Genetic monitoring of the endangered Castor fiber birulai is hindered by its elusive behavior and by the logistical challenges of collecting fecal samples in aquatic environments. This proof-of-concept validation assessed salivary DNA retrieved from feeding traces on Salix turanica twigs as a non-invasive source for species-level genetic detection. Using a controlled rescue setting with one individual, we evaluated DNA persistence across a 0–12 h post-feeding timeline under low-temperature conditions (< 10 °C). To reduce PCR inhibition caused by willow bark secondary metabolites (e.g., tannins and polyphenols), we used a soil-based DNA extraction protocol selected for inhibitor removal. The mitochondrial cytochrome b (cytb) fragment was amplified successfully from all samples collected within 0–10 h, whereas amplification failed after 12 h. Spearman’s rank correlation revealed significant negative relationships between exposure time and both DNA concentration and purity. These findings define a practical freshness threshold for field sampling under favorable low-temperature conditions and indicate that feeding trace-derived saliva can provide a complementary, species-specific source for non-invasive genetic monitoring of this endangered subspecies.
Leporids play an important ecological role by contributing to seed dispersal and serving as prey for a wide range of predators. However, several leporid species are threatened by habitat degradation, highlighting the need for genetic resources that can support studies of wild and managed populations. The SRY gene, located on the Y chromosome in most male mammals, is a useful marker for paternal lineage studies and may provide information relevant to conservation genetics. Here, we generated partial Sex-determining region Y (SRY) sequences from 14 male leporids: 12 zoo-managed Romerolagus diazi, one wild R. diazi carcass, and one wild Sylvilagus floridanus carcass. Three unique consensus sequences were obtained and deposited in GenBank: PV612454 and PV612455 for R. diazi, and PV612456 for S. floridanus. The analyzed fragments comprised 703 base pairs (bp) in R. diazi and 741 bp in S. floridanus, including approximately 173 bp of the 5′ regulatory region and a partial transcriptional unit covering the first 176 codons, including the high-mobility group box domain; the complete SRY coding region was not recovered. Comparisons with available leporid SRY sequences showed that the regulatory region had lower guanine-cytosine content than the transcriptional unit, whereas R. diazi and S. floridanus showed relatively high guanine-cytosine values in the regulatory and 3′ SRY regions. Polymorphic sites were detected in the regulatory and coding regions, and amino acid variation was concentrated outside the conserved high-mobility group box domain. A distance-based, single-locus comparison provided a preliminary paternal-lineage framework among the leporid sequences analyzed.
Identifying hybrid individuals is essential for conservation management of threatened species and delimiting species boundaries. The MONGRAIL 2.0 software package applies Bayesian inference to classify individuals as purebred or hybrid/backcrossed using genome sequences of putative hybrids and pure individuals from two reference populations. The program evaluates six genealogical classes spanning two generations: purebreds from each population, F1 hybrids, F2 hybrids, and backcrosses to each parental population. MONGRAIL 2.0 uses a posterior predictive distribution that accounts for uncertainty in population haplotype frequencies, and correctly marginalizes over haplotypes while modeling linkage and recombination. It can use the exact likelihood for linked variants or highly accurate approximations that increase the number of SNPs that can be simultaneously analyzed per chromosome. The software accepts either VCF or custom genotype formats and outputs posterior probabilities for each genealogical class. MONGRAIL 2.0 is implemented in C for computational efficiency and is freely available at https://github.com/mongrail/mongrail2 .
The growing integration of advanced technologies in molecular studies has highlighted the need for efficient and reliable DNA extraction methods, as the accuracy and success of these technologies are highly dependent on DNA purity and integrity. Efficient isolation of high-quality genomic DNA is a prerequisite for next-generation sequencing (NGS) and other molecular applications, yet remains challenging in woody species owing to high levels of phenolics, polysaccharides, and secondary metabolites. The present study aimed to optimize a reliable DNA extraction protocol for B. wallichiana. Five different protocols – conventional CTAB (CCP), activated charcoal (ACP), modified CTAB (MCP), modified activated charcoal (MACP), and double buffer protocol (DBP) were evaluated using leaf tissues. The comparative analysis using various parameters such as DNA yield, purity, integrity, and storage stability demonstrated that MCP was most effective. Notably, this protocol consistently produced higher yields (301 to 850 ng/µL) with stable purity (A260/280 = 1.82–2.01), and intact DNA, outperformed other methods that showed variability, contamination, and degradation. Further validation through polymerase chain reaction using five Inter Simple Sequence Repeat (ISSR) primers produced clear and reproducible amplification, confirming that MCP is suitable for downstream genetic analysis. The study provides a reliable, efficient, and cost-effective approach for molecular characterization of B. wallichiana and will facilitate broader application in conservation genetics of Himalayan woody species rich in secondary metabolites and phenolics. By enabling stable yield and purity of genomic DNA, the protocol will contribute in developing informed conservation strategies and support sustainable management of its genetic resources.
