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Results for “Conservation Genetics Resources”

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At least 55 records · Page 3Linked to original sources
USGS2014

Development of 13 microsatellites for Gunnison Sage-grouse ( Centrocercus minimus ) using next-generation shotgun sequencing and their utility in Greater Sage-grouse ( Centrocercus urophasianus )

Gunnison Sage-grouse are an obligate sagebrush species that has experienced significant population declines and has been proposed for listing under the U.S. Endangered Species Act. In order to examine levels of connectivity among Gunnison Sage-grouse leks, we identified 13 novel microsatellite loci though next-generation shotgun sequencing, and tested them on the closely related Greater Sage-grouse. The number of alleles per locus ranged from 2 to 12. No loci were found to be linked, although 2 loci revealed significant departures from Hardy–Weinberg equilibrium or evidence of null alleles. While these microsatellites were designed for Gunnison Sage-grouse, they also work well for Greater Sage-grouse and could be used for numerous genetic questions including landscape and population genetics.

Conservation Genetics Resources↗
USGS2015

Development and characterization of polymorphic microsatellitemarkers for the crested caracara, Caracara cheriway

We isolated novel microsatellites from the crested caracara ( Caracara cheriway ) with a shotgun pyrosequencing approach. We tested 80 loci for polymorphism among 20 individuals from the threatened Florida population. Fourteen loci were polymorphic. The mean number of alleles was 2.21 (range 2–3) and the mean observed heterozygosity was 0.41 (range 0.15–0.65). None of the 14 polymorphic loci exhibited significant linkage disequilibrium nor did they deviate significantly from Hardy–Weinberg expectations. We also report 16 monomorphic loci.

Florida↗
USGS2015

Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated from two three-hour runs on the Ion Torrent PGM can generate a sufficient number of nuclear and mitochondrial markers to improve understanding of the evolutionary and ecological dynamics of non-model and in particular, invasive species.

European Russia↗
USGS2015

Development of twelve microsatellite loci in the red tree corals Primnoa resedaeformis and Primnoa pacifica

A suite of tetra-, penta-, and hexa-nucleotide microsatellite loci were developed from Roche 454 pyrosequencing data for the cold-water octocorals Primnoa resedaeformis and P . pacifica . Twelve of 98 primer sets tested consistently amplified in 30 P . resedaeformis samples from Baltimore Canyon (western North Atlantic Ocean) and in 24 P . pacifica samples (Shutter Ridge, eastern Gulf of Alaska). The loci displayed moderate levels of allelic diversity (average 7.5 alleles/locus) and heterozygosity (average 47 %). Levels of genetic diversity were sufficient to produce unique multi-locus genotypes and to distinguish species. These common species are long-lived (hundreds of years) and provide essential fish habitat ( P . pacifica ), yet populations are provided little protection from human activities. These loci will be used to determine regional patterns of population connectivity to inform effective marine spatial planning and ecosystem-based fisheries management.

Conservation Genetics Resources↗
USGS2015

Isolation and characterization of microsatellite DNA loci in the threatened flat-spired three-toothed land snail Triodopsis platysayoides

The hermaphroditic flat-spired three-tooth land snail ( Triodopsis platysayoides ) is endemic to a 21-km stretch of the Cheat River Gorge of northeastern West Virginia, USA. We document isolation and characterization of ten microsatellite DNA markers in this at-risk species. The markers displayed a moderate level of allelic diversity (averaging 7.1 alleles/locus) and heterozygosity (averaging 58.6 %). Allelic diversity at seven loci was sufficient to produce unique multilocus genotypes; no indication of selfing was detected in this cosexual species. Minimal deviations from Hardy–Weinberg equilibrium and no linkage disequilibrium were observed within subpopulations. All loci deviated from Hardy–Weinberg expectations when individuals from subpopulations were pooled. Microsatellite markers developed for T . platysayoides yielded sufficient genetic diversity to (1) distinguish all individuals sampled and the level of selfing; (2) be appropriate for addressing fine-scale population structuring; (3) provide novel demographic insights for the species; and (4) cross-amplify and detect allelic diversity in the congeneric T. juxtidens .

