On Monday, which was actually a holiday - the mid-Autumn Festival - we spent the great bulk of the morning putting together all the solutions that would be used throughout the rest of the extraction procedures. I should say I'm using the word "we" loosely, as I didn't do much but catch up on data entry while "they" put together the solutions.
But, for those who are interested, here is a list of a few things "they" made up on Monday morning:
DEPC Water (actually made up before we left)
1 M Tris (pH 8) (100 ml) FW: 121.14
0.5 M EDTA (pH 8) (100 ml)
4 M NaCl (200 ml) FW: 58.44
CTAB for RNA (200ml) (need ~ 100 ml)
Just to name a few. How much fun is THAT!! It's a lot of fun, that's how much!
After all the solutions were made, we went out to lunch while the autoclave sterilized everything (for about 2 hours).
When we got back, we could finally get down to business. The hardest part was grinding the samples. For those of you who have never tried to grind small pieces of wood into a fine powder using liquid nitrogen and a mortar and pestle, well, my friend, you just haven't lived! Actually, you can easily simulate the process by putting on leather gloves, and grinding a short piece of cinder block into concrete for about 15 minutes, and putting a couple of bags of ice around you. Oh, but only use one arm for the grinding. Repeat that six times. How much fun is THAT! Too much fun, right there.
After the samples were ground, it was mostly just a lot of waiting. For those who have never done any sort of extractions, the majority of it involves a lot of pipetting, pouring, loading of centrifuges, and waiting. The DNA extraction protocol actually involves a total of 4 hours of waiting (if you follow the entire protocol). So, by the time we got all of our samples ground, it was about 3pm. We were in the lab until about 7:30 that night.
We got through six of our 18 samples on the first day, because those were the ones we were going to use for DNA extraction. Then, both those and the rest were going to be for RNA extraction. The RNA folks finished their protocol, and Katie and I stopped our DNA protocol at the iso-propanol precipitation step, where the solutions could be left to sit overnight at -20C (they should be left for at least 1 hour).
All our samples, floating piece-fully (ha!) in RNALater. The stem samples are on the left. The leaf samples on the right were only taken from the healthy trees. We would grind those on Wednesday to use as backups for DNA extractions from the stem tissue.
Andy pours some things and lets some stuff mix up and junk.
Two finches, the labs mascots. They were actually quite nice to have in the lab. At first, I thought it was something electronic, but they were very real. And cute!
Kathleen and Amelia label microcentrifuge tubes. The DNA extractions alone take 42 tubes for he first six samples. Having them pre-labeled helps the process go much more smoothly. I believe Amelia is doing the DNA tubes and Kathleen is prepping the ones for RNA.
Bill and Andy work on grinding samples. It's definitely the most difficult part of the process. The dewer of liquid nitrogen is in the middle.
Frozen lab bench, frozen mortar, and frozen stem powder. This is the product after the stem grinding process. A small amount was used for RNA extractions, another small bit for the DNA extractions, and then a final small amount for some metabolomics that Amelia was working on. We only used about 3/4 of the powder that was made.
The trip normally takes 3 hours and involves several very long tunnels, including one that is 18km long. They even have plants growing in 3 places in that tunnel! It was raining as we left Ankang, which can make for dangerous driving conditions, but we thought we'd already finished with the worst! We were wrong.
Turns out that our trip through the mountains had loosened up some fittings in the joinings between the axle and the right front tire. I'm not sure if you've ever had that happen, but basically this makes your vehicle swerve and turn almost uncontrollably. So, as we entered our third or so tunnel, we started drifting sideways, and then hit the inside of the tunnel on the right front part of our van.
Thankfully, the accident was not a bad one, but one that did require repair. All of us are just incredibly thankful the accident didn't happen while we were in the mountains, especially on the twisty, turny road at 5800 feet in elevation!
Today, we are to visit the city wall of Xi'an and relax the rest of the time. Tomorrow, we will visit the Terra Cotta Soldiers and then depart for Beijing. We have all of our samples, so we'll get down to extractions on Monday.
The van. AFTER it was fixed. But also imagine this thing going around those mountain roads. The feeling is really indescribable, and probably personal in nature. Kathleen might describe that feeling as "sheer terror" where I might describe it as "fun when you don't think about what could happen".
Our group at the Xi'an City Wall. Mr. Wong, Mr. Lin (our MOA trip interpreter), Bill, Kathleen, Katie, Andy, Amelia, and me.
On our second day in the mountains, we were told we would be able to collect samples of C. mollissima. We did not collect these on Nan Gong mountain, but instead traveled about 20 miles south and then about 40 miles north and east of that site to find large ban li.
