04/2/14

Lodgepole pine DNA extractions

If you’ve read any of my previous posts, you may be aware that I have a truck-load of lodgepole pine samples collected last summer that are destined for SNP genotyping in the near future. I am close to wrapping up all of my DNA extractions (fingers crossed) for sending out, and because I tinkered a bit with the standard extraction protocol, I thought I’d discuss it here. Before I get there though, I have to give huge, gigantic, endless thanks to Kristin Nurkowski and Robin Mellway for all of their help. Most of these adjustments are a direct result of their insight and suggestions. They also taught me the standard protocol, how to use the lab’s robot, and saw me through lots and lots of troubleshooting.

To start at the beginning, I have needle samples collected on silica gel. In the field, my assistants and I tried to take the freshest tissue possible (new needle growth at branch ends), which was then kept on silica gel in coin envelopes within sealed ziplocs. When it was not possible to collect new needles, older ones were taken, and we even sampled some clearly dead trees (brown needles) in the off chance they’d work and some dying trees (recently fallen tree, needles not yet brown and brittle, but clearly dead); I’ll discuss this again a bit later. My collections in the Yukon were much later in the summer, so new growth was not new anymore, and we ran into a couple rainy days here which is a real pain when storing on silica. Other notable points were that these samples then sat in tupperwares in the back of a truck for at most 2 weeks while we continued collecting, so they definitely experienced some warm temperatures, but I have seen no correlation in the time since collection and the quality of DNA.

For the standard extraction, we used Machery-Nagel kits with NucleoSpin filters. These come with two different protocols using either PL1 lysis buffer or PL2 lysis buffer. From an initial test of both protocols and various weights of tissues, I found for my samples that the PL2 protocol worked better. PL1 is based on a CTAB extraction while PL2 is SDS based and has an additional step of a protein precipitation using potassium acetate (PL3). 20mg of tissue (dried) also seemed to be best, with less not giving enough DNA, and more also decreasing in yield. Presumably one could increase the amounts of buffers and reagents used per reaction relative to increase in tissue if more tissue wanted to be used, but at these volumes the filters were filled quite high with supernatant at the binding step, so eventually there is a limit. Except where I have pointed out exceptions, I followed the protocol outlined in the manual and used the volumes given, and just upped my lysis incubation to 45 minutes at 65C and my protein precipitation to 20 minutes at -20C (on ice, and in the freezer).

Initially I stuck with this protocol and it worked well. I had about 80% success per plate, which I was pleased with considering the quality of some of my tissue samples. And believe it or not, some of the dead needles we had collected yielded DNA with enough to meet the cutoff! After completing eight plates of extractions, we had some issues crop up with our robot, and then extractions after that started failing. It is unclear if it was a product of the robot or a bad set of reagents or something entirely unknown, but it was clear nothing in the protocol had changed. It was also clear that it was not alone due to the quality of samples being used as some were trees from the same sites and provenances as samples that had already worked successfully. I did a lot of troubleshooting at this point to try to get things back up and running and eventually we found that adding PVPP to the PL2 buffer got us back up to an 80% success rate.

I used a 1% PVPP buffer for a while and then increased to 2% which improved it further. Because PVPP is mainly insoluble in liquid, I didn’t see the point in increasing past this as it was clear that there was undissolved PVPP in my buffer. I added 1 gram of PVPP to 50 mL of the PL2 buffer (preheated at 65C) plus 10 ul of antifoaming agent since the PL2 is quite bubbly, and incubated this overnight at 65C with 1 mL of RNaseA being added just before starting the extraction. A shaking incubator might be nice to use here since in the water bath, a lot of the PVPP just settles at the bottom of the tube. Using the PVPP improved my 260/230 from about 1.75 on average before to 2.33 on average.

Additionally, it seemed that the second plate from each extraction was on average coming out with poorer quality — the robot can do two plates at once. The robot is not incredibly fast at pipetting, so with a second plate, it sits there a little longer than the first before it is acted upon. This can be helped in the beginning by not removing the second plate of ground tissue from the freezer until just when the robot is ready to add lysis buffer to it. Because I did not have a very large number of plates to do, I instead began single plate extractions and simply kept an old used plate, filter, and elution plate to balance the centrifuge at the necessary steps. It is worth pointing out here that this was not the most efficient route. Though I have not raced the robot, if I had had access to a multichannel pipette, it is clear that doing this protocol sans robot would have been faster and easier. The robot is by no means a requirement for this extraction protocol, but it does have the added benefit of freeing you up to do other work while it pipettes away. I have even heard that newer models of robots have arms that will move the plates around for you! And the robot will definitely be a huge help for the plate normalization step once I have all my samples.

