Chapter 8. Fun and Fights with Fungi, Part I

Derek Jacoby

This was supposed to have been a nice little article on barcoding mushrooms. There was a simple protocol, and lots of folks in the DIYbio community have done all sorts of barcoding so it should have just gone easily, right? Well, not really. Unlike debugging computer code, where everything is right there to see, when debugging biology there’s a whole lot of inference involved. So even though I don’t have a huge set of exciting mushroom barcoding results to share, I thought sharing my experiences so far might be of some general interest.

This story starts back in October 2013 when BioCoder contributor Noah Most (Chapter 9) was visiting Victoria, BC, Canada. I dragged him and my friend Andy around to a few of the mushroom shows that spring up in the fall in British Columbia. The goal was to take pieces of the mushrooms on the show tables, which had already been identified by experts, and do a genetic identification. This would result in one of three possibilities. First, the mushroom could exist in the barcoding database and agree with the expert identification. Yay, concordance! Second, the mushroom could exist in the database, but be different than what the expert identified. This would also be interesting. Finally, the mushroom could be absent from the barcoding database, meaning we could put it in! That’s the really fun part, when something I’ve discovered turns out to be new and adds to our shared pool of scientific knowledge.

So where’s this barcoding database? And how does this all work? DNA barcoding is a process of using a predefined section of an organism’s DNA to identify it. Different kingdoms use different segments of DNA—the barcode region for animals is not the same for plants, which is not the same for bacteria. But in each case, the concept is the same. The barcoding region is an area of the genome that is not under evolutionary selection pressure, so over evolutionary time it is subject to random drift. This means that each species you want to identify will have random variations in the barcoding region. As more species are collected into the barcoding database, it becomes possible to construct a phylogenetic tree of the species based on genetic similarity. The central place on the Web for DNA barcoding covers a lot of things we’re not directly interested in for fungal barcoding, so we use a more specific site.

One of the first things that must be done in barcoding is to identify the set of primers that will be used. Since we want to use a small segment of the genome and not have to sequence more than we need to, the first step in barcoding is to extract that DNA from the fungal cells and amplify our barcoding region using polymerase chain reaction (PCR.) The primers determine which region it is that we will amplify. Originally choosing the primers in fungi took a lot of work and a lot of cross-species comparison. Through the process, a number of different barcoding regions were suggested. The most common barcoding region currently used is between locations ITS1 and ITS4, so we’ll use primers ITS1F and ITS4R to amplify our barcoding region, leaving us only approximately 800 base pairs to be sequenced. (A more detailed look at other fungal barcoding regions is available online.

But I’ve skipped over a part, and it’s a part that needed a lot of debugging. How do you extract the DNA from the fungal cells? Many of you have probably seen the demonstration of getting DNA from a strawberry—if not, it’s a great instructional activity and a lot of fun. There’s a complete protocol online. Unfortunately, most fungi are not as easy as strawberries. Trying to extract DNA with the dishwashing detergent, salt, and water mix that works well on strawberries isn’t effective on most fungi. This step of the DNA prep consists of breaking the cell open—through mechanical and chemical means with a lysis buffer—then precipitating the proteins and spinning down the precipitate in a centrifuge (this leaves the DNA in the supernatant—the liquid on top of the pellet of protein). Then, with the proteins gone, you perform an isopropanol extraction of the DNA and an ethanol wash. Finally, the DNA is rehydrated and ready to be used. I started out trying to use the cell lysis buffer from a Carolina Biological barcoding kit, but that seemed to be a protocol geared mostly toward plants, and none of the first three mushrooms I tried in that protocol produced any template DNA. Frustrated, I ordered a DNA prep kit from Feldan (kit number 9K-006-0016) and tried that. It still didn’t work very well. Finally, using the cell grinding and mechanical disruption from the Carolina kit and the lysis buffer from the Feldan kit, I began to get results—at least, from some of the mushrooms. There remain some mushrooms that I am not very successful in getting DNA out of: specifically, the ascomycetes, which include mushrooms such as morels, seem to be very resistant to DNA extraction using this protocol. In the end, I suspect I will have to use more forceful mechanical disruption techniques, perhaps sonication or something else that would release more DNA. I would also like to find a cheaper DNA prep solution—perhaps guanidine for a lysis buffer instead of depending on kits that end up costing 75 cents or so per prep.

If all has gone well in the DNA extraction, you see an 800-base pair band when you run a gel of the PCR product. (Running a gel consists of pulling negatively charged DNA through a gel matrix using electrical potential in order to sort the DNA fragments by size.) Before sending things away for sequencing, it’s important to verify that you have produced a nice sharp band on the gel, indicating that the barcoding region was properly amplified. Here was the place where a second round of debugging was needed. Gel electrophoresis is a basic procedure that is used in all sorts of molecular biology, including protein and DNA identification. When using gel electrophoresis for identifying DNA fragments, you generally run a lane of the gel with a size standard. This is known as a DNA ladder, and allows you to compare your unknown DNA fragments with DNA fragments of known size. When I ran the first gel I didn’t see anything, not even the ladder DNA. This told me that something was wrong with my gel.

Historically, gels have been run using a DNA binding dye called ethidium bromide (EtBr.) It’s very effective, but EtBr is a mutagen and requires hazardous waste disposal, so most labs try to move away from using it. One of the gel dyes that I like is called GelGreen, but it had been a few months since I last used my stock of GelGreen. Apparently it is not as shelf-stable as would be ideal. When I saw nothing, I ran a gel on EtBr and at least saw my ladder bands, so I knew something was there. One of the other folks in the lab, Vince, had the bright idea to use a UV laser (405 nm) to excite the GelGreen rather than the set of blue LEDs that we normally use. When viewed through an orange filter, the laser illumination let the dim bands pop out, whereas they were not visible at all under our normal illumination. We ordered new GelGreen and will go back to our normal gel procedures soon, but maybe we’ll get Vince to write up an article on his super-sensitive laser gelbox in a future BioCoder issue. Seeing the ladder bands gave me confidence in the lack of amplification of the samples and led me back to debugging either the PCR process or the DNA extraction process, and concluding in this case that it was an extraction problem.

At this point I have a couple species of mushroom sent off for sequencing, but no results yet (see Figure 8-1). The experts identified them as Sparassis crispa and Pseudohydnum gelatinosum, but we’ll see if that’s what they really are. Then there are about 40 more samples to run through that we collected from the mushroom shows. We’ll go through the bioinformatics in the next issue, after I get results back. My current passion happens to be fungi, but DNA barcoding is applicable to any living organism you might be interested in. I bet there are new discoveries to be made right in your backyard!

Gel electrophoresis of the two samples, Pseudohydnum gelatinosum and Sparassis crispa, in lanes 13 and 14. S. crispa is showing a double band and had to be rejected for sequencing, but P. gelatinosum was properly identified by sequence results.
Figure 8-1. Gel electrophoresis of the two samples, Pseudohydnum gelatinosum and Sparassis crispa, in lanes 13 and 14. S. crispa is showing a double band and had to be rejected for sequencing, but P. gelatinosum was properly identified by sequence results.