichs, Maria Brandes, Lena Koehler. In front, Bernhard von Hagen and Dirk Albach. Photo by Gerhard Zotz.

Photo of Dirk Albach’s working group, outside our office and lab space at the Universtiy of Oldenburg, May 2013. Standing, left to right, Heidi Meudt, Eike Mayland-Quellhorst, Vera Mageney, Niklas Buhk, Katarzyna Palinska, Thomas Schmidt, Jane Looschen, Jennifer Nolzen, Lillian-Lee Müller, Imke Notholt, Simon Pfanzelt, Ute Friedrichs, Maria Brandes, Lena Koehler. In front, Bernhard von Hagen and Dirk Albach. Photo by Gerhard Zotz.

Lab outings and field trips are a great way to get to know each other. One day last October, we took a trip to the nearby North Sea coast to the Wattenmeer (Wadden Sea), which is a UNESCO World Heritage Site.

At the Wattenmeer (Wadden Sea) along the North Sea coast in Germany in May 2013. From left to right Petr Kosachev, Eike Mayland-Quellhorst, Dirk Albach, Carolina García, Simon Pfanzelt. Photo by Heidi Meudt.

At the Wattenmeer (Wadden Sea) along the North Sea coast in Germany in May 2013. From left to right Petr Kosachev, Eike Mayland-Quellhorst, Dirk Albach, Carolina García, Simon Pfanzelt. Photo by Heidi Meudt.

Then in February, one afternoon our lab took a “Grünkohlfahrt” (literally, “kale walk”), which is a regional custom involving walking around with your friends or colleagues while eating, drinking, and playing special, regional games together. Oldenburg claims to be the kale capital of Germany.

Here I am on our Grünkohlfahrt ("kale walk") taking my turn at the northern German sport called "Boßeln", which is essentially outdoor road bowling! Feb 2013. Photo by Silvia Kempen.

Here I am on our Grünkohlfahrt (“kale walk”) taking my turn at the northern German sport called “Boßeln”, which is essentially outdoor road bowling! Feb 2013. Photo by Silvia Kempen.

At the end of the Grünkohlfahrt, we sat down together to share some excellent regional cuisine…

Our traditional northern German dinner after the "kale walk", including two types of German sausages (Pinkel and Kochwurs), potatoes, and (of course!) kale! Feb 2013. Photo by Silvia Kempen.

Our traditional northern German dinner after the “kale walk”, including two types of German sausages (Pinkel and Kochwurs), potatoes, and (of course!) kale! Feb 2013. Photo by Silvia Kempen.

And earlier this month, we had a very exciting special visitor, Radio New Zealand journalist Veronika Meduna, who came to interview Dirk and me about our collaborative research. You can hear the resulting interview here.

Dirk Albach and his son Felix, Veronika Meduna, and Heidi Meudt in the University of Oldenburg glasshouses during our interview. There is one Veronica plant in a pot in front of us, and several kale plants behind us. April 2013. Photo copyright Veronika Meduna, Radio NZ National.

Dirk Albach and his son Felix, Veronika Meduna, and Heidi Meudt in the University of Oldenburg glasshouses during our interview. There is one Veronica plant in a pot in front of us, and several kale plants behind us. April 2013. Photo copyright Veronika Meduna, Radio NZ National.

On both professional and personal levels, our experience in Germany so far has been at times enlightening, challenging, surprising, and overwhelming. Germany is a great place to do scientific research, and there are countless opportunities to learn about and experience its fascinating culture and history. Our first 9 months have certainly qualified as an adventure so far, and I look forward to experiencing what the next 9 months will bring.

Bis dann!

A big thank you to my whanau for supporting and accompanying me in this adventure. Here they are in the Schlossgarten (Palace Garden) in the winter snow! The main church of Oldenburg, Lambertikirche, is in the background. Jan 2013. Photo by Heidi Meudt.

A big thank you to my whanau for supporting and accompanying me in this adventure. Here they are in the Schlossgarten (Palace Garden) in the winter snow! The main church of Oldenburg, Lambertikirche, is in the background. Jan 2013. Photo by Heidi Meudt.

DNA sequences reveal unexpected fern relationships

Recently I have been obtaining DNA sequences from some of the fern samples collected by Te Papa Botany curator Leon Perrie on his recent trip to New Caledonia. We aim to determine the relationships of these New Caledonian ferns to other ferns around the world, including those from New Zealand.

One sample, however, gave us a surprising result. Two of the New Caledonian samples had previously been identified by Leon as members of the fern genus Dryopteris, based on their morphology. The genus Dryopteris has not previously been recorded from New Caledonia, so Leon was quite excited by these finds.