Some final points to make for anyone following along: from nanodropping my samples and qubiting a subset of them, most had about 40% less DNA per ul than was shown by the nanodrop, and I found no significant correlation of this with either my 260/230 or 260/280, though others in the lab have found that to be related for samples extracted from fresh, frozen tissue under the PL1 protocol, and with less of a decrease in DNA quantity between the two.

For samples that didn’t meet my cutoff criteria in terms of quality and quantity, performing the same extraction protocol a second time was successful about 60% of the time. I would speculate that the first failure in these cases was due to either the robot picking up some of the pellet with the supernatant which happened on rare occasions, or due to differences between different needles collected from the same tree.

And lastly, for those samples that did not succeed the first or second time around, it is now time to perform the more labor-intensive but hopefully cleaner and higher-yielding CTAB extraction protocol! Most of my samples that fall into this category are those I collected from the Yukon, so a product of the tissue being older and additionally not drying as quickly from the rainy day extractions. So for anyone out there planning a field season, I definitely advise lots and lots of extra silica gel and the youngest tissue possible — two already-well-known points that cannot be emphasized enough. If I have any serious alterations to the CTAB protocol, I may update this post or add another, but until then I hope that some of this information might be useful to anyone else out there working with conifers and their pesky secondary compounds!

08/27/13

Lodgepole pining

Another summer is drawing to a close and another school year starting, meaning that a few of us have wrapped up some successful field seasons! I spent a good chunk of my summer travelling across BC and the Yukon collecting lodgepole pine needles for DNA samples in my project looking at the genetic basis of local adaptation to climate, a ground-truth of the main AdapTree project. I’ve returned with 2,585 trees sampled from 122 different provenances grown across 16 different test sites I visited, as set up by the BC Ministry of Forests in the Illingworth provenance trial as well as a smaller provenance trial set up in the Yukon Territory.

I just want to share a few photos and fun stories to give a feel of what my field season was like, but first I have to acknowledge a lot of very great people. I had a lot of help along the way, and these are the people to whom I owe many thanks! First off, Sally, Ian, and Kristin, head of and in the Aitken lab, for all the help given in terms of advice for the field, getting gear together, and letting me borrow gear, supplies, and a great 4WD field truck. Second, to Nick Ukrainetz and Vicky Berger with the BC Ministry of Forests for their immense amount of help providing me detailed information on all of the provenance trial sites I visited, teaching me good safety practices for driving on logging roads, and providing me with a VHF transceiver. Third, a huge nod to my advisor Mike Whitlock for all of his support in making these collecting trips possible and imparting knowledge over the phone of what to do when we were being swarmed and chased for >20km by what we thought were angry bees in the woods. Fourth, to Anne Berland, graduate student at the University of Victoria for joining us in the field, not only being great company, but also an excellent navigator to finding our sites and to letting me borrow a second pruning pole from their lab. As well as to Stilianos Louca, UBC grad student, who without having been in a car accident in the Yukon (no one hurt) I would not have been able to visit test sites in the Yukon for sampling and received his help in exchange for helping on the long drive back to Vancouver. And lastly and also most greatly to my two amazing field assistants for my main sampling trip: Evan Cronmiller and Warren Neuvonen, UBC undergraduates, who endured hot days, cold nights, hard ground, tall trees, biting insects, scratching branches, swarming flies, long drives, slingshot fatigue, and in the face of it all remained happy, friendly, energetic, hard-working, and continued to keep me laughing and smiling on our whole trip. I really could not have done it without all of these people and have the good fortune of being able to now spend my fall semester extracting DNA from all of my samples.

So, on to the fun stuff. What is it like to sample lodgepole pine for 3 weeks in the woods?

First off, you see lots of trees, and lots of logging roads.

You drive around a lot, and often it is hard to find a site (the sites were established in 1974, so just under 40 years old!), but sometimes it is actually quite easy!

And you encounter many logging trucks along the way.

The loggers would often talk to us on the radio, curious about what we were doing in our truck way out in the middle of nowhere.

Short tree from a provenance in Yellowstone

The trees can be very tall, or they may be very short. We clearly could see effects of local adaptation going on in the field.

Holding a 40-year-old tree.

