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American Pika Research Shows Value of NGS in Conservation Genomics Studies of Elusive Species

NEW YORK (GenomeWeb) – Based on next-generation sequencing analysis, University of British Columbia researchers were able to identify nearly 4,000 SNPs in samples collected from the elusive American pika and found that genetic variation in the species varies with elevation.

Conservation studies of endangered and elusive species like the American pika, Ochotona princeps, often have to rely on non-invasively collected samples, such as DNA found in hair, feathers, or even in droppings they've left in their wake. As these sorts of samples often contain only a small amount of DNA, whether next-generation sequencing-based approaches could be applied to them has been questionable, the researchers noted.

As they reported in PeerJ today, the researchers were able perform genotyping-by-sequencing on hair samples from the American pika, a small mammal that lives in mountainous regions in the western US and Canada that is threatened by climate change, and found that pikas living at lower elevations had lower levels of genetic diversity.

"We were able to collect genome-wide data from natural populations of the elusive and climate-sensitive American pika on a scale unheard of just a few years ago," Michael Russello, an associate professor at the University of British Columbia, said in a statement.

Russello and his colleagues collected hair samples using non-invasive snares from 12 pika individuals at four sites along two elevation transects, Pyramid Peak and Thornton Lakes, in the North Cascades National Park in Washington State, for a total of 96 samples.

The mean starting DNA concentration obtained from these hair samples was 0.55 ng/μl, the researchers reported, with some samples providing as little as a nanogram in total.

They converted the DNA from these samples into nextRAD genotyping-by-sequencing libraries and yielded a mean 1,863,634 sequencing reads per sample.

To ensure that the DNA they had was actually from the pika, they mapped their reads back to the O. princeps genome, and from this found 16 samples had a good portion of reads that matched other small mammals that live in the area, two samples that matched humans, and one that was from corn.

In the 67 remaining samples, the researchers uncovered 3,803 SNPs, and outlier detection revealed 37 loci along one transect and 18 along the other. Five of these could be annotated to genes of known function, including the ROR2 gene, which is involved in skeletal and neuronal development as well as cell movement and cell polarity; the HEPHL1 gene, which appears to be a ferroxidase; and TMX4, a transmembrane protein that may act as a reductase in the clanexin folding complex.

These outlier loci, the researchers said, could be candidate gene regions that exhibit signs of divergent selection.

The researchers noted that the proportion of polymorphic loci varied across the sampling sites. The lower elevation sites, they reported, had lower numbers of polymorphic loci than the mid- and high-elevation sites. They noted a similar trend in gene diversity along the transects as well.

"We found genetic variation to be significantly correlated with elevation, with sites at the lower fringe of American pika distribution in North Cascades National Park exhibiting substantially lower levels of gene diversity," the researchers said in their paper.

Significant inbreeding was also uncovered at the low- and mid-low elevation site along the Pyramid Peak (PP) transect, but not at the higher elevation sites, though all sites along the Thornton Lakes (TL) transect exhibited evidence of inbreeding.

"The detection of significant genome-wide evidence of heterozygote deficit at low elevation sites in both transects further suggests inbreeding may be leading to the observed patterns, a particular concern for PP1, TL1, and TL2 given their apparent distinctiveness from higher elevation sites," they added.

Despite their ability to uncover genetic variation within the pika, the researchers noted that non-invasive sampling of endangered and elusive species remains challenging. For instance, sampling protocols must try to limit the non-target DNA collected, though how that can be done varies with sample collection method, and the sequences obtained need to be assessed to be sure they are from the species of interest.

"Here, we have shown that with careful consideration, genomic data collection is compatible with the non-invasive sampling required in practice for many conservation-related studies," the researchers added.