The DNA sequences established that one of these samples is indeed a Dryopteris, thus confirming that this genus is present in New Caledonia. However, the other sample unexpectedly grouped with another, albeit related, fern genus!

Watch this space as we do more work to try and establish the identity of this mystery fern.

The mystery New Caledonian fern that looks remarkably like a Dryopteris. Photo credit: Leon Perrie

The mystery New Caledonian fern that looks remarkably like a Dryopteris Photo credit: Leon Perrie.

Using DNA forensics to determine the past distribution of the brown kiwi species rowi.

Yesterday was a special day for 20 rowi (a species of the flightless kiwi) who were flown from the South Island to their new home on Mana Island, near Wellington. It was reported that this was the first time that this species of kiwi had been in the North Island for over a century.

So how do we know that rowi used to be in the North Island?

Kiwi researcher Kristina Ramstad holding a rowi. Photo by Rachael Abbott.

Kiwi researcher Kristina Ramstad holding a rowi. Photo by Rachael Abbott.

Today kiwi are absent from large areas of New Zealand, including the southern North Island (North Island brown kiwi occur from the central North Island northwards).  We know that kiwi used to occur in the southern North Island because their bones have been found in caves and other deposits.  However, trying to identify kiwi species just by looking at the shape and size of their bones is tricky.

Little spotted kiwi is the only species that can be identified from its bones because they are much smaller than the other kiwi species.  The bones of great spotted kiwi and the three species of brown kiwi (rowi, North Island brown kiwi and tokoeka) can’t be identified to species because they overlap in size and shape.

This is the kind of puzzle that DNA can solve. As part of my PhD I examined the past distribution of each kiwi species by sequencing DNA from kiwi bones that had been collected from throughout New Zealand.  Some of these bones were up to several thousand years old, but they still contained small amounts of DNA!

Surprisingly I found that the bones in the southern North Island were most closely related to rowi, rather than the geographically closer North Island brown kiwi.  Today rowi only naturally occur in one small population at Okarito on the West Coast of the South Island and they are the rarest species of  kiwi.  My DNA work showed that they used to occur as far north as the southern Hawke’s Bay. You can read the published results here.

NZ fern colonises Australia, twice

Asplenium hookerianum

Hooker's spleenwort fern. Near Levin, New Zealand. (c) Leon Perrie.

It is not just people crossing the ditch – a little New Zealand fern has also emigrated to Australia, and not just once but twice.

This is the first known case amongst ferns or seed plants of the same species dispersing twice across the Tasman Sea.

Hooker’s spleenwort fern, or Asplenium hookerianum, is a close relative of the hen & chickens ferns. Hooker’s spleenwort is widespread and common in New Zealand, but rare in Australia, with only a few, small populations in each of Victoria and Tasmania.

DNA analyses of the populations of Hooker’s spleenwort were carried out by researchers from Te Papa, Massey University, and the University of Melbourne.

26 genetic variants were found in New Zealand, but only one each in Victoria and Tasmania. Not only are the Australian variants at the tips of the genetic family tree, they are more closely related to variants in New Zealand than to each other.

This research was recently published in the journal Australian Systematic Botany.  Email me if you would like a copy of the paper: leonp@tepapa.govt.nz

Many plant species are known to have dispersed across the Tasman Sea, in either direction. Numerous New Zealand species also occur in Australia (about 50% in ferns), and more have close relatives there. But, it remains an open question how common multiple dispersals within a species are.

Big travels for little ferns

Lindsaea are small dainty ferns that are easily overlooked. Three species are indigenous to New Zealand.

Recent DNA-based research (Lehtonen et al. 2010) implies that each got here independently; i.e., there were three separate dispersal events. This is because the three species in New Zealand are each more closely related to an overseas species than to each other.

The three indigenous New Zealand species are:

Lindsaea trichomanoides (also in Australia) is related to L. rufa of New Caledonia.

Lindsaea trichomanoides. (c) Leon Perrie.

Lindsaea viridis (only found in New Zealand) is related to a group of species occurring from Madagascar through Malesia to western Polynesia.

Lindsaea viridis. (c) Leon Perrie.

Lindsaea linearis (also in Australia and New Caledonia) is related to L. microphylla of Australia.

Lindsaea linearis. (c) Leon Perrie

This is a common pattern. Many New Zealand ferns are also indigenous elsewhere. Of the species only found in New Zealand, many are more closely related to overseas species than to other New Zealanders. This indicates a comparatively high level of immigration and emigration.