We used slingshots to sample the unreachable branches on the tallest trees. By the end of the trip, it was no problem to shoot a tree and get a branch to fall in one shot. The slingshots got so much use that we had to buy replacement straps halfway through the trip. But we sometimes resorted to extreme measures to sample when trees were too sparse for the slingshot.

Warren and Evan became pros.

These included throwing the pole to reach a just-out-of-reach branch or climbing a tree.

We had a lot of really, really buggy days where the incessant buzz in your ears almost makes you go mad.

But you manage to find your peaceful and pretty moments.

And enjoy some amazing campsites, like this one on the Bluewater River in the Canadian Rockies.

We often got covered in pollen, walking out of the woods with yellow boots and pants.

And I spent a lot of time each evening filling my samples with silica gel to dry them out for the long trip back to the lab.

And in the end, I even was able to see the Yukon and sample there!

Stilian with the pruning pole

So thanks again to everyone for all of the help and making this a productive and memorable summer. Stay tuned in the future for the results of my project!

05/17/13

Like a moth to the flame or a beetle to the tree

I have had the Mountain Pine Beetle (MPB) on my mind recently for many reasons.

First and foremost, my field work planning is highly complicated by trying to choose sites that will have the least amount of MPB kill. Second, I recently watched an episode of The Nature of Things on CBC about MPB (here) which was an excellent intro to the beetle and the current situation, and also featured UBC forestry professor Dr. Allan Carroll. And third, I have just returned from attending the annual CSEE conference which was in Kelowna, BC this year, where I saw a few talks on the topic. Jasmine Janes from the University of Alberta gave a talk on the genetic signature of MPB range expansion, and I also attended a talk by Mathias Kaiser from the University of Calgary who talked about beetle stridulation, and the different noises the MPB makes to communicate with other beetles.

There is so much that can be said about the beetles, so I just want to highlight some of the things I found interesting after what will be a short introduction. Mountain pine beetle (Dendroctonus ponderosae) is a bark beetle native to western North America. One beetle is about the size of a grain of rice. In the past few years, these beetles, which inhabit many pine tree species in the west, have exploded in population size and caused devastation to the lodgepole pine.

A lodgepole pine forest in “red phase”, where needles are still retained after MPB attack. They then enter gray phase where needles drop from the dead trees.

Entire forests of lodgepole pine have been killed across BC. MPB has been around in the past and has co-evolved with western pine species, but their increased numbers leading to this explosive destruction seem to be due to two main causes: warmer winters from climate change and reduction of natural fire regimes in forests.

When beetles attack an individual tree, they overwinter under the bark. With warmer winters due to climate change, more beetles started to survive than in the past, allowing populations to expand much more than they had historically. And larger population sizes have more of an implication than one might think initially, a point I will come back to momentarily. Historically, lodgepole pine is a fire-associated species, too. Serotinous cones open during extreme heat and reseed forests after fire in a natural situation. This also used to help control the beetles and keep populations down, however human efforts to suppress fires have decreased this effect.

So what are the cool things about MPB that make its larger populations so deadly? Allan Carroll was kind enough to have a chat with me and explain some of the finer details.

It takes many beetles to kill one tree, and the tree doesn’t go down without a fight. An individual beetle may land on a tree, find a good spot and chew in through the bark and into the phloem. The pine, in an effort to stop the beetle, has a constitutive defense where its resin canals kick in to try to flush the beetle out just with physical force. Once the beetle gets far enough into the tree to start chewing through live tissue, the tree has an induced response where it starts releasing toxins within the resin as a chemical defense against the beetle. (You can see this in action if you watch the CBC show; there is very cool footage.) The next neat thing that happens though from the beetle’s point of view, is that while this toxic resin is being produced, it really has no choice if it wants to continue other than to just ingest it. And once it starts ingesting the toxins, it begins to release what is called an aggregation pheromone. This aggregation pheromone is picked up by other nearby beetles and acts as a signal that “hey this tree is under attack right now”. If nothing further happened at this point, the individual beetle would be hopeless and die alone, and the tree would prevail. But, with other beetles in the area, they come to the tree like moths to a flame when they smell the pheromone. With many beetles attacking at once, the tree has a much harder time defending, and will eventually lose and be killed. The beetles will eat to their heart’s desire, lay their eggs, and come next spring, the next generation of beetles will go off and do this again.

Another cool MPB fact I learned was that one female can have around 60 offspring in a generation. Not only this though, but they also do not have 50:50 sex ratios! As an evolutionary biologist, this is such an interesting point. With an altered sex ratio (more females than males) the population can grow at a much faster rate than otherwise. And a larger population means higher likelihood of beetles picking up the pheromone and coming in for the attack. All of which is bad news for the pines.

