The consequences of the expansion of human development have been especially felt in California where human activity has eradicated several apex predators, like the California Grizzly and gray wolf. Unlike these predators, the puma, also called the mountain lion or cougar, has continued to populate the state, though their population is threatened by the continued development of intercoastal mountain ranges1. As a result of this development and attempted human avoidance, populations of pumas have driven themselves into regional isolation, which could lead to inbreeding and depletion of genetic variance, ultimately resulting in the eradication of the puma1. Genome sequencing data from pumas in different geographical locations show that while there is a lack of genetic variation within the geographical groups due to this inbreeding; it is possible to strategically reintroduce variation, as there is less homozygosity among different groups1.
To collect this genomic information, 354 tissue samples were collected from deceased pumas which were located throughout the state, excluding desert regions1. DNA was extracted using Omega Bio-tek’s Mag-Bind® Blood & Tissue DNA HDQ 96 Kit (M6399) in conjunction with an automation protocol for the KingFisher Duo Prime which was developed in-house at Omega Bio-tek. This kit is designed for the isolation of genomic DNA from various samples including fresh or frozen animal cultured cells and tissues. The Mag-Bind® paramagnetic particles provide high-quality DNA that is suitable for a myriad of downstream applications, including Next Generation Sequencing. After isolation, 48 samples were pooled using a library construction scheme, and their DNA concentrations were normalized. Double-digestion was performed using restriction enzymes, the library was cleaned-up following ligation, and finally, the PCR products were cleaned-up after amplification1.
Results and Discussion
The PCR products resulting from the DNA isolated using the Mag-Bind® Blood & Tissue DNA HDQ 96 Kit (M6399) were used for data analysis to determine the genetic diversity of the pumas. Principal Component Analysis (PCA), an unsupervised machine learning algorithm, was used to cluster the genetic libraries of the populations by grouping them based on similarity1. From this, it was found that there were four broad genetic clusters across California that correspond geographically with isolated groups of pumas. The geographic landmarks corresponding with these groups include the Sierra Nevada, North Coastal, Central Coastal, and Southern Coastal mountain ranges1. Of these clusters, the Sierra Nevada population was found to be the most genetically diverse, though it is still threatened by the increasing demand for urban development1. Even the most isolated and inbred populations of pumas do not share genetics with other populations located in different areas. This analysis allows conservationists to strategically plan programs for the reintroduction of genetic variation to these populations by providing information on the genetic makeup of pumas in various regions throughout the state.
The current social climate surrounding environmental conservation lends itself to emphasizing the importance of conserving animal populations threatened by the expansion of human development. As illustrated here, the genomes of inbred puma populations are not shared among other inbred populations, making the reintroduction of genetic diversity possible. However, even the most diverse populations remain at risk of becoming unviable and unsustainable if urban expansion into these mountain ranges continues. The discussed methods of genetic characterization and analysis of puma populations in California allow conservationists to make informed decisions about safeguarding these populations. Research regarding the protection of the environment and conservation of genetic diversity will continue to be of utmost importance for the foreseeable future, making efficient and high-quality nucleic acid extractions an integral part of this research.
For more information about the Mag-Bind® Blood & Tissue DNA HDQ 96 Kit and to request a sample kit, visit our website here
 Gustafson KD, Gagne RB, Buchalski MR, et al. Multi-population puma connectivity could restore genomic diversity to at-risk coastal populations in California. Evol Appl. 2022;15(2):286-299. Published 2022 Jan 27. doi:10.1111/eva.13341