At the first tree, we had to hike up about 1/4 mile to a farmer's house. He said the tree was fairly large when he was a kid, and our guides estimated it at about 100 years old. Again, it was dangling on the side of a mountain, and it took some work to get that sample.
From there, we drove another 2 hours, up through some of the most gorgeous country I've ever seen. The terrain was much like the Rockies, but vegetated. We climbed from about 1000 ft elevation up to almost 5800 ft elevation, all the while twisting and turning around Nan Gong Mountain. We were basically going from west to east through the National Park on a concrete road.
We kept going, and going, and going. We finally came to a small town where we met with some local officials and ate lunch. They mostly took shots of rice wine. We mostly ate, and wondered where we were going!
Back on the road, we took a right across a creek, and started back up a mountain. Unfortunately, our path all the way up was blocked by a very bad rock slide, so we had to back down the mountain. We found a small, about 20 year old ban li that we sampled. While we sampled, the bus tried to turn around. Those efforts were unsuccessful until about 30 minutes later and some excavation of a small rock wall. The bus was finally turned around and we were back on the road again.
Since we'd only collected from 2 trees, we wondered again where we were going. How far would it be until we would find another ban li? Although C. mollissima is quite plentiful in Shaanxi province, and much of northern China, the trick is finding a LARGE tree that is also "wild". Most Chinese chestnuts that you actually come across are quite young, and most are grafted into orchards.
So, we finally reached another area where we pulled into a farmers house alongside the road. We climbed up about 500 feet and found an area where someone had grafted Chinese chestnuts amongst some rather large and older C. mollissima. Here, we were able to collect the final samples of our trip.
Oh. Who cares if the road is washed out a little bit and the van will barely squeeze through?
Or that there are giant rocks in the way! (picture by Andy Newhouse)
Of course we forged on to find wild ban li! The C. mollissima is the large tree on the left hand of the frame. Andy is in the middle of the picture, getting ready to take the samples.
In this picture, Bill has taken over cutting up the stem segments and putting them in the test tubes with RNALater. Andy is in the background using a cork borer. It was at this site that I dropped my camera about 100 feet down the slope. In retrieving it, I had the misfortune of finding that Chinese stinging nettle acts the same as it does here in the US. Fun for me!
Town of Zoujiaping where we made our first C. mollissima collection. The mountains of southern Shaanxi province are just spectacular.
And on through the bamboo forest, up a hill (we were always either going up or down a hill. No flat land here!!) to our last collection site for C. mollissima.
We probably spent a good 6 hours just driving on this day, but the terrain is really spectacular. Andy was able to take this picture out of his window as we were making our way over NanGong Mountain from the west to the east. We went up about 4500 vertical feet in 6 miles and then back down the same way, just twisting and turning the whole way. In the middle of this picture, you can see the concrete road winding it's way around the mountains. An how about those "guardrails", huh? I'm kind of just glad we made it back to 1500 feet elevation in one piece, and with all four wheels on the road (most of the time!).
And, for fun, I though you guys might like to see a map of all the twists and turns we made looking for 6, that's right, 6, Chinese chestnuts in China. You'd think it wouldn't be that difficult . . . !
Our tour guide this time was Mr. Chen, whom you may remember from our trip to the same mountains on our last trip. We again went up to Nan Gong Mountain, and also ate lunch at the same location where we met the mayor last time.
We were able to collect all of our samples of Castanea seguinii (mao li) and C. henryi (zhui li) here, but had to wait until the next day to collect the C. mollissima (ban li) samples.
At this point, you may wonder about our sample collection methods. The idea for this trip is to collect different tissue material from all three species. For each of the three species, we want three fully healthy trees and three cankered trees (preferably ones with active cankers). This makes a total of 18 trees all together that we need to find.
When we find a tree, Andy uses a large cork borer, about 1/2" in diameter, to collect two cores of cambium tissue. He scrapes away much of the bark before coring into the tree. For the cankered trees, we also want material from both the healthy part of the tree as well as the active canker margin. By doing this, we can see if there is differential expression between the tissue around the blight and the other tissue in the tree. If done right, this can allow us to find activated areas of the genome that may be related to blight-resistance. For the healthy trees, we want to collect tissue from the trunk as well as leaf tissue. The leaf tissue will be used for genomic DNA extractions.
For the cores that are taken, two cores from each type of tissue, cankered and healthy, are taken. Each core is then cut into four wedges using a knife and the wedges are placed into a 50mL tube containing RNA later. Because we have no way to get ice, and keep it ice, while out in the mountains, the RNA later will act to keep the RNA and DNA from our samples from degrading until we can get them back to the lab in Beijing for extractions.