As the beetles have expanded their range northward and up in elevation, it also seems like the “naive” trees that did not have to deal with MPB in the past are less able to defend themselves. There is strong selection for the trees to be able to make the toxins (monoterpenes) in their resin, but this seems to be less strong or less common in naive populations.

And lastly, to return to the point about the fire regime, MPB prefers older trees of about 60-80 years in age or greater. These trees are bigger and thus a better food source and better for surviving through the winter. As the trees age, they also have decreased ability to fight off the beetles. When fire was more common for lodgepole, stands of trees would not reach this age as often; forests would regenerate more frequently and on average have a younger age structure. Lack of fire has created an important change in the landscape that is another token of misfortune for the trees in the face of mountain pine beetle.

There are multitudes of other things that can be said about the mountain pine beetle, but I personally found the above facts most interesting as well as new for me to learn. And here’s hoping that the beetles have held off from the sites I am visiting this summer to collect DNA samples!

01/24/13

Adventures in the field

After completing the first year of my PhD studies here at UBC and having developed a good general idea of the projects I hoped to pursue, I embarked last summer on several forays out of the office, out of the lab, and out of the city to do some field work!

I am a grad student in the zoology department working with Mike Whitlock, and one of my thesis projects will be looking at adaptation in the lodgepole pine (Pinus contorta), a focal species in the AdapTree project. My background now being stated, I will reveal that before starting this project I knew next to nothing about trees. I will admit that I used to call any type of evergreen tree a “pine tree”, and thankfully have since been corrected into saying “conifer”. My preliminary field work thus proved to be extremely educational in terms of my specific project goals, my exposure to field work in BC (I am from the east coast of the US, which is where the bulk of my previous field work experience accrued), my knowledge of trees, and my personal growth as a researcher. I have many people to thank along the way for this, especially those who were willing to go into the field with me to help out when I had nothing in return to offer except some beautiful scenery, fresh air, exercise, and trees. Lots of trees.

I began my first trip to the woods with a lot of planning. And I will admit that one of my favorite parts of doing scientific field work is the strange assortment of equipment any researcher needs.

I enlisted the help of two grad students from the Aitken lab: Ian, who has taught me nearly all of my conifer identification skills so that I can now proudly say I am a pro at IDing lodgepole pine, and Katha who was a visiting student from Spain and very experienced from her own field work with trees.

We headed three hours north of Vancouver, past the town of Pemberton in search of some trees to sample DNA, cores, and take some phenotypic and environmental measures. My first time being “in the middle of nowhere” in BC was quite the adventure. Upon arrival, I laid on the truck horn a bit to ward off the grizzlies that I was probably overly worried about being everywhere. We took in the beautiful view on a sunny day, latched on our bear spray, and were off!

Fortunately, no grizzlies were encountered, but unfortunately, we trudged up a very dense and steep slope with only Douglas fir in sight. With no motivation lost however, we returned to the road to practice our data collection skills on some roadside lodgepole we’d passed on the way up. Coring trees proved to be the favorite activity and measuring local density the least favorite. Though we may not have accomplished the most efficient and worthwhile sampling trip, this first outing proved vital in getting a real feel for how time-consuming different methods were, which data seemed most worthwhile, and how feasible my then-thought-out sampling scheme was. I returned to Vancouver with confidence in the skills needed to both find my species and collect the necessary data from trees.

It was thus time for field trip number 2. My labmate Katie volunteered to help out with her willing husband and dog for a trip to the interior of BC, a first for all four of us! We headed east of Hope, up the Coquihalla highway where we found lodgepole a-plenty, along with Mountain pine beetle damage and death abounding. We had two extremely successful days of sampling, covering two nice elevational transects in what we found to be a beautiful area for wandering around in the woods. My driving skills were also significantly improved on some little-used logging roads. I am no stranger to 4-wheel-drive on unsteady terrain, but it took a little bit of adjusting to get used to this when on a narrow road with trenches every hundred yards or so as drainage, dense tree and brush cover right up against the truck on one side, and essentially a cliff on the other. I was surprised to learn what kinds of roads in BC merit indication on a map.

