Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner.

The genome of the American groundhog, Marmota monax

We sequenced the genome of the North American groundhog, Marmota monax, also known as the woodchuck. Our sequencing strategy included a combination of short, high-quality Illumina reads plus long reads generated by both Pacific Biosciences and Oxford Nanopore instruments. Assembly of the combined data produced a genome of 2.74 Gbp in total length, with an N50 contig size of 1,094,236 bp. To annotate the genome, we mapped the genes from another M. monax genome and from the closely related Alpine marmot, Marmota marmota, onto our assembly, resulting in 20,559 annotated protein-coding genes and 28,135 transcripts. The genome assembly and annotation are available in GenBank under BioProject PRJNA587092.

Hair cortisol analyses in different mammal species: choosing the wrong assay may lead to erroneous results

Wild animals are faced with a broad range of environmental stressors and research is needed to better understand their effect on populations. Hormone analysis based on enzyme immunoassays (EIAs) can provide valuable information on adrenocortical activity (stress), and assessment of cortisol in hair may allow the quantification of cortisol production. To validate hair hormone analysis, we compared two EIAs based on antibodies against cortisol-3-CMO-BSA and cortisol-21-HS-BSA for hair glucocorticoid (hGC) measurements in Egyptian mongoose, Iberian lynx, Alpine marmot, Asiatic black bear, spotted hyena and cheetah, with results obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Both EIAs were also characterized by HPLC immunograms. Our results revealed that the cortisol-21-HS EIA measured 2.3- to 12-fold higher hGC concentrations than the cortisol-3-CMO assay. In dependence of the species, high-performance liquid chromatography (HPLC) immunograms showed that up to 70% of immunoreactivities determined by the cortisol-21-HS constituted of unknown unpolar compounds leading to an overestimation of hGC. The cortisol-3-CMO EIA expressed a better specificity, with 32.1–67.4% of immunoreactivity represented by cortisol and cortisone. The LC-MS/MS analyses (gold standard) revealed that the cortisol-3-CMO EIA also resulted in an (up to 3-fold) overestimation of hGC, but EIA results were correlated with LC-MS/MS in the mongoose, the lynx, the spotted hyena and the marmot. No correlation was obtained for Asiatic black bears. As a result of our study, we strongly recommend to test any cortisol EIA for its specificity towards extracted hair components. In all analyzed species, except the Asiatic black bear, cortisone and cortisol were simultaneously present in hair extracts; consequently, an appropriate EIA should cross-react to these two glucocorticoid hormones and express negligible affinity towards substances with less polarity than corticosterone. Choosing the wrong EIA for hGC analyses may lead to overestimations of hGC or—in the worst case—to results that do not mirror real adrenocortical activity.