At the Frontier of Wolf Conservation Research

Robert Kraus advances field efforts using SNP genotyping

“If SNP genotyping technology by Fluidigm becomes daily business, we can create bigger 
DNA biobanks from really precious samples.”—Robert Kraus, PhD 

If wolves routinely stopped by the lab for a blood draw, Robert Kraus’s work would be a simpler task. Since wolf subjects are less than cooperative, Kraus and other scientists monitoring wildlife for conservation research extract genomic data from fur, urine, and other biological calling cards the wild lupines leave on their trail. The primary concern with these nonintrusive samples is their scant genomic content, which poses a significant hurdle to genotyping studies. Current advances in single-nucleotide polymorphism (SNP) genotyping technologies enable preservation scientists to extract high-quality data from miniscule amounts of DNA found in challenging samples. 

In a 2014 study published in Molecular Ecology Resources, Kraus—an assistant professor at Konstanz University in Germany and a research scientist at the Max Planck Institute for Ornithology—and colleagues developed a SNP-based genotyping assay for tracking wolves using noninvasive samples. This study is significant in the field for validating SNP genotyping as a viable alternative to standard microsatellite markers. SNPs offer key advantages due to their predictable mutation modes and high genomic abundance. SNP data is also easy to standardize and share among labs, which has been another barrier to global conservation efforts. 

The Fluidigm EP1 system enabled Kraus to do more with less: he genotyped the usual  noninvasive animal samples and obtained far better data than he could have using  microsatellites. An efficient solution for SNP genotyping and end-point PCR applications, EP1 is uniquely suited for challenging sample types. Exclusive integrated fluidic circuits (IFCs) fully automate reaction setups, and on a miniaturized, nanoliter scale. The result is a simplified workflow with fewer pipetting steps, which translates to high-throughput SNP genotyping with better results.

Kraus genotyped 158 samples collected from wolf fur, urine, scat and tissue for 192 SNPs. He designed two sets of 96 SNP Type assays for use on the 96.96 Genotyping IFC and performed reaction setup, genotyping and data analysis on the EP1. He observed a “missing data” rate of less than 10 percent and a genotyping error of less than 1 percent—an order-of-magnitude improvement over other published methods. Based on the data, Kraus and colleagues selected 96 optimal SNPs for wolf genotyping studies. 

This study provides evidence that SNP genotyping technologies meeting specific needs of wildlife conservation researchers are available today. The Fluidigm SNP genotyping workflow eliminated high error rates and replication needs associated with microsatellite markers and vastly improved upon costly, labor-intensive protocols. The protocol’s low DNA-input requirement also saved precious resources for downstream applications. The newly developed Juno system further addresses this key pain point by pushing the limits of sample requirement for high-throughput genotyping to as little as 5.5 ng total DNA. 

Kraus believes the field will see a shift in the near future as more labs adopt SNP genotyping. He recently shared some of his recent research news and insights with Fluidigm and also discussed the 2014 study he carried out at his former Conservation Genetics Group lab at the Senckenberg Research Institute.

Q: You make a strong case for switching from microsatellite markers to SNP genotyping; why haven’t more scientists switched? 

A: There are strong incentives to stick to an established technology, especially because  researchers want as much of their ongoing data collection to be compatible with data sets collected in the past. Additionally, it requires investments of time, effort and money to switch from one technology to another. Wildlife conservation doesn’t have lots of investment to devote to risky enterprises like new technology. Installing a new technology thus needs to confer a significant advantage to the daily business of a  conservation research group.

Q: What can you do now with the Fluidigm genotyping workflow that wasn’t previously possible? 

A: The types of samples that we genotype are the same as the ones we did with microsatellites. But for my work, the Fluidigm benefit is that it promises to save time, effort and money, and it may be more precise and scalable to bigger projects.

Q: Regarding sample conservation, what are some of the downstream studies you perform on samples after genotyping?

A: Conservation genetics based on SNP genotypes provides answers such as the extent to which wildlife populations are genetically impoverished. We can infer demographic and geographic population structure to delineate management units. Furthermore, we can use our markers as a genetic fingerprint system to follow particular individuals during their lifetime to learn about the pack structure of wolves, to provide useful data about their spatial range and pedigrees. Forensic applications interest me, too. For example, using genetic fingerprinting with our marker system for a Bulgarian case study, we identified a brown bear that may have  attacked a hunter. By genetic means, we can tell if a wolf or a dog killed a specific sheep, which determines whether a farmer is compensated for the loss. Sample conservation means we can work on multiple projects and dig out samples from the freezer as soon as new questions arise that we can address using stored material. If SNP genotyping technology by Fluidigm becomes daily business, we can create bigger DNA biobanks from really precious samples.

Q: In your methods study, you used the Fluidigm SNP Type assay and EP1 system to  develop a SNP genotyping methodology for a wolf species. What is the current status of wolves in the wild? 

A: In Europe, the wolf is strictly protected by EU regulations, and its monitoring is a binding action for all countries. Here wolves live in the wild, mostly outside large national parks. They were hunted and driven to extinction in huge areas here in Europe, but there has been a stunning comeback in the past 20–30 years as people’s attitudes toward nature have changed. In Germany, there hasn’t been a single wolf wandering the land since the last one was killed in 1904. The first established territory by a female wolf was recorded in East Germany in 1998, with the first reproduction in 2000. By 2010 we had seven packs, and by 2012 there were 16 packs. 

Q: Are you using this platform to genotype other animals?

A: Yes, I am collaborating with my former Conservation Genetics Group lab at the Senckenberg Research Institute to genotype wildcats, beavers, lynxes, brown bears, hamsters and endangered mammals. We hope to install SNP-based genetic monitoring for all of them. 

Q: What are some studies you have planned that take advantage of the SNP genotyping methodology discussed in your paper?

A: Once installed and fit into daily routine, my former lab (that I still collaborate with) can deliver faster turnaround times for contract work, since the method works faster and a single person can oversee more projects, and also interrelatedness among projects. We also hope to build a database where other labs can store their SNP genotyping data to create a transboundary network of wildlife monitoring. This is important because animals don’t know our borders, and nature conservation has to be considered on a continental scale for many of the large and mobile species. This international database would be paramount for many future projects.