Conservation Genetics Resources↗
USGS2017

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae , which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Conservation Genetics Resources↗
USGS2018

Ethanol and sodium acetate as a preservation method to delay degradation of environmental DNA

Environmental DNA (eDNA) samples that are collected from remote locations depend on rapid stabilization of the DNA. The degradation of eDNA in water samples is minimized when samples are stored at ≤ 4 °C. Developing a preservation technique to maintain eDNA integrity at room temperature would allow a wider range of locations to be sampled. We evaluated an ethanol and sodium acetate solution to maintain the integrity of the DNA samples for the time between collection and lab testing. For this evaluation, replicate water samples taken from a tank housing Asian carp were placed on ice or held at room temperature. At both temperatures, water samples were left untreated or were preserved with an ethanol and sodium acetate solution (EtOH–NaAc). Every day for 6 days following collection, a subset of the samples was removed from each preservation method and DNA was extracted and nuclear and mitochondrial markers were assayed with qPCR. Results showed comparable persistence of DNA between iced samples without the EtOH–NaAc treatment and samples that received EtOH–NaAc treatment that were kept at room temperature. We found that DNA can be amplified from preserved samples using an EtOH–NaAc solution after up to 7 days at room temperature.

Conservation Genetics Resources↗
USGS2019

Development of two quantitative PCR assays for detection of several Cottus species from environmental DNA in Pacific coast watersheds of North America

We developed two quantitative PCR assays for use with environmental DNA (eDNA) to detect numerous species in the genus Cottus that are indigenous to the Pacific coast watersheds of North America. We conducted in vitro assay validations on ten Cottus species and 32 potentially co-occurring non- Cottus species. We demonstrate the efficacy of these assays by field testing eDNA samples collected from streams inhabited by Cottus . These assays will be particularly useful for detecting Cottus in habitats where one (or more) of several Cottus species could be present.

Pacific Northwest↗
USGS2020

A conservation-oriented SNP panel for Smallmouth Bass (Micropterus dolomieu), with emphasis on Interior Highlands lineages

The Smallmouth Bass ( Micropterus dolomieu ; SMB) is a widely distributed black bass species, but the southwestern edge of the species range within the Interior Highlands contains some of the most divergent ecotypes. The Neosho subspecies ( M. d. velox ) inhabits tributaries of the Arkansas River within the Ozark Mountains and a second lineage is reported from drainages of the Ouachita Mountains. We sought to develop a single nucleotide polymorphism (SNP) panel to (1) diagnose hybridization with sympatric Spotted Bass ( Micropterus punctulatus ; SPB) and non-native Northern SMB ( M. d. dolomieu ) stocked in the region, and (2) delineate population structure within the ranges of the Neosho and Ouachita SMB lineages. We obtained 76 individual SMB samples from across their range but concentrated within the Interior Highlands ( n = 50). We also included 3 SPB to allow for hybrid detection and 3 Shoal Bass ( Micropterus cataractae ) as an outgroup. Phylogenetic trees constructed with the generated SNP data corroborated the existence of at least three major lineages of SMB (Northern, Neosho, and Ouachita), each containing varying degrees of differentiation among major drainages. Simulation analyses revealed that chosen SNPs had high power (> 0.9) to assign SMB × SPB hybrid categories and similarly high power (> 0.8) for Northern SMB × Interior Highlands SMB hybrids. Clustering methods delineated major inter-basin population structure within the native ranges of Neosho and Ouachita SMB with chosen SNPs. Anticipated uses of the resulting 192-loci SNP panel include conservation planning, fisheries management assessments, and ecological investigations of the Neosho and Ouachita SMB lineages.