The grey wolf Canis lupus is legally protected in Europe under the Habitats Directive and the Bern Convention, requiring population monitoring for conservation issues. In France, long-term monitoring relies on the collection of non-invasive samples genotyped at 22 microsatellite loci to identify individuals and estimate population size. To avoid bias in subsequent capture-recapture models, this study assessed the accuracy of the mismatch method used for individual identification and recapture detection. Based on simulations from true allelic frequencies and sensitivity analyses incorporating genotyping quality, recapture rates, and assignment methods, the three-mismatch method threshold showed high accuracy, with only 0.82
Hucho bleekeri is a Class I protected animal in China and is listed as Critically Endangered in the International Union for Conservation of Nature Red List. This study aimed to perform metagenomic (mainly for bacteria and aquatic organisms) and amplicon sequencing (12 S rRNA for fish) of water samples collected from the upper reaches of the Dadu River, including 12 historical distribution areas of H. bleekeri and one breeding site. Metagenomic gene-catalog diversity in the breeding base was significantly lower than that observed in the other 12 wild habitats. The dominant phylum in water was Pseudomonadota, followed by Actinomycetota. Correlation analysis of environmental factors revealed that species belonging to 31 and 3 phyla were significantly correlated with dissolved oxygen and temperature, respectively. We also identified 115 species of algae, with some species uniquely present in either the wild or farmed environments. Linear discriminant analysis effect size was used to identify organisms with significant differences in samples from different environments. The species in the phyla Pseudomonadota, Bacteroidota, and Actinomycetota exhibited the greatest influence. Amplicon sequencing revealed that H. bleekeri was distributed at all sites examined, with higher abundance observed at its releasing sites. Furthermore, the distribution of some prey fish species of H. bleekeri had the trend to be positively correlated with H. bleekeri distribution, but the correlation was not statistically significant. This study represents the first application of environmental DNA to monitor the wild resources of H. bleekeri and provide critical insights for the conservation and management of this critically endangered species and other rare fish within their habitat.
Sibship inference is commonly performed for conservation and management purposes in polyploid taxa like sturgeons. While no program has been developed for compatibility with polyploid genetic markers, the program Colony can effectively analyze polyploid genotypes converted to pseudo-diploid dominant format (PDDF). This approach has largely been applied to microsatellites and was also demonstrated to be effective with the novel GTseq panel of tetrasomic SNPs developed for the white sturgeon (Acipenser transmontanus). These two polyploid marker types have not been directly compared with respect to sibship inference accuracy. Additionally, the biallelic nature of SNPs enables their conversion to pseudo-diploid codominant format (PDCF), which has not been tested for sibship inference. Utilizing the white sturgeon microsatellite and GTseq SNP panels, we tested these three converted marker classes (microsatellite PDDF, SNP PDDF, and SNP PDCF) for sibship inference in a captive-spawned offspring group of known sibship structure and further tested the two classes of converted SNPs in a second such offspring group. Broadly, we found that both microsatellites and SNPs in PDDF could produce complete accuracy, although the latter performed less accurately in one of the offspring groups. Inputting inflated mistyping rates was necessary to maximize accuracy for these two converted marker classes. In both groups, SNPs in PDCF yielded poor accuracy. Our results demonstrate how to employ tetrasomic SNPs for sibship inference most effectively and are applicable to polyploid taxa with tetrasomic loci more broadly.
Morphological identification of stomach contents is the most common methodology used for shark diet reconstruction. However, this approach often limits prey identification to low taxonomic levels (i.e., order or class) and is prone to inaccuracies due to subtle morphological differences among prey. Additionally, varying digestion rates can lead to the underrepresentation of soft-bodied prey, which degrade and become unrecognizable more quickly than hard-shelled organisms. We used both visual morphological analysis and DNA barcoding on each prey item collected from the stomachs of four coastal shark species (i.e., Atlantic sharpnose [Rhizoprionodon terraenovae], blacknose [Carcharhinus acronotus], blacktip [Carcharhinus limbatus], and bonnethead [Sphyrna tiburo]) in North Carolina. Stomachs were collected from 49 sharks, yielding 118 prey items for analysis. We then compared the results of both visual and genetic methods to assess whether DNA barcoding increased taxonomic resolution of prey. Each prey item was also assigned an equivalency category to quantify the level of agreement between the two methodologies. DNA barcoding more than doubled the number of prey identified to the species level compared to morphological identification, and dietary composition significantly differed between methods for all shark species. Additionally, equivalency categories were significantly correlated with shark species and prey taxa. Bonnetheads had the highest proportion of prey classified as a match between methods due to their dietary specialization on Atlantic blue crabs (Callinectes sapidus). The hard carapace of this species digests more slowly, making Atlantic blue crabs easier to identify during morphological analysis. Blacknose sharks had the highest proportion of prey with their comparative equivalency categorized as ‘unidentified general morphological’, where morphological and DNA results aligned, but prey remained unidentifiable through morphology alone. Incorporating DNA barcoding into dietary studies provides an inexpensive, accurate, and effective approach to improving prey identification in sharks and other taxa. Improved resolution of shark diets strengthens our understanding of their ecological roles and supports ecosystem-based management approaches for shark and marine ecosystem conservation.