In addition to collecting the tissue samples, we also took several data points on each tree including 1) height; 2) dbh; 3) health (1-5, like last time); 4) form (1-5, like last time); and 5) GPS coordinates. Each tree was coded based on species and health, and we also took some general notes on the condition of the tree such as whether it was multi-stemmed, growing on the side of the road, or anything else of note.
Terrain in this part of China is very tough, which makes finding trees to sample somewhat more challenging. Because we wanted trees in very specific condition - large and healthy or large and with active cankers - Mr. Chen and I spent some time walking around and trying to find the most appropriate trees for sampling.
Some pictures are below. We'll be typing in the data when we get back to Beijing for the extractions. I'll be sure to update everyone on what all we sampled once we get that done.
A message from the hotel in Lan Gao where we stayed for one night. We used this as our base of operations between driving around the mountains of southern Shaanxi province.
How samples were taken in the field. Here we are in NanGongShan Park, just off the road at about 3500' in elevation. Andy is in the background taking stem sections from the tree with a large cork borer. Bill is sitting, labeling tubes filled with RNALater. Kathleen is on the left, dipping her knife into alcohol, sterilizing it so that she can use it to cut up the samples Andy takes from the tree (samples need to be small for the RNALater to work). On the right is Katy's hand w/ a lighter which she uses to flame the tools for sterilization. Amelia is not in the picture, but is helping to interpret our guide, Mr. Chen, as he finds new trees for sampling.
In this picture, we are all holding on for dear life on the slope. Here is a cankered C. henryi that Andy is getting ready for sample collection - as you can see he hangs on to the tree to stay steady. Kathleen had a hard time finding a flat spot for her cutting board where she would cut the stem disks into 4 pie segments. Bill is handing the labeled tube up to Kathleen so she can put the samples into it.
In the foreground, you can see the needles of a tree that is often found in these mountains, which we think is a species of Araucacia.
Here you can see the tally sheet along with what is left behind on the trees once a sample is taken. Both stems here are from a C. seguinii. The one on the left is quite cankered while the one on the right is healthy. Two tubes, then, were taken from this one tree and used for both DNA and RNA extraction.
Our group from the collections. Katie D'Amico, Bill Powell, Mr. Chen, (still getting name), Andy Newhouse, Ameila Zhang, Kathleen Baier, (still getting name).
Once we started on our three hour drive, we found out why! There were many, many, many rock slides, mud slides, and areas where the road had been washed out. It certainly made for an interesting ride, and put some amount of trepidation about heading up into the steep terrain of the mountains where we'd be getting chestnut samples.
One of the many rock / mud slides we encountered on the way to Ankang.
Our group has now all been in China for two days. Katie D'Amico and I have been here for about 5 days, having spent a few days in Shanghai for general sightseeing (more on that later. Shanghai is awesome!)
So, I suppose I should introduce our group! We've got me and five folks from SUNY-ESF: Dr. Bill Powell, Amelia (Bo) Zhang, Kathleen Baier, Andy Newhouse, and Katie D'Amico. Yesterday, Katie and I flew in to Beijing from Shanghai and met the group. If anyone followed my blog from last time, I bet you can guess what we did as our first activity ... EAT!!
I had a couple of firsts yesterday: squashed chicken (picture below) and Peking duck. Surprisingly, we didn't have any last trip; then again, we weren't in Beijing very long. We also had Peking duck again today, and it was still very good. I can definitely say that, when in Beijing, you need to get the Peking duck. I didn't realize that you don't just eat the duck, but wrap it in something like a wheat tortilla w/ a thick sauce, cucumber and scallion strips. Yum.
As for work, we met up today with a couple of our main collaborators, including Dr. He Wei, a host from last time, and a new host, Dr. Jiang. Both Dr. He and Dr. Jiang are from Beijing Forestry University, so we took some time to walk around and visit the University's lab spaces.
As for sightseeing, we've had a bit to do. We did get to see the Olympic Village (pic of group below). Tonight, we saw a very good show at a Middle Eastern type restaurant. The entertainment was an excellent band that sounded much like the Gypsy Kings. Among other songs, they played "Volare" (of course!) and a cover of "My Way" and "Perhaps, Perhaps, Perhaps". The band played about 3-4 songs per set, in between which we had two belly dancers. It was definitely a new experience in Beijing!
Tomorrow, we head out to Xi'an, and hopefully then on to Ankang. We are still unsure of our exact location of field work, but we do know it's raining there, and scheduled to continue for the next couple of days. Hopefully it will let up by the time we hit the field!