Lehtonen S, Tuomisto H, Rouhan G, Christenhusz MJM (2010) Phylogenetics and classification of the pantropical fern family Lindsaeaceae. Botanical Journal of the Linnean Society 163: 305-359.

More on the origins of New Zealand’s ferns.

What’s it like to be a MSc student in systematic botany? Just ask Jessie…

My name is Jessie Prebble and I am the current (2009) recipient of the Te Papa MSc Scholarship in Molecular Systematics. I’m studying at Victoria University, looking at the evolution of the plant genus Wahlenbergia in New Zealand and Australia. I’m using various molecular techniques to try to determine how reliable the current taxonomy of the New Zealand species is, and whether I can infer how many times the genus invaded New Zealand, where from, and when.

Jessie and Wahlenbergia albomarginata subsp. olvina on the ultramafic Dun Mountains near Nelson, New Zealand.

Me and Wahlenbergia albomarginata subsp. olvina on the ultramafic Dun Mountains near Nelson, New Zealand. Photo © Jessie Prebble.

Here I am finding Wahlenbergia gloriosa in an alpine herbfield on Mt Kosciuszko, New South Wales, Australia. Photo © Jessie Prebble.

Here I am finding Wahlenbergia gloriosa in an alpine herbfield on Mt Kosciuszko, New South Wales, Australia. Photo © Jessie Prebble.

I love my research. I spent last summer exploring the country collecting specimens in beautiful locations from the Garvie Mountains in Southland to Muriwai Beach north of Auckland. I even got to head over to New South Wales to hunt down some of the Australian species.  I then spent a few weeks mounting and processing all of my collections, and now they’re stored in the Te Papa Herbarium.

This is the common South Island alpine plant Wahlenbergia albomarginata subsp. albomarginata, which grows profusely on the slopes of Mt Robert, Nelson Lakes area, New Zealand.

This is the common South Island alpine plant Wahlenbergia albomarginata subsp. albomarginata, which grows profusely on the slopes of Mt Robert, Nelson Lakes area, New Zealand. Photo © Jessie Prebble.

Wahlenbergia ceracea growing in an alpine bog on the slopes of Mt Kosciuszko, New South Wales, Australia.

Wahlenbergia ceracea growing in an alpine bog on the slopes of Mt Kosciuszko, New South Wales, Australia. Photo © Jessie Prebble.

Currently I’m dividing my time between the lab, where I extract and sequence short fragments of my specimens’ DNA, and the computer lab, where I puzzle my head over numerous types of data files. I have selected three regions to sequence, two from the chloroplast (trnL-F and trnK-psbA) and one nuclear ribosomal region (ITS). I explore my sequence data by forming alignments of the sequences, then creating phylogenetic trees to tease out the relationships between the species.

Results are starting to trickle in, and so far I can tell that all of the New Zealand species are very closely related, which most likely points to recent and rapid evolution here.  Further results to follow…

The beautiful coastal plant Wahlenbergia congesta subps. haastii growing on sand dunes on the South Island’s west coast, by the mouth of Ship Ck. Photo © Jessie Prebble.

The beautiful coastal plant Wahlenbergia congesta subps. haastii growing on sand dunes on the South Island’s west coast, by the mouth of Ship Ck. Photo © Jessie Prebble.

DNA-fingerprinting fierce lancewood

Aside from ferns, my main research interest is the group of trees known as Pseudopanax, for which I collaborate with Lara Shepherd from the Allan Wilson Centre.

Blog posts on ferns

Blog posts on Pseudopanax

Lara at the Allan Wilson Centre

Pseudopanax includes the lancewoods and five-fingers. Several of the species are popular in cultivation, including fierce lancewood (Pseudopanax ferox). This species is so named for having bigger ‘teeth’ along the margins of its juvenile leaves than lancewood (Pseudopanax crassifolius).

Blog post on lancewood and its hybridisation with coastal five-finger

Paper in Molecular Phylogenetics & Evolution about the relationships of Pseudopanax

Fierce lancewood, Pseudopanax ferox. Juveniles (left) and adults (right) have very different leaves and habits. Both images by Leon Perrie, Curator. © Museum of New Zealand Te Papa Tongarewa.

Fierce lancewood, Pseudopanax ferox. Juveniles (left) and adults (right) have very different leaves and habits. Both images by Leon Perrie, Curator. © Museum of New Zealand Te Papa Tongarewa.

