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Establishing genome sizes of focal fishery and aquaculture species along Baja California, Mexico

Genome size—the total haploid content of nuclear DNA— is constant in all cells in individuals within a species, but differs among species. Consequently, the genome size is a quantifiable genetic signature that not only characterizes a species, but it can reflect chromatin modifications, which play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. This characteristic makes the genome size a crucial parameter for genetic research on endemic aquatic species and for genetic manipulations in aquaculture species. Technologies for genetic assessments and improvements applied to fishery and aquaculture species use genome size values as a means by which hybrids, polyploids, and sex can be identified, when sex chromosomes exist. The objectives of this study were to determine genome sizes of aquatic species with economic and biological importance along the Pacific coast of Mexico, as well as to identify the appropriate reference standards for use in this study. Blood, hemolymph or milt were collected from 10 species occurring along the coast of Baja California: Sablefish Anoplopoma fimbria , Black Snapper Lutjanus novemfasciatus , California Halibut Paralichthys californicus , Pacific Sardine Sardinops sagax , Flag Rockfish Sebastes rubrivinctus , Starry Rockfish Sebastes constellatus, Totoaba Totoaba macdonaldi , Whiteleg Shrimp Litopenaeus vannamei and two Yellowtail Seriola lalandi and S. dorsalis . Nuclear DNA was stained with propidium iodide solution and the genome size was determined by flow cytometry, with results ranging from 0.61 pg (1.22 pg/diploid cell) to 2.59 pg (5.18 pg/diploid cell), with the smallest value in Sablefish and the largest in the Whiteleg Shrimp. No significant differences were detected ( P ≤ 0.05) among individuals of the same species; the likely reason behind any dissimilar DNA content values with those from the literature were differences in methodologies or variations in genetics. Red-ear Slider Turtle Trachemys scripta elegans 2.65 pg (5.30 pg/diploid cell) and Red Junglefowl Gallus gallus 1.27 pg (2.54 pg/diploid cell) were chosen as the standards for reference values. These results establish the basis for the Mexican National Aquatic Genetic Resources project supporting genetic improvements for aquaculture and conservation status parameters for fisheries species.

Baja California

Development of 20 TaqMan assays differentiating the endangered shortnose and Lost River suckers

Accurate species identification is vital to conservation and management of species at risk. Species identification is challenging when taxa express similar phenotypic characters and form hybrids, for example the endangered shortnose sucker ( Chasmistes brevirostris ) and Lost River sucker ( Deltistes luxatus ). Here, we developed 20 Taqman assays that differentiate these species (19 nuclear DNA and one mitochondrial DNA). Assays were evaluated in 160 young-of-the-year identified to species using meristic counts. Alleles were not fixed between species, but species were highly differentiated ( F ST = 0.753, P < 0.001). The assays developed herein will be a valuable tool for resource managers.

Conservation Genetics Resources

A single nucleotide polymorphism panel for identifying North American species and hybrids in the genus Morone

Four species of the fish genus Morone exist in North America and are often the focus of management actions, including propagation and stocking of their hybrids. We have developed an amplicon-based single nucleotide polymorphism (SNP) genotyping panel that provides the ability to distinguish all four species and the commonly stocked M. chrysops x M. saxatilis hybrid using a set of 47 SNPs. This resource provides a means of cost effective, high confidence genetic species identification that may aid management and conservation efforts for North American Morone species.

North America

Implementation of genetic conservation practices in a muskellunge propagation and stocking program

Conservation of genetic resources is a challenging issue for agencies managing popular sport fishes. To address the ongoing potential for genetic risks, we developed a comprehensive set of recommendations to conserve genetic diversity of muskellunge ( Esox masquinongy ) in Wisconsin, and evaluated the extent to which the recommendations can be implemented. Although some details are specific to Wisconsin's muskellunge propagation program, many of the practical issues affecting implementation are applicable to other species and production systems. We developed guidelines to restrict future broodstock collection operations to lakes with natural reproduction and to develop a set of brood lakes to use on a rotational basis within regional stock boundaries, but implementation will require considering lakes with variable stocking histories. Maintaining an effective population size sufficient to minimize the risk of losing alleles requires limiting broodstock collection to large lakes. Recommendations to better approximate the temporal distribution of spawning in hatchery operations and randomize selection of brood fish are feasible. Guidelines to modify rearing and distribution procedures face some logistic constraints. An evaluation of genetic diversity of hatchery-produced fish during 2008 demonstrated variable success representing genetic variation of the source population. Continued evaluation of hatchery operations will optimize operational efficiency while moving toward genetic conservation goals.

Fisheries

PumaPlex100: An expanded tool for puma SNP genotyping with low-yield DNA

The original PumaPlex is a high-throughput assay developed to genotype 25 single nucleotide polymorphisms (SNPs) in pumas ( Puma concolor ). Here, we describe the development of PumaPlex100 – an expanded version of the original assay that now genotypes > 100 SNPs. We tested 142 candidate SNPs and developed a panel of 101 polymorphic loci, which are spread across four multiplexes and suitable for genotyping of non-invasive samples. This panel will provide researchers a set of standardized markers, that can be analyzed with minimal bioinformatic skills, for the assessment of population structure and genetic diversity. These SNPs will serve as an important resource for the continued genetic monitoring of this species, especially monitoring through non-invasive sampling.