Mmmm. Squashed chicken. It actually was VERY good, but had a lot of elements that seemed to be repeated on this trip. 1) Lots of bones in the food; 2) head attached and/or included w/ meal; and 3) Yummy!
Kathleen and Katie review the materials at Dr. Jiang's lab that were ordered prior to our arrival. We spent part of the day putting all these materials together and prepping for our trip to the field.
.Dr. He shows off isolates taken from Dalaoling and the NanGongShan Mountains, both sites we visited back in 2008.
Our group at Bird's Nest Stadium. Amelia, Sara, Kathleen, Katie, Bill, and Andy. The Water Cube is behind the photographer, on his right (Picture from Andy Newhouse).
Beijing Forestry University is pretty close to where Beijing hosted the Olympics. In the background is the Bird's Nest Stadium, which I'm sure you guys have all seen plenty off. Off to the left, then, would be the Water Cube, which we also walked by. In the foreground is a really big piece of jade.
Our first dinner out as a group, and we went out for Peking Duck! In the picture are our bus driver while in Beijing, then Mr. Lin, our interpreter, and Bill, our fearless leader. The tortilla-like wraps for Peking duck are there in the middle. The sliced duck is the upper-left of them. A thick soup of duck fat and duck pieces can be found in the upper-right of the picture. And, no Chinese meal would be complete without a head of some kind. You can see the duck head, split in half, there to the lower-right of the wraps. Mmmm. Duck head. (Picture courtesy Andy Newhouse).
The crew this time will be mostly composed of members of Dr. Bill Powell's lab from SUNY-ESF. These are the guys who have been focused on the molecular side of chestnut breeding, from the mapping of chestnut genomes to transgenic work. With such a different point of view for this trip, our group will be visiting most of the places we did last time, but instead of visiting each area for several days, we will stay only long enough to collect samples. We will then take those samples back to a host's lab at Beijing Forestry University for DNA/RNA extraction.
Keep your eyes out for updates, possibly over the upcoming weekend, or sometime next week!
Before we left the Lan Gao area, we would make one last stop to look at some chestnut trees. This site would again be on very steep slopes but this time would primarily have Castanea mollissima. We could not confirm whether these trees were wild or naturalized, but they were likely not planted.
We spent less than an hour at the site so that we could get back to Ankang on time. Xi'an is a 5 hour - at least - drive from Ankang, so we wanted to leave shortly after lunch. We were making pretty good time, but the roads were very twisty and turny - it took us quite some time to get onto the road that's much like a Turnpike. We did have to stop to get gas before we got on the major Road. And Fred needed a Slim Jim and a Coke. The Coke was there, but he had to settle for a bag of Chinese crackers.
Most of us took little naps - it had been a long trip. But after about 2 hours, we were stopped by a police blockade. We were about the 4th or 5th vehicle to be stopped. Thankfully, we were stopped right by a rest area. We learned traffic was stopped because of a bad accident in a tunnel a few miles up the road. Turns out there were several very long tunnels up ahead. So, we stayed there for about 3 hours while the accident was cleared.
It wasn't such a bad time, actually. What was refreshing about this wait is that everyone around us seemed to be taking the delay in stride. A snack stand, complete with drinks and snacks (including two types of chicken feet), opened up outside the rest area. The folks beside us whipped out some moon cakes, pears, and started playing cards. Most people milled about outside their cars chatting. Noone seemed upset. What a different scene I would suspect we would see in the US.
Fred pondered if his extra stop to get a drink and snack saved us from an accident. You never know . . .
Measuring Castanea mollissima at forest plot in Bashan mountains. Having climbed down to this tree, I can tell you the slope was pretty darn steep, the steepest I'd went down so far. I think Kim and Songlin measured it at 93%. I had on my boots. Mr. Liao, to my right, was wearing loafers. I am not kidding. They were pretty nice shoes. It looked like he hardly even got them dirty.
Not the most flattering pose, but we all got in a nap or two during the duration of our trip, espeically going from Ankang to Xi'an.
The group waits for the traffic jam to clear up.
A magical bottle of Coke and Chinese crackers??? At least we got stopped next to a rest stop and not in the middle of nowhere next to an 8km long tunnel on up the road. We'll give the snacks at least this special citation for their possible role in keeping us comfortable, if not safe and alive.
After meeting the mayor and eating much, much, much more food than is really good for any one person, we set off into the park. Our first stop would be up to the temple in the park. Unfortunately, about half-way up the mountain, we encountered some nasty fog which lasted the all the way to the top. So our view of the temple and anything from the mountain was quite obscured.