DNA isolation from oil and fat samples has long been a technical challenge, mainly due to hydrophobic substances and some interfering compounds in PCR. We describe an optimized protocol enabling DNA extraction from Python oil and fat. Using a modified lysing and purification technique, we managed to recover approximately 300 bp PCR amplifiable DNA. This approach presents the potential genetic identification of the species from oil and fat-derived from python/reptiles for population genetics and forensic studies.
Hornbills (Bucerotidae) and ground-hornbills (Bucorvidae) are families of birds found in Asia and Africa. At least 29 species are considered Near Threatened or above by the IUCN, with hunting for trade being the main threat to these birds. As many species have a unique feature on the top of their beaks called a casque, they are most commonly legally or illegally traded for their skulls or, in the case of the Helmeted Hornbill (Rhinoplax vigil), ivory. Hornbill casques are sexually dimorphic, vary in size within species across age classes, juvenile individuals can look like females, and, further, can be convergent across species. As not all hornbill species are subject to CITES regulation, the use of morphological identification guides is essential for efficient and quick identification especially of often difficult to identify non-adult male individuals In this study, we evaluated the performance of multiple DNA sex primers for the ZW system in birds, focusing on the chromo-helicase-DNA binding protein gene (CHD-ZW-common/CHD-W-specific/P2; M5/P8; P2/P8; 2250 F/2718R; CHD1F/CHD1R). The amplification of these markers was performed using a polymerase chain reaction (PCR), followed by visualisation via capillary electrophoresis. The primer sets CHD1F/CHD1R and 2250 F/2718R were found to be the most effective, together successfully amplifying in 18 hornbill species from diverse genera. Both sets also exhibited functionality with diverse tissue types (muscle, liver, whole blood, dried blood, and bone) and varying DNA quantities (1.991–23.356 ng/µL). CHD1F/CHD1R and 2250 F/2718R further benefit from the trait that the female-determining allele is smaller. Consequently, the potential for a misidentification resulting from degraded samples can be ruled out. By using the CHD1F/CHD1R and 2250 F/2718R markers, the sex of hornbills can be reliably determined in morphologically referenced specimens, which provides documentation of the casque variability within a species from adult males, juvenile individuals, and females. Such a guide can account for interspecific and ontogenetic variation and support relevant authorities in the rapid identification of hornbill species in both legal and illegal trade.
Species from the genera Bellator and Prionotus (subfamily Prionotinae), commonly known as “searobins”, are marine ray-finned fish with unique motility and prey-detection capabilities, making them ideal evolutionary genetic models. Despite their significance, their phylogeny remains poorly understood. The only existing molecular study on this group classified Prionotus as paraphyletic, in contrast to traditional morphological taxonomy. To definitively resolve their taxonomic status, we conducted the first phylomitogenomic analysis of the group, utilizing 22 complete mitogenomes, including three newly assembled mitogenomes for Bellator militaris, Prionotus alatus, and Prionotus stephanophrys. These three new mitogenomes exhibited a typical vertebrate organization, with the exception of P. stephanophrys, which contains an extra tRNA-Leu and an additional non-coding region. Comparative analysis revealed a minimal mitogenomic divergence (K2P = 0.001) between P. rubio and P. tribulus, suggesting potential mitochondrial introgression, conspecificity, or ongoing divergence, warranting further research to clarify taxonomic boundaries. Our fine-scale partitioned Bayesian Inference and Maximum Likelihood phylogenetic analyses yielded a highly supported congruent topology (BPP = 1.0; UFBoot2 = 100) where Bellator and Prionotus were recovered as reciprocally monophyletic sister groups. These findings definitively resolve the generic status within Prionotinae, providing valuable insights into their evolutionary history, which is fundamental for both evolutionary genetic research and the informed management of these economically valuable species.