Arkansas, Missouri, Oklahoma↗
USGS2021

Efficient genotyping with backwards compatibility: Converting a legacy microsatellite panel for muskellunge (Esox masquinongy) to genotyping-by-sequencing chemistry

Microsatellites have been a staple of population genetics research for over three decades, and many large datasets have been generated with these markers. Microsatellites have been used, for example, to conduct genetic monitoring and construct large multigeneration pedigrees as well as genotype thousands of individuals from a given species to create high-resolution baselines of spatial genetic structure. However, the capillary electrophoresis (CE) approach used to genotype microsatellites is inefficient compared to newer genotyping-by-sequencing (GBS) approaches, and researchers have begun transitioning away from CE. Backward compatibility between GBS and CE would facilitate a seamless transition to a more efficient chemistry, while ensuring that research based on CE panels could continue. Here, we explore the feasibility of converting a legacy panel of 15 microsatellites developed for muskellunge ( Esox masquinongy ) from CE to GBS chemistry. Muskellunge are an important sportfish in the Great Lakes region, and the existing microsatellite panel has been used to genotype thousands of samples to develop a region-wide baseline of genetic structure. We successfully converted all 15 microsatellites to GBS chemistry. GBS produced high genotyping rates (98%) and had high concordance with CE microsatellite genotypes (99%). Conversion to GBS required redesign of some primers and pairs to shorten amplicon length and adjust melting temperatures, optimization of primer concentrations, and comparisons with CE genotypes to optimize GBS genotyping parameters; however, none of these steps were especially onerous. Our results demonstrate that it is highly feasible to convert legacy CE panels to GBS, ensuring seamless continuation of important, often long-term research.

Conservation Genetics Resources↗
USGS2022

Rapid SNP genotyping, sex identification, and hybrid-detection in threatened bull trout

We developed new bull trout genetic markers using Restriction-site Associated DNA sequencing (RAD-seq) to improve our ability to address questions important for their conservation and management. Samples from across the species range were sequenced and 5020 high quality single nucleotide polymorphism (SNP) loci were discovered, including hundreds with high heterozygosity ( H > 0.30). We developed 63 high-heterozygosity bull trout polymorphic SNPs and one sex-identification SNP and tested them on range-wide samples. In addition, we tested previously published SNP assays including 11 species-diagnostic SNPs differentiating bull trout from brook trout and 3 brook trout variable SNPs on a broad set of range-wide samples. Genotypes from the sex-identification SNP showed 95% agreement with the field sex identification across 113 samples. The eleven species-diagnostic loci reliably discriminated between known brook trout, bull trout, and F 1 hybrid control samples. These SNP assays will facilitate genotyping of partially degraded museum fin clips, and tissues with low DNA content such as scales and otoliths. Finally, these loci will allow rapid genotyping for improved resolution of bull trout population structure, sex ratios, movement patterns, and introgressive hybridization with non-native brook trout for a wide range of management questions.

British Columbia, Idaho, Montana, Nevada, Oregon, ↗
USGS2022

Development of a ddPCR assay for the detection of the Smoky Madtom (Noturus baileyi) from eDNA in stream water samples

The Smoky Madtom Noturus baileyi is a federally endangered species, whose native distribution includes lower Abrams Creek in Great Smoky Mountains National Park (GRSM) and Citico Creek in nearby Cherokee National Forest. Due to challenges for bio-monitoring posed by its nocturnality and cryptic life history, an environmental DNA (eDNA)-based approach for detection would be useful to complement existing electrofishing and seining efforts to better understand the distribution of this species. We developed a probe-based droplet digital PCR (ddPCR) assay to detect Smoky Madtoms from non-invasively collected water samples. The assay was specific to N. baileyi and did not amplify concentrated genomic DNA of 16 co-occurring or regional fish species, including the yellowfin madtom N. flavipinnis and stonecat N. flavus . The assay limit of detection (LOD) was determined to be 4.18 copies (95% CI: 3.95, 4.41). Several 2 L water samples collected from throughout various streams in GRSM in 2016 and 2017 were tested for the presence of N. baileyi using the ddPCR assay. N. baileyi was detected at two different sites in 2016 and 2017 within Abrams Creek previously known to contain N. baileyi , but no novel detections in other sampled streams were observed. This assay should prove useful for continued surveys of N. baileyi in GRSM.