We didn't have any time to tour the temple since it was already early afternoon. With the fog settling in, we knew - and were told - that it would be too dangerous to try and scale the mountains of the area to take data on chesntut trees. The combination of 80 degree (!!) slope, skree, and pea-soup fog was just too risky. So we decided to make our way to a fog-free zone to see what we could find. We actually did see a lot of trees.
In fact, it was here that we saw what we could absolutely, positively be certain were wild Chinese chestnut trees. But it was also at this point that our hosts told us something a little sad, at least for this trip. Mr. Chen basically said that if we really wanted to see a lot of wild Chinese and possibly large chestnut trees, that we would need at least a week in the area. He noted that we would need to backpack in about a day, camp out, and explore a couple of different sites.
Sign me up!!
But we were able to take some general measurements on about 10 more trees before it was too dark to continue. At that point, we would head back to Lan Gao where we would eat another huge meal and stay for the night.
Meeting the mayor of Ankang at Nan Gongshan National Park. The mayor, Mr. Fang wei Feng is the white shirt between Kim and Fred P.
After coming down from the temple side, we started our data collection at a small set of Castanea henryi. These trees appear to have been damaged during recent road construction.
Larger Castanea henryi just down the road from the trees above. This tree was in very good health and quite large for the area, about 65' tall and around 13-15" dbh.
Rock pile leading to C. henryi above. The slopes here were very steep, approaching 80°+ in places. As shown in this picture, leaves on this tree were right down to the ground and easily accessible for appropriate species identification.
Burs of Castanea mollissima, just down the road from C. henryi above. Again, this tree is located down a very steep and rocky slope that leads to a stream below. As I recall, the slope made this tree inaccessible for measurement.
This is a view across the ravine from the main road along which we were measuring trees. Across the ravine, from what we were told, is a forest just full of chestnuts, especially Castanea mollissima and C. henryi. If you click on this picture and zoom in, you may be able to pick out the chestnuts by looking for trees with burs on them. They were probably 20% of the trees we could view from the road on the other side.
Fog settling in over Nan Gongshan Park. It tracked us all the way down throughout the afternoon and early evening.
The landscape from Ankang to Lan Gao was slighly different than what we had seen between Yichang and Dalaoling. The major difference was the slope of the mountains - very, very steep! Even steeper than what we encountered at Dalaoling. As a result, there was not as much cultivation (although there was still a lot of it). We hardly saw any tea planted here, most likely because of a difference in climate (too cold, we imagined).
In Lan Gao, we picked up the Director of Forestry for the area, Mr. Liao. Between Lan Gao and Nan Gaoshan, we briefly stopped at an area where reforestation efforts with Chinese chestnut ("ban li") were underway. We asked why Chinese chestnut. They replied that the species would not only provide stability for the mountainside (vs. planting corn or rice), but that it would also provide a crop at the same time. Although the trees would be weeded (by hand) and planted in rows much like in an orchard, because the trees were being planted as part of an afforestation project, they would be overseen by the local forestry administration, no agriculture.
Before lunch, we would also travel to an area where some local people were cutting Castanea henryi for firewood. As we climbed up the muddy road, the sound of chainsaws and axes grew louder and louder. As we approached a local home, we met several local loggers actively cutting both Castanea henryi and Castanea mollissima. We stopped to chat and measure a couple of the felled Castanea mollissima.
After meeting the loggers, we continue to climb up the mountain, past the house and through their garden, to an area where the loggers had already cut several specimens. It was actually somewhat lucky to have them cut as we had an opportunity to look at the growth and count their ages.
We took data on a handful of trees and then had a decision to make. We could either hike on up about 2 miles to see a large stand of Castanea seguinii or we could make our way to the Park to begin looking at the trees there. Having eaten so much food as we had, my vote was for the hike, but I was overruled (and for good reason - we would see more Castanea mollissima in the Park). We would make our way to the park and, with it, lunch!
More farming with terracing. Terracing is even more necessary in this area. And notice the vegetables growing right along the side of the road. Someone on the bus quipped about how this is one place [China] where you could definitely get hit by a car while gardening.
Castanea henryi leaves and burs. The leaves of C. henryi are more like those of Castanea crenata (Japanese chestnut) than any other species. C. henryi leaves are typically more narrow (especially true of sun-leaves) and are totally glabrous (without hairs) on the underside. C. crenata has many stellate hairs and 9-celled trichomes on the underside.
Songlin and Fred work their way up through the fallen trees. All of those trees are chestnuts, either C. henryi or C. mollissima, cut primarily for firewood. These trees were all approximately 28-32 years of age.
Cut portion of Castanea mollissima with Kim's pen.