Horse mackerels (Trachurus spp.) of the family Carangidae (Perciformes) are small pelagic migratory fishes widely distributed in temperate to subtropical seas and represent one of Türkiye’s most commercially important groups, contributing about 5.2
The study aimed to develop and standardize a reliable, cost-effective, and reproducible protocol for isolating high-quality total genomic DNA suitable for chloroplast marker amplification from two Dendrobium hybrids—Dendrobium ‘Sonia Earsakul’ and Dendrobium ‘Singapore White’—and to assess its suitability for downstream molecular applications, such as PCR amplification using selected chloroplast markers. Leaf tissues were collected from healthy plants and subjected to three different DNA extraction methods: (i) a modified cetyltrimethylammonium bromide (CTAB) method, (ii) a modified sodium dodecyl sulfate (SDS) method, and (iii) the DNeasy Plant Mini Kit. The optimized CTAB protocol incorporated polyvinylpyrrolidone (PVP) and β-mercaptoethanol to counteract secondary metabolites present in orchid tissues. DNA yield and purity were measured using a Colibri + Microvolume UV-Vis spectrophotometer, while integrity was assessed by agarose gel electrophoresis. The extracted total genomic DNA was further validated through PCR amplification using three widely applied chloroplast markers: matK, trnL-F, and trnH-psbA. The standardized CTAB method produced the highest yield and purity, with DNA concentrations of 145–150 ng/µL, A260/A280 ratios of 1.88–1.92, and A260/A230 ratios of 2.03–2.11. In contrast, the SDS method and DNeasy kit produced lower yields and showed signs of contamination. Agarose gel electrophoresis confirmed intact, high-molecular-weight genomic DNA suitable for downstream plastid marker amplification. Successful amplification of all three chloroplast markers confirmed the suitability of the isolated DNA for downstream molecular studies. The optimized CTAB-based protocol provides a reliable and cost-effective method for genomic DNA extraction from Dendrobium hybrids. Its higher DNA yield and purity values observed in this study compared with the SDS method and the commercial kit suggest its potential utility in DNA barcoding, phylogenetics, population genetic analysis, and species authentication, which are essential tools for conservation genetics, monitoring illegal orchid trade, and biodiversity conservation of Dendrobium species. It is important to note that the protocol yields total genomic DNA; however, the quality obtained was sufficient for robust amplification of chloroplast markers without the need for chloroplast enrichment procedures.
We present novel genetic data for the rare deep-water shark Pseudotriakis microdon, including sequences for COI, 16S rRNA, NADH2, and nuclear ITS2 from specimens sampled in the North Atlantic Ocean. These data, combined with all publicly available sequences, reveal clear genetic structuring: Atlantic and Indian Ocean populations form a cluster clearly separated from West Pacific individuals. The ITS2 marker, previously unreported for this species, appears particularly divergent, reflecting a poorly sampled phylogenetic branch and representing a valuable genomic resource. These findings lay the groundwork for future conservation and taxonomic studies of P. microdon populations.
The accelerating loss of global biodiversity necessitates proactive conservation strategies that integrate genetic diversity with projected climate impacts, especially in biodiversity hotspots such as the Hengduan Mountains. While previous phylogeographic studies in this region primarily relied on a few DNA fragments, plastid phylogeography has seldom been integrated with ecological niche modelling for conservation planning. Here, we combined plastid phylogeography and ecological niche modelling using data from 107 individuals across 14 populations of Acanthochlamys bracteata, a vulnerable alpine species and monotypic genus, to identify genetically distinct populations and predict potential climatic refugia. We identified three genetically distinct groups (Groups A to C), all characterised by low nucleotide diversity but high haplotype endemism. Notably, Group C comprised a single population from Luhuo County, Sichuan Province, and represents an independent evolutionarily significant unit due to its pronounced genetic distinctiveness. Most genetic variation occurred among populations, with one population showing relatively high diversity, while three lacked variation. A northward range shift and expansion of suitable habitats are projected under future climatic scenarios. Three climatic refugia were identified, characterized by divergent genetic lineages and numerous private haplotypes, and supported by the persistence of suitable habitat through time. The observed phylogeographic structure is likely driven by both geographical distance and relict persistence. We recommend that conservation efforts should focus on protecting genetically distinct and diverse populations through an integration of in situ, ex situ, and assisted migration measures. This study advances our understanding of the evolutionary history of Acanthochlamys bracteata and could serve as a model for conserving other rare and isolated plants under climate change and human disturbance.
