While most people have heard of DNA, not everyone knows how important it is. Every living thing on planet Earth has DNA, or deoxyribonucleic acid if we are being formal. DNA is the chemical compound that contains the instructions needed to make proteins that are essential to the survival of just about every organism. An organism’s entire collection of DNA is called its genome. Basically, it’s the blueprint of life and every cell in every organism contains a complete copy of its particular genome. You might be thinking “Wow, that’s a lot of blueprints for just one organism.” Yes, it is, but that’s to be expected. Life is complicated, and it requires a lot of instructions.
So, what does this have to do with the mangrove rivulus? Well, it has everything to do with the mangrove rivulus! In the Earley lab, we care about the genetic makeup, or the “blueprint,” of each of our fish. Why? Because without this information we would not be able to study them the way we do. Before we can get into the nitty gritty of this ‘genetic makeup’ stuff, and why we study it, there are a couple of things you should know. Every organism’s genome is made up of a collection of genes. Simple enough, right? Wrong. In most organisms, these genes are present in two alternative forms, or alleles, that can be identical or different. Organisms, including us, are said to be heterozygous if they have two different alleles for a given gene and homozygous if they have identical alleles for a given gene. In a previous article (see “Let’s Talk About Sex”), I mentioned that the mangrove rivulus, specifically the hermaphrodites, can fertilize themselves. This is especially cool if the hermaphrodite is completely homozygous, a situation where the animal has identical alleles for all of its genes. In this case, self-fertilization will result in offspring that also have identical alleles for all of their genes, and the exact same alleles as the parent. Thus, self-fertilization effectively produces clones! For our experiments, you can imagine why homozygous offspring would be very useful. We can expose individuals of each homozygous clonal lineage to different environmental conditions and see how it affects their behavior, physiology, morphology, or any other trait of interest. Since there is no genetic variation among siblings, the only differences we might see among them are the direct result of environmental factors. Remember, there might not be variation among siblings in one clonal lineage, but there definitely is variation among lineages. One homozygous ‘parent’ might have a different genetic composition than another homozygous ‘parent,’ leading to clonal lineages that are distinct from one another. All this talk of homozygotes might have led you to believe that heterozygotes don’t exist in mangrove rivulus populations. Well, they do. Unlike homozygotes however their offspring are not genetically identical. But, how are there both homozygotes and heterozygotes in natural populations? Instead of fertilizing themselves, hermaphrodites also can mate with male members of the population. This is known as outcrossing and it introduces new, different genetic information, and heterozygosity, to the offspring. Therefore, the kids are not all genetically identical and some might have inherited some different versions of a trait that proves to be advantageous.
Considering our research and how I mentioned that we really like to make clonal lineages from homozygotes, you might be wondering how we can tell if a fish we acquire from a natural population is a heterozygote and homozygote. Well here is where being able to actually figure out the fish’s blueprint, or at least part of it anyways, comes in handy. In order to do these types of studies, we have to decipher each fish’s blueprint before we can actually do anything with it. But how the heck can we do that? Science, duh! It’s complicated, but definitely not impossible.
So, here’s the rundown. The process of deciphering the ‘foreign language’ that makes up an organism’s genome is called “sequencing.” Technically, there are many different types of sequencing methods that can be used to determine the degree of variation between different individuals in a population, but today we will just focus on one: DNA fingerprinting. DNA fingerprinting does not require the researcher to decipher the fish’s entire genome in order to find out if it is a homozygote or a heterozygote. Instead of taking the time and resources to determine an individual fish’s genome, we can instead take only a portion of that genome, sequence it and use it to determine how heterozygous an individual is, and also levels of heterozygosity in an entire natural population. The process involves collecting a little piece of the fish’s fin, 1 millimeter by 1 millimeter in size, which is then used to determine their genotype, as well as if they are homozygous or heterozygous.
Interesting stuff, but how do we know what portion of the genome we should be comparing? Can we just take any random part of the fish’s genome and determine its heterozygosity from there? Well, no. The thing about genomes, and especially genomes from members of the same species, is that there are always parts that are the same within a species. That means that every individual mangrove rivulus, regardless of who its parent or parents are, shares part of their blueprint with other members of the species. This also applies to heterozygotes and homozygotes. Lets take a look at this from a human perspective. Part of my genome is identical to part of your genome. So how are we not exactly identical? Well, only part of our genome is conserved. Other parts of our genome might be very different from each other, and it is these differences that make me uniquely me, and you uniquely you. So with this idea in mind, lets get back to the fish. Researchers of the mangrove rivulus have identified 32 neutral loci, a fancy word for different positions in the fishes genome that do not encode a protein and that, when amplified, studied and compared, can tell us whether these fish are heterozygotes or homozygotes. These loci are called “microsatellites” and, because they do not encode proteins, they are ‘free’ to vary in their sequence without having any adverse effects on the organism. That’s exactly why these regions often have more variation than other loci; the fish can afford it! These microsatellites typically consist of tandemly repeated nucleotide bases. The genetic code is just a bunch of A’s, T’s, C’s, and G’s (nucleotides) strung together in a particular order; microsatellites can be identified because they have repeated sequences of nucleotides. For example, a microsatellite might have ATC repeated multiple times; you might have ATCATC, and I might have ATCATCATC.
Now, remember that this fingerprinting is being done on fish collected in the wild. We use these tests to find homozygotes so we can collect them and create lineages of genetically identical fish (isogenic lineages) for our experiments. The fact that homozygotes as well as heterozygotes exist in natural populations raises a very interesting question: if the homozygotes are able to successfully produce offspring in a completely self-sufficient manner, why should they ever want to deal with a male? Good question, and one that many researchers are interested in answering. One idea is that reproducing with males introduces new combinations of the blueprint into the population. If these new genotypes confer some sort of benefit to the fish in terms of survival or reproduction, then this new genotype will persist, and some combination of self-fertilization and outcrossing with males will be favored evolutionarily. It has also come to light that homozygotes collected in the field are actually more susceptible to parasites than their heterozygote counterparts (Ellison et al. 2011). This could be one possible benefit that heterozygotes have over homozygotes. Other studies have shown that male heterozygotes are often larger than homozygotes. This could be another reason we observe heterozygotes in natural populations, especially if you consider the competitive edge a larger male could have over a smaller male when looking for love or when fighting with other individuals (Molloy et al. 2011).
Wow, while this may seem like a lot of information it is really just the tip of the iceberg when it comes to what we can do with genomes, as well as genome sequencing. Being able to decipher the language of the rivulus’ genetic blueprint affords us endless opportunities for studying these little fish with a lot to offer. For more information about how examining our fish’s blueprint can provide novel insights into its biology, visit the pages of our collaborators. Dr. Andrei Tatarenkov and Dr. John Avise at the University of California, Irvine are experts in identifying microsatellites and using them (as well as an arsenal of population genetic analyses) to address fascinating questions in mangrove rivulus and loads of other organisms! Dr. Joanna Kelley at Washington State University is diving into the mangrove rivulus genome to determine how our fish’s blueprint governs many aspects of its extraordinary biology!
Ellison A, Cable J, Consuegra S (2011). Best of both worlds? association between outcrossing and parasite loads in a selfing fish. Evolution 65: 3021–3026.
Molloy PP, Nyboer EA, Côté IM. Male-male competition in a mixed-mating fish. Ethology. 2011;117:1–11.