Great Smoky Mountains National Park↗
USGS2023

Validation of a species-specific probe-based qPCR assay for the threatened meltwater stonefly, Lednia tumana, in environmental samples

A probe-based quantitative real-time PCR assay was developed to detect meltwater stonefly ( Lednia tumana ) environmental (e)DNA in water samples. The limits of detection and quantification, respectively, were 12.1 and 58.4 gene copies for calibration standards and these values were similarly low in a relevant environmental sample matrix (8.6 and 174.2, respectively). The assay’s utility was demonstrated in situ on water samples with concomitant manual invertebrate surveys from a wide range of alpine streams across L. tumana ’s native range.

Conservation Genetics Resources↗
USGS2023

Development of a quantitative PCR assay for detecting northwest salamander (ambystoma gracile) in environmental DNA samples

We developed a primer and probe based quantitative PCR assay for use with environmental DNA to detect Northwest salamander ( Ambystoma gracile ), a species endemic to the temperate Pacific coastal region of North America. The assay targets a region in the mitochondrial DNA (mtDNA) D-loop gene. Tests of the assay were performed in silico (using the NCBI BLAST tool), in vitro (using DNA extracted from A. gracile and related species) and in situ (water samples collected from a lake with a known population of A. gracile ). This assay will be useful in efforts to monitor the distribution and occupancy of this species.

Conservation Genetics Resources↗
USGS2024

Development of an 8K SNP chip to assess adaptive diversity and hybridization in polar bears

The polar bear ( Ursus maritimus ) is a species particularly vulnerable to the effects of climate change. As the climate warms, polar bears will be forced to move to more suitable habitats which are likely to shrink, adapt to the new conditions, or decline in population size. However, the genomic diversity within and among all 19 subpopulations of polar bears, and therefore their adaptive potential, is currently unknown. In addition, warmer climates are likely to result in more frequent contact between polar bears and grizzly bears ( U. arctos ), with which they can hybridize. Here we describe the development, quality control, and application of the Ursus maritimus V2 SNP chip. This 8 K SNP chip contains loci explicitly selected to assess both RAD-derived and transcriptome-derived loci, as well as SNPs to detect hybridization between species. A total of 7,239 loci (90.3% of those printed) were successfully genotyped, with over 99% genotype concordance for individuals typed in duplicate on this chip, and between individuals typed here and on the Ursus maritimus V1 SNP chip. Using simulations, we demonstrate that the markers have high accuracy and efficiency to detect hybridization and backcrosses between polar bears and grizzly bears. However, empirical analysis of 371 polar bears, 440 grizzly bears, and 8 known hybrids found no novel instances of recent hybridization. The Ursus maritimus V2 SNP chip provides a powerful tool for monitoring the adaptive potential of this species along with assessing population structure, quantitative genomics, and hybridization in polar bears.

Conservation Genetics Resources↗
USGS2025

Development of species-specific primers for the identification of Atlantic and shortnose sturgeons

Atlantic ( Acipenser oxyrinchus oxyrinchus ) and shortnose sturgeon ( Acipenser brevirostrum ) are broadly distributed along the Atlantic Coast of North America, where they use rivers, estuaries, and coastal habitats. In order to support management under the U.S. Endangered Species Act, it is important to understand when and where these fish occur. However, this presents a challenge as the two taxa are sometimes misidentified and some life stages (e.g., eggs) are challenging to identify with confidence. In this study, we used cytochrome b sequences to develop molecular primers to confirm the identity of shortnose and Atlantic sturgeon samples. We tested these primers using reference DNA samples for these two species. The results suggested that the primers were able to positively identify and distinguish Atlantic and shortnose sturgeon. The accuracy of the Atlantic sturgeon primers was 95.34%, whereas the accuracy of the shortnose sturgeon primers was 90.7%. Even though there were some individuals that were not positively identified as their corresponding species (false negatives), we did not observe any false positives. Our paper does not aim to develop eDNA markers; rather, the objective of our study was to create species-specific, unlabeled, and cost-effective primers which can be amplified using conventional PCR. The amplification product can be observed in a 2% agarose gel run through electrophoresis. This entire procedure is relatively inexpensive and involves basic instruments found in most conservation genetics laboratories.

