Konec, M. | bioRxiv Science

bioRxiv2026

Workflow for multiplex microsatellite panel development and sample preparation for robust amplicon sequencing of low-template and degraded DNA: validation for non-invasive genotyping in three large carnivore species

High-throughput amplicon sequencing has transformed microsatellite (STR) genotyping by overcoming many of the limitations of fragment-length analysis, enabling more accurate, cost-effective, and standardized genotyping. Yet, protocols specifically designed for high-throughput sequencing (HTS)-based STR genotyping from low-template and degraded DNA remain scarce, despite the prevalence of these challenging sample types in ecological and conservation contexts. We present a methodology for the de novo development of robust STR multiplex panels together with a laboratory protocol for efficient and reliable STR genotyping by sequencing with low quantity and quality DNA samples. The protocol comprises (i) an automated bioinformatic pipeline to design large sets of short tetranucleotide markers optimized for multiplex amplicon sequencing of degraded and low-template DNA; (ii) guidelines for efficient in vitro optimization of multiplex amplification using directly low quantity/quality template DNA; and (iii) a library preparation procedure that improves detection of low-level allele signal while enabling quality assessment of STR amplicon sequencing under limiting DNA conditions. We demonstrate the approach by developing and validating STR panels for non-invasive genotyping of three large carnivore species: a 44-plex for the grey wolf (Canis lupus), a 41-plex for the Eurasian lynx (Lynx lynx), and a 30-plex for the brown bear (Ursus arctos). Multiplex performance was high, with [≥]91% of samples successfully genotyped at [≥]50% of loci (allele size range 28-110 bp across panels) and correctly assigned to known individuals, negligible levels of noise in the controls, and high discriminatory power (PIDsibs [≤]2.4 x 1e-12), also owing to sequence variation among same-length alleles at 15-50% of loci. The approach is broadly applicable to animal and plant species, a wide range of sample types, and large-scale analysis such as genetic monitoring. Our study reinforces the value of STR amplicon sequencing for ecological and conservation applications while highlighting the importance of marker design and laboratory workflows tailored to HTS-based genotyping for accurate and efficient implementation.

ecology↗

bioRxiv2024

Saving the Dinaric lynx: multidisciplinary monitoring and stakeholder engagement support large carnivore restoration in human-dominated landscape

Translocations are central to large carnivore restoration efforts, but inadequate monitoring often inhibits effective conservation decision-making. Extinctions, reintroductions, poaching and high inbreeding levels of the Central European populations of Eurasian lynx (Lynx lynx) typify the carnivore conservation challenges in the Anthropocene. Recently, several conservation efforts were initiated to improve the genetic and demographic status, but were met with variable success. Here, we report on a successful, stakeholder-engaged translocation effort to reinforce the highly-inbred Dinaric lynx population and create a new stepping-stone subpopulation in the Southeastern Alps. We used multidisciplinary and internationally-coordinated monitoring using systematic camera- trapping, non-invasive genetic sampling, GPS-tracking of translocated and remnant individuals, recording of reproductive events and interspecific interactions, as well as the simultaneous tracking of the public and stakeholders support of carnivore conservation before, during and after the translocation process across the three countries. Among the 22 translocated wild-caught Carpathian lynx, 68% successfully integrated into the population and local ecosystems and at least 59% reproduced. Probability of dispersing from the release areas was 3-times lower when soft-release rather than hard-release method was used. Translocated individuals had lower natural mortality, higher reproductive success and similar ungulate kill rates compared to the remnant lynx. Cooperation with local hunters and protected area managers enabled us to conduct multi-year camera-trapping and non-invasive genetic monitoring across a 12,000-km2 transboundary area. Results indicate a reversal in population decline, as the lynx abundance increased for >40% during the 4-year translocation period. Effective inbreeding decreased from 0.32 to 0.08-0.19, suggesting a 2- to 4-fold increase in fitness. Furthermore, successful establishment of a new stepping-stone subpopulation represents an important step towards restoring the Central European lynx metapopulation. Robust partnerships with local communities and hunters coupled with transparent communication helped maintain high public and stakeholder support for lynx conservation throughout the translocation process. Lessons learned about the importance of stakeholder involvement and multidisciplinary monitoring conducted across several countries provide a successful example for further efforts to restore large carnivores in human-dominated ecosystems.

ecology↗