So the previous project I described is what I worked on most of my junior year and is in the process of being transformed into a paper (which I’ll likely get to be first author on – huzzah!). What other lab work have I been doing recently?
This wasn’t even meant to be a full project, but really started out of curiosity (like all good science does). One of my professors, Prof W, was doing some collection in the krat’s breeding season (Novemberish) instead of our usual July. During his trapping he was lucky enough to discover some copulatory plugs.
What the hell are copulatory plugs, you ask? Basically it’s a secretion the male deposits during mating that hardens into…well, a plug that takes time to remove. This makes it harder for the female to mate with another male later for obvious reasons. Or to steal a good analogy from Wikipedia, it’s a biological chastity belt.
Most rodents and some insects use copulatory plugs but they’re still a bit of a mystery. There’s not much literature on them in rodents, and virtually none on kangaroo rats. So we thought, what the heck can we do these things? Hmmm, let’s chop them up and genotype them!
We cut the plug into four segments, so one segment 1 would be the most internal in the female, and segment 4 would be the most external. We digested the plug and extracted DNA, then genotyped the DNA using various genetic markers. We then compared the results for these markers to the genotypes of the females we retrieved the plugs from. The tricky thing here is that you have a mixed sample, something they have to deal with a lot in forensic cases. Think of a rape case – you may have a semen sample, but it’ll have DNA from the female in it too. How do you know which belongs to which when you’re looking at something on a screen?
This is an example of what you would see (each number represents an allele):
Female: 130, 142
Mixed sample: 130, 136, 138, 142
You know that 130 and 142 most likely came from the female, and the new 136 and 138 came from the male. So the male is 136, 138 right? Well…it’s a bit more complicated. Maybe there are two males, and one is 130, 136 and the other is 138, 142. Or maybe one is 136, 138, and the other is 138, 142. What if there are three males?
Thankfully, there are ways around this. One is by comparing the relative strengths of each allele (not going to explain that, sorry). Another is using multiple markers. Another is assessing the probability of the combination using statistics. And finally, you can use the exclusion principle – see what males absolutely cannot possibly have contributed those alleles, and see who’s left and how the puzzle fits together.
I think you can imagine that this project is the ultimate puzzle. It can be a pain in the butt deciphering everything, but it’s really rewarding once you crack the code. And what have we figured out so far? Well, we have a pretty good guess of what male contributed to the plug, and in some cases more than one male appears to have contributed to the same plug, with their contributions separated by location in plug. Aka, the male that got their first formed the most internal part of the plug, and the second male formed the most external.
And before I start talking too much about rodent sex, I’m going to leave the implications of that up to your imagination.