Conservation Genetics Resources↗
USGS2025

Quantitative PCR detection of endangered diamond darter Crystallaria Cincotta in environmental DNA: Employing locked nucleic acids and blocking probe for specificity

This study presents a quantitative PCR (qPCR) assay for the detection of the endangered diamond darter Crystallaria cincotta from environmental DNA (eDNA) in water samples. The assay design is based on an alignment of mitochondrial cytochrome b DNA sequences from 58 individuals representing 25 percid species. Leveraging genetic differences, a species-specific qPCR assay was designed, incorporating alocked nucleic acid (LNA)-enriched probe and a secondary blocking probe to enhance specificity. The assay targets a 93-base pair fragment that includes a diagnostic single nucleotide polymorphism in the probe region; combined with multiple primer mismatches, this provides specificity for distinguishing C. cincotta from other sympatric percid species. Specificity was validated by testing genomic DNA from 16 percid species and synthetic templates, confirming no cross-reactivity. Performance metrics, including the standard curve, qPCR efficiency, limit of detection, and limit of quantification, are reported. The qPCR assay exhibited sufficient sensitivity to detect C. cincotta eDNA in environmental water samples collected from occupied riverine habitats. This study illustrates the effectiveness of LNA-enriched and blocking probes in developing species-specific qPCR assays for eDNA applications, demonstrating their utility in accurately distinguishing closely related species within diverse fish communities.

Conservation Genetics Resources↗
USGS2004

Cryopreservation of Sperm from the Endangered Colorado Pikeminnow

We developed methods for the cryopreservation of sperm of the endangered Colorado pikeminnow Ptychocheilus lucius. Sperm were collected from a captive broodstock population of Colorado pikeminnow reared and maintained at the Dexter National Fish Hatchery and Technology Center. Our objectives were to (1) evaluate the effects on sperm motility of 24-h storage in Hanks' balanced salt solution (HBSS); (2) characterize sperm motility and duration; (3) examine the relationship between sperm motility and osmotic pressure; (4) examine the effect of four cryoprotectants (dimethyl sulfoxide [DMSO], dimethyl acetamide [DMA], glycerol, and methanol [MeOH] at two concentrations [5% and 10%]) on postthaw motility; and (5) compare the effect of two cooling rates (40??C/ min and 4??C/min) on postthaw motility. The sperm samples diluted with HBSS retained higher motility (mean ??SD, 77 ?? 22%; n = 9) than did undiluted samples (12 ?? 30%; n = 9) after 24 h of storage. When exposed to HBSS at 274 mosmols/kg or more, few sperm became motile (???1%). Exposure to HBSS at 265 mosmols/kg elicited threshold activation (defined as 10% motility), and maximum motility (>95%) was observed at 93 mosmols/ kg. The maximum motility of sperm was observed within 10 s after activation with deionized water, and sperm remained motile for 57 s. The sperm that were cooled at a rate of 40??C/min and cryopreserved with 5% MeOH retained higher postthaw motility (56 ?? 13%) than did sperm cryopreserved with DMSO, DMA, or glycerol (at 5% and 10%). When the sperm samples were cooled at a rate of 4??C/min, sperm cryopreserved with MeOH (5% or 10%) or DMSO (5% or 10%) retained the highest postthaw motilities (???14%). The use of cryopreserved sperm can assist hatchery managers in the production of fish, provide for the long-term conservation of genetic resources, and assist in the recovery of endangered species such as the Colorado pikeminnow.

North American Journal of Aquaculture↗