 

How are fierce lancewood populations related? In the wild, fierce lancewood has a very discontinuous distribution, with some populations being very isolated. We want to know how the various populations are related to one another. We are using a DNA-fingerprinting method known as “microsatellites” to determine how the various populations are related to one another. The approach is analogous to criminal forensics.

Microsatellites are highly variable regions of DNA. Each microsatellite has a number of variants which differ in length. We determine how many DNA nucleotides long the microsatellite variants are in each individual sampled. This tells us how the individuals and the populations they came from are related.

 

At each different kind of microsatellite, each individual has two copies, one inherited from its mother and the other from its father. The two copies in an individual can be the same or different lengths. This is a figure of one particular kind of microsatellite for two individuals. In the upper individual, the two copies are of different lengths: length 129, which is quite uncommon, and length 135 which is common and widespread. In the lower individual, the two copies are both of length 135, which is why there is only one large peak.

At each different kind of microsatellite, each individual has two copies, one inherited from its mother and the other from its father. The two copies in an individual can be the same or different lengths. This is a figure of one particular kind of microsatellite for two individuals. In the upper individual, the two copies are of different lengths: length 129, which is quite uncommon, and length 135 which is common and widespread. In the lower individual, the two copies are both of length 135, which is why there is only one large peak.

 

Our preliminary analyses suggest there are four principal genetic groups within fierce lancewood. There has probably been very little gene-flow between these groups for some time.

The four principal genetic groups detected by microsatellite DNA-fingerprinting in fierce lancewood are indicated by different colours. The small grey circles are populations that we haven’t sampled, but which are represented by specimens in the herbarium collections of Te Papa, Auckland Museum, and Landcare Research.

The four principal genetic groups detected by microsatellite DNA-fingerprinting in fierce lancewood are indicated by different colours. The small grey circles are populations that we haven’t sampled, but which are represented by specimens in the herbarium collections of Te Papa, Auckland Museum, and Landcare Research.

 

One of the four groups, that in the southern North Island (the brown dot), comprises a single population on one hillside!

On the other hand, another of the four groups encompasses most of the South Island (from Kaikoura southwards; green dots). The absence of strong genetic subdivision within this group suggests its populations have been more recently connected by gene-flow. This may indicate that fierce lancewood has recently been more continuously distributed in the central and southern South Island, perhaps even until the widespread clearance of lowland forests by humans.

Our preliminary analyses also suggest that the central and southern South Island populations of fierce lancewood may have survived the Last Glacial Maximum of the ice-age more or less in situ, rather than being derived from one or a few major refugia. This is consistent with the hypothesis that there was widespread survival of New Zealand’s forests during the ice-age, as we have previously inferred from genetic analyses of the forest fern Asplenium hookerianum.

 Paper in Molecular Ecology about the ice-age survival of New Zealand’s vegetation

 

More rare maidenhair spleenwort.

The rare, tetraploid maidenhair spleenwort  (Asplenium trichomanes subsp. quadrivalens) has only recently been rediscovered in New Zealand.  Several people have contacted me with possible additional sightings. As described by the Scoop website, Jack Ritchie had a maidenhair spleenwort self-sow on a rock used to construct a water feature in his nursery, Tree Guys, in Otane.

Jack took us to the local farm where the rock was sourced from, and without too much effort we found a good population: about 70 plants growing on limestone outcrops in pasture.

Asplenium trichomanes subsp. quadrivalens

Tetraploid maidenhair spleenwort.

These rocks are host to several plants of tetraploid maidenhair spleenwort.

These rocks are host to several plants of tetraploid maidenhair spleenwort.

Lara Shepherd (Massey University) and I collected a few samples, and confirmed through analyses of their spores and DNA that they were the tetraploid maidenhair spleenwort rather than the common hexaploid maidenhair spleenwort.

DNA sequence data. The highlighted position is one of several DNA sites found by Lara that differ between the tetraploid (upper two samples) and octoploid (lower two samples) maidenhair spleenworts.

DNA sequence data. The highlighted position is one of several DNA sites found by Lara that differ between the tetraploid (upper two samples) and hexaploid (lower two samples) maidenhair spleenworts.

There is plenty of similar habitat in the region, so the tetraploid maidenhair spleenwort could well be much more widespread. We need to find more than 250 individuals to lift it out of the Nationally Critical conservation category, which I am hopeful we will achieve with more searching.  Ideally, it will turn out to be sufficiently common that it doesn’t even need to be on the threatened list.

Thanks to Jack Ritchie and everyone else who has contacted me about the maidenhair spleenwort. I have several other promising leads to follow-up when I am next able to escape the office.

