We often refer to DNA as the building block of life. But when we expand our view of DNA to examine the whole genome, it starts to look more like a storybook that tells us all the history, plot, and relationships of an organism’s existence. There are so many questions we can ask just by diving into different pieces of genetic information within the genome. What is the genetic variation that drives diversity and phenotypic variation we see across species? How are different groups of organisms related? These questions are especially interesting for organisms that are adapted to flourish in extreme conditions, like deep-diving cetaceans. Are any genes undergoing selection or genome regions that are important for how dolphins and whales adapt to deep diving environments? 

 

To explore these questions, scientists have developed a rad technique called RADseq, or restriction site-associated DNA sequencing. This method falls into the “reduced-representation” category of high-throughput sequencing, which aims to make genome sequencing operations more efficient and cost-effective. “Reduced-representation” means RADSeq only targets and sequences a smaller subset of DNA fragments within the genome.The innovation of RADSeq truly revolutionized fields of ecological, evolutionary, and conservation genetics - in 2010, Science even listed RADseq as the “Breakthrough of the Year.”

 

So how does RADSeq work? The process relies on a special class of enzymes known as restriction enzymes. Once DNA is extracted from tissue samples, these enzymes are designed to cut the DNA strand at specific nucleotide sequences, giving researchers the flexibility to sample many markers (also known as loci) across the genome. Researchers can also control how many snipped DNA fragments are created and how long they are, ultimately determining how much DNA is read into useful data. For instance, an 8-cutter enzyme will cut the DNA more infrequently at around every ~65,000 base pairs, compared to the ~4,000 base pair interval of a 6-cutter. 

 

The DNA fragment sequences can then be aligned with a reference genome. Think of the reference genome as a blueprint map that someone has already pieced together, base pair by base pair, and annotated with street signs marking out where gene-coding sequences begin and end. This allows us to navigate to the locations of interesting genetic landmarks. Once we match up our RADSeq loci to know exactly where in the reference genome it sits, we can then pinpoint if any single-nucleotide polymorphisms (SNPs) exist, where a nucleotide differs between the individual we just RADsequenced and the reference genome. If we sampled many individuals, we can use statistical techniques to determine how those individuals group together based on how similar or dissimilar the occurrence of SNPs are in their genomes. 

 

There are several advantages to using RADSeq. Sequencing the entire genome of an organism - which would involve reading every single basepair in the billions - is incredibly time-intensive and expensive. So RADSeq can be particularly valuable in analyzing non-model organisms that we don’t have existing genetic information for, such as the many marine mammal species we work with on this project. It is also advantageous when studying species with large genomes or conducting population-level studies with a large number of individual samples needing to be processed. 

 

This was exactly the case for our study looking at the genetic variation in dolphin populations on the southeast U.S. coast. The 2020-2021 team created a RADSeq “library” from the DNA of 96 bottlenose dolphins that the team collected skin samples from. RADSeq allowed us to identify SNPs that were outliers in terms of how different they were between the inshore dolphins and offshore dolphins. This year, we went back to our reference genome map, dropped those outlier SNP coordinates into the equivalent of a genetic StreetView, and looked around to see if any genes were within the vicinity of the SNPs. By searching within a window of 200,000 base pairs around each SNP, we found 11 genes that regulate different aspects of inflammation and cell-death. These could signify important cellular and physiological responses when a dolphin is holding its breath and diving deep. 

 

However, RAD-seq only gives us a thousand-feet view of the genome. So far we’ve identified the general neighborhoods containing genes that may be driving the differentiation between in-shore, shelf, and offshore pelagic dolphins. But what if we want to zoom in to find the exact loci under selection between dolphins that like to spend time close to the shore, versus dolphins that hang out beyond the continental shelf? This is where whole-genome sequencing comes in. We are fortunate to have funding through the Bass Connections program at Duke to complete whole-genome sequencing on a select number of dolphin DNA samples. The ReSeq analysis will also help us determine whether the genetic variation we observe in the dolphin populations is different from “neutral” variation. A paper published in October 2021 conducted a similar analysis using whole-genome sequencing on bottlenose dolphins. Marie Louis et al. found that ancestral genes across similar dolphin ecotypes observed across the world - both in-shore pelagic vs. offshore ones - played a crucial role in dolphin expansion into these coastal habitats. Our genome data for dolphins on the East Coast can help tell the story of how these different groups of dolphins evolved and adapted to their current environments.

 

More Reading: 

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Andrews, K. R., Good, J. M., Miller, M. R., Luikart, G., & Hohenlohe, P. A. (2016). Harnessing the power of RADseq for ecological and evolutionary genomics. Nature Reviews Genetics, 17(2), 81-92. https://www.nature.com/articles/nrg.2015.28 

Louis, M., Galimberti, M., Archer, F., Berrow, S., Brownlow, A., Fallon, R., ... & Gaggiotti, O. E. (2021). Selection on ancestral genetic variation fuels repeated ecotype formation in bottlenose dolphins. Science advances, 7(44), eabg1245. https://www.science.org/doi/10.1126/sciadv.abg1245