We returned with a truck full of pine cones, pine needles, tree cores, data sheets, GPS points, sappy fingers, a tired dog, and 3 slightly dehydrated people. All in all boding well for the future. My third foray to the field took us to a site even further into the interior of BC, just outside of Vernon. I had chosen an elevational transect along a road leading up to Silver Star Mountain, which proved to be more difficult sampling than foreseen. There was little public land here, being in a more developed area, a provincial park near the top of the mountain, and a resort at the very top. Yet we still managed the full transect and remained law-abiding citizens. One of my favorite parts of the day was actually talking to the resort staff, slowly finding my way through their chain of command to get permission to sample on the property. They turned out to be extremely helpful, letting me sample wherever I liked and providing me with a local map.

However, as grad students and advisors alike know, developing projects change, sometimes quite drastically. And after this third field trip, so did mine. A major realization from my field work to this point was that I was going to be very hard-pressed to find natural stands of lodgepole pine. Every tree I had sampled so far was within logging stands, and if I wanted naturally growing trees, I would need permits to sample in a park. As it turns out though, there is an invaluable resource available for the lodgepole pine, in the form of a long-term provenance trial established in the 1970’s: the Illingworth provenance trial. With the turns my research project was taking, this resource was the perfect tool.

This provenance trial contains trees collected from 169 provenances (or sources) across the range of lodgepole pine and planted out at 60 test sites across BC. Useful phenotypic data is already available for these trees, so the only thing lacking on my end was DNA samples! Summer was drawing to a close and classes had started, so there was time left for one last weekend of field work to visit an Illingworth test site and see if my plans for this data set were feasible. I again was fortunate to have labmates volunteer for help in the field, and was happy to give my theoretician friends a chance at true field work.

We embarked to seek out the closest test site to Vancouver,somewhat southeast of Princeton, a site called Pettigrew Creek. I received copious preparatory help from Nick Ukrainetz, the lodgepole pine breeder for the BC Ministry of Forests, who provided detailed access notes and site maps. As one might expect however, nearly 40-year-old research plots are not as easy to find as they once were. We succeeded in finding one out of two blocks planted at this site after much driving and searching through the woods. At one point, I am entirely certain that we left the road to find the trial site, walked around and past it, and managed to return to the road entirely looping around the research plot. Luckily, on the second go-around, we spotted an old sign, half buried in the dirt, identifying that we were on the right track.

We fanned out into the woods, and like finding buried treasure, spotted man-made posts, labelled trees, and painted signs. Eureka!

We spent the rest of the day successfully sampling tree heights, DBH’s, and collecting cones and needles for DNA samples. The highlight of this trip would definitely be the testing of a new method for collecting needles from trees with branches too high for the pruning poles to reach: slingshot. I had spent a good deal of time prior to this trip asking around different forestry labs how they collected DNA from very tall trees, and as I did not want to invest in either a rifle or an expensive, multi-telescoping pole, slingshot became the tool of choice. It proved somewhat time-consuming and dangerous to shoot nearly straight in the air, with pieces of gravel ricocheting off of branches and trickling back down to where we stood, but it was fun and we obtained every single DNA sample we sought which I call a success.

I found this summer of field work, despite some hitches, to be priceless. My advice to any beginning grad students out there seeking to do field work of their own is this. Definitely plan your own preliminary outings or tests. Something always goes wrong when working in the field, so you just need to be prepared. I do not feel as if any of my time in the field was wasted, and even if all of my samples will not end up being analyzed for my thesis, they also provide me with material to test my genetic methods on to ensure that no issues arise on that front. There is no amount of planning that can replace the actual experience of being in the field, because that is where you can learn the most about your system. And with the proper preparation and hands-on experience, when the time comes for the real work, your stress level will hopefully be much diminished knowing that you are capable of the task at hand. I now look forward to the coming summer knowing what I can expect to find for the most part.

Lastly, I want to end this post with a few last thoughts that struck me. It truly amazed me how much land there really is in BC, as well as how much logging. Hearing numbers and acres in my head was nothing at all like seeing an entire mountainside swept clear.

As opposed to the cow pastures and corn fields I am accustomed to, it is clear that the resource here is timber. It is also clear that BC has vast areas of unpopulated land, which was the strangest experience for me. Where I am from, you are hard-pressed to drive more than an hour without seeing a gas station or a rest stop or a town. Here, you can drive half a day or more and see no signs of habitation. BC is full of beautiful untamed areas, and I feel like my home state’s motto of “Wild and Wonderful” is just as fitting here.

The Modern Forest

Does my forest look good in these genes?

Author Archives: kimgilbert

Lodgepole pine DNA extractions

Lodgepole pining

Like a moth to the flame or a beetle to the tree

Adventures in the field