Expanding brackish and semi-brackish water aquaculture is an essential strategy to improve land-use efficiency and alleviate spatial constraints in freshwater aquaculture. However, current efforts are constrained by the limited availability of high-quality, salt-tolerant fish species. Grass carp (Ctenopharyngodon idella), a widely farmed freshwater species in China, has had limited success in brackish-water environments due to its low tolerance to salinity. In this study, genome-wide association analysis (GWAS) was conducted on 60 grass carp individuals that survived under two salinity conditions (10‰ and 15‰). The study identified 12 significant SNPs across seven chromosomes and annotated 71 candidate genes. Among these, nos1, slc26a3 were closely associated with osmoregulation and ion transport. Enrichment analyses indicated that salinity tolerance in grass carp is primarily related to immune stress responses, membrane remodeling, and energy metabolism. This study comprehensively identified genetic loci and candidate genes associated with salinity tolerance in grass carp at the genome-wide level, providing valuable information for developing molecular markers and improving salt tolerance in this species.
Identification of undocumented or unrecognized indigenous livestock populations is essential for accurate inventory of animal genetic resources and effective conservation planning. However, nomenclature-based taxonomy in developing countries often conflates genuinely distinct populations with casual phenotypic variants, potentially obscuring true genetic diversity. This study systematically explored and identified undocumented goat genetic resources in Tamil Nadu, India, and verified their distinctiveness using standardized breed identification criteria. An integrated survey-verification methodology was employed combining an online questionnaire of 3,876 practicing veterinarians (272 responses, 198 containing population information) and systematic phenotypic observations at 22 livestock markets across five agro-climatic zones. Survey respondents and market observations identified 10 unique putative goat populations. Targeted field visits to 83 villages across 21 districts and verification using FAO and NBAGR breed identification criteria revealed that only three populations (30
Largemouth bass (Micropterus salmoides) is a globally significant species in freshwater aquaculture with sex-specific differences in growth patterns. To develop a non-invasive, early-stage molecular sex identification tool for selective breeding. We identified a male-specific 90-kb structural variant (SV) hotspot on chromosome 10 through comparative genomics of telomere-to-telomere (T2T) genome assemblies of both sexes. A unique 2.2-kb repetitive region (SVER-6) within this SV was targeted to design sex-specific PCR primers. Validation included non-lethal caudal fin clipping from 73 samples from two populations, followed by gonadal histology as phenotypic reference.
Fecal samples are valuable for wildlife genetic studies where invasive tissue sampling is difficult, expensive, or negatively impacts welfare. However, fecal DNA is challenging to work with due to high amounts of exogenous (non-host) DNA and PCR inhibitors. For conservation and management of caribou (Rangifer tarandus), feces may be used as a DNA source, but optimal extraction protocols remain unclear. We compared combinations of cell isolation methods (using a swab or toothpick, washing, or incubating in lysis buffer) and QIAGEN DNA extraction kits (QIAamp DNA Mini, Fast DNA Stool Mini, PowerSoil Pro) on fecal samples from a boreal caribou population in British Columbia, Canada. We assessed protocols based on total and target (host) DNA yields and PCR amplification success using a new qPCR assay. The most successful methods were further tested on samples from seven additional populations across three caribou ecotypes. All methods yielded at least 10 ng of caribou DNA per pellet, sufficient for most PCR-based genotyping protocols. In boreal caribou, the incubation isolation and QIAamp DNA Mini kit yielded the highest DNA quantities, while the wash isolation with Fast DNA Stool and PowerSoil kits yielded the highest host DNA proportions. Post-hoc validation, however, revealed that the wash isolation in combination with the QIAamp DNA Mini kit was more reliable for minimizing PCR inhibitors across populations while maximizing DNA yield. These results will inform feces-based genetic workflows in caribou, and offer a transferable framework for refining similar protocols in other species to advance non-invasive genetic monitoring more broadly.
We propose an approach of 6 STR-loci to one-stage species identification of four species of the Canidae family, the red fox (Vulpes vulpes), the arctic fox (Vulpes lagopus), the raccoon dog (Nyctereutes procyonoides), and the wolf (including the grey wolf (Canis lupus lupus) and the domestic dog (Canis lupus familiaris), based on differences in manifestation of microsatellite loci during cross-species amplification. The test system was optimized for interspecies differentiation within the Canidae family rather than identification of individual animals. Additionally, we present an approach to DNA identification of the subspecies of the Canis lupus, the grey wolf (Canis lupus lupus) and the domestic dog (Canis lupus familiaris), based on variations in the copy number of the amylase gene (Amy2b). This comprehensive study of canids makes it possible to reliably differentiate forensic samples by species and subspecies before genetic identification of individuals.