If you would like to see a tetraploid maidenhair spleenwort for yourself, then visit Jack Ritchie. He is a very knowledgeable and enthusiastic plant-person, and his tetraploid maidenhair spleenwort is the only one I know of in ‘captivity’. Jack’s Tree Guys nursery is in Otane, on the main road between Waipukurau and Hastings.

We have DNA

DNA of Pseudopanax on agarose gel after electrophoresis

DNA of Pseudopanax on agarose gel after electrophoresis

The first step after collecting samples for genetic analyses is to extract the DNA. Lara and I do this for lancewood and five-finger plants (Pseudopanax) by:

  • freezing a small piece of leaf tissue in liquid nitrogen (-196 degrees C !) and grinding it as finely as possible.
  • adding a detergent to release the DNA from the cells of the leaf tissue.
  • adding chloroform.  The detergent and chloroform do not mix (like oil and water), but proteins and other things we do not want are drawn into the chloroform while the DNA is left in the detergent.
  • the detergent layer is removed, and alcohol is added to it.  This precipitates the DNA (i.e., makes it turn into a solid), and we can actually see it.  I don’t have any pictures of Pseudopanax DNA, but precipitated DNA all looks much the same – see this link.

It is possible to extract DNA using household items (see this link).

In order to analyse the DNA further we have to make it go back into solution. The alcohol is tipped off, and a small amount of salt solution is added; the DNA ‘dissolves’ in this.

To test the quality and quantity of the extracted DNA, we run a small amount of the DNA solution on an agarose gel in a process called electrophoresis (see link).

In the gel above, each lane corresponds to a separate sample, except the right-most lane which is a ‘ladder’ for sizing the DNA samples. A negative charge was applied at the top and a positive charge at the bottom. DNA is negatively charged, so its moves towards a positive charge.

The bright blobs indicated by the green arrow indicate that we got high quality (the DNA is in big pieces, as it hasn’t moved very far) and quantity (a brighter stain indicates more DNA) for most of these samples, which is great!

The sample labelled 5957 (my collection number) is a bit weak, while we didn’t get anything for sample 5964.

These DNA extractions are all from samples of fierce lancewood (Pseudopanax ferox), except 5966 which is P. macintyrei.

Adult tree of fierce lancewood, Pseudopanax ferox.

Adult tree of fierce lancewood, Pseudopanax ferox.

 

Pseudopanax macintyrei.

Pseudopanax macintyrei.

The next step in assessing the relationships of these plants is to genetically ‘fingerprint’ them.

Lancewood hunting

Field-work is one of the best aspects of working as a Natural Environment curator at Te Papa. I get to spend about three weeks a year in the field collecting plant specimens.

tepapa4wd2

Te Papa’s 4WD. If seen outside Wellington, there is a good chance this vehicle is being used to collect plants, whales, or fossils.

I’ve recently returned from ten days field-work in the South Island, collecting samples for our research on lancewood (horoeka, Pseudopanax crassifolius) and fierce lancewood (P. ferox). This is in collaboration with Lara Shepherd from Massey University. Lancewood is a common forest tree and we are using DNA analyses to determine where it survived in New Zealand during the last glacial period. This follows our previous research on the forest fern Hooker’s spleenwort (Asplenium hookerianum), which seems to have survived throughout New Zealand, and conflicts with evidence that Metrosideros trees (rata and pohutukawa) were confined to only a few refugia.

Fierce lancewood, named for its bigger ‘teeth’ on the leaf margins, is more sparsely distributed that lancewood. Given the discontinuous distribution of fierce lancewood, we expected each population to exhibit its own diagnostic set of genetic variation. Preliminary results suggest this might be true for Auckland and Wellington populations, but, at this stage, we can’t genetically distinguish populations from the southern South Island, indicating the geographic discontinuity there is a geologically-recent phenomenon. We collected specimens to augment our existing sampling (the northern South Island, in particular, was a bit of a gap for us for both lancewood and fierce lancewood).

pseudopanax_ferox_juvenile_leaf1
Apex of a leaf from a juvenile fierce lancewood, showing the curious ‘paint-splash’ coloration. I have heard it hypothesised that the prominent white splashes draw attention to the marginal ‘teeth’, themselves a putative defence against moa herbivory.

The trip was largely successful, with the weather good and the plants cooperative (in that we could find them where they were supposed to be). We now have to process the samples in the laboratory, which isn’t nearly as much fun but still necessary if we are to address the questions we’re interested in.

pseudopanax_ferox_juvenile2

A juvenile of fierce lancewood.

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