Sonora

A GT-seq panel for walleye (Sander vitreus) provides important insights for efficient development and implementation of amplicon panels in non-model organisms

Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals. Development of GT-seq panels is nontrivial, but studies describing trade-offs associated with different steps of GT-seq panel development are rare. Here, we construct a dual-purpose GT-seq panel for walleye ( Sander vitreus ), discuss trade-offs associated with different development and genotyping approaches, and provide suggestions for researchers constructing their own GT-seq panels. Our GT-seq panel was developed using an ascertainment set consisting of restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We conducted simulations to test the utility of all loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We then performed three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (i) inexpensive Chelex extractions performed well for genotyping; (ii) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was probably unnecessary; and (iii) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings and suggestions should aid in their construction in myriad taxa.

Minnesota, Wisconsin

Nine microsatellite loci developed from the octocoral, Paragorgia arborea

Paragorgia arborea, or bubblegum coral, occurs in continental slope habitats worldwide, which are increasingly threatened by human activities such as energy development and fisheries practices. From 101 putative loci screened, nine microsatellite markers were developed from samples taken from Baltimore canyon in the western North Atlantic Ocean. The number of alleles ranged from two to thirteen per locus and each displayed equilibrium. These nuclear resources will help further research on population connectivity in threatened coral species where mitochondrial markers are known to lack fine-scale genetic diversity.

Baltimore Canyon

Development of ten microsatellite loci in the invasive giant African land snail, Achatina (=Lissachatina) fulica Bowdich, 1822

A suite of tetra-nucleotide microsatellite loci were developed for the invasive giant African land snail, Achatina (=Lissachatina) fulica Bowdich, 1822, from Ion Torrent next-generation sequencing data. Ten of the 96 primer sets tested amplified consistently in 30 snails from Miami, Florida, plus 12 individuals representative of their native East Africa, Indian and Pacific Ocean regions. The loci displayed moderate levels of allelic diversity (average 5.6 alleles/locus) and heterozygosity (average 42 %). Levels of genetic diversity were sufficient to produce unique multi-locus genotypes and detect phylogeographic structuring among regional samples. The invasive A. fulica can cause extensive damage to important food crops and natural resources, including native flora and fauna. The loci characterized here will be useful for determining the origins and tracking the spread of invasions, detecting fine-scale spatial structuring and estimating demographic parameters.

Conservation Genetics Resources

Development of a quantitative PCR assay for detecting Egeria densa in environmental DNA samples

Brazilian elodea ( Egeria densa ) is an invasive freshwater plant that demonstrates widespread ecological impacts in freshwater ecosystems and causes substantial economic damage. Here, we developed an environmental DNA assay for detection of E. densa to provide resource managers with a tool for early detection, identification, and monitoring of invasive populations.

Conservation Genetics Resources

Characterization of microsatellite loci from two-spotted octopus Octopus bimaculatus Verrill 1883 from pyrosequencing reads

We characterized 22 novel microsatellite loci in the two-spotted octopus Octopus bimaculatus using 454 pyrosequencing reads. All loci were polymorphic and will be used in studies of marine connectivity aimed at increasing sustainability of the resource. The mean number alleles per locus was 13.09 (range 7–19) and observed heterozygosities ranged from 0.50 to 1.00. Four loci pairs were linked and three deviated from Hardy–Weinberg equilibrium. Eighteen and 12 loci were polymorphic in Octopus bimaculoides and Octopus hubbsorum , respectively.

Conservation Genetics Resources

An amplicon genotyping panel suitable for species identification and population genetics in sauger (Sander canadensis) and walleye (Sander vitreus)

Sauger ( Sander canadensis ) and walleye ( Sander vitreus ) are closely related North American fish species that are often managed by fishery agencies throughout their ranges. However, genotyping resources for sauger are presently limited to a small set of microsatellite loci. We evaluated whether primers in an existing walleye genotyping-in-thousands panel could amplify single nucleotide polymorphism loci (SNPs) in sauger. We identified 71 primer pairs that amplify 118 SNPs in both species. Allele frequency differences were large enough to confidently distinguish the species and identify hybrids. Additionally, we identified 41 loci with observed heterozygosity > 0.1 in sauger; these markers may be useful for simple population genetic analyses and parentage analysis when few contributors are present and for differentiating highly structured populations.

Minnesota, Wisconsin

Using redundant primer sets to detect multiple native Alaskan fish species from environmental DNA

Accurate and timely data regarding freshwater fish communities is important for informed decision-making by local, state, tribal, and federal land and resource managers; however, conducting traditional gear-based fish surveys can be an expensive and time-consuming process, particularly in remote areas, like those that characterize much of Alaska. To help address this challenge, we developed and tested five multi-species environmental DNA (eDNA) primer sets for the simultaneous detection of up to 37 target fish species in a single sample. Using these primer sets can reduce the cost and time needed to perform future studies of fish communities. Our results comparing multiple samples from multiple lakes and streams using multiple next-generation sequencing runs show the efficacy and reproducibility of these primers.

Alaska

Novel RAD sequence data reveal a lack of genomic divergence between dietary ecotypes in a landlocked salmonid population

Preservation of heritable ecological diversity within species and populations is a key challenge for managing natural resources and wild populations. Salmonid fish are iconic and socio-economically important species for commercial, aquaculture, and recreational fisheries across the globe. Many salmonids are known to exhibit ecological divergence within species, including distinct feeding ecotypes within the same lakes. Here we used 5559 SNPs, derived from RAD sequencing, to perform population genetic comparisons between two dietary ecotypes of sockeye salmon ( Oncorhynchus nerka ) in Jo-Jo Lake, Alaska (USA). We tested the standing hypothesis that these two ecotypes are currently diverging as a result of adaptation to distinct dietary niches; results support earlier conclusions of a single panmictic population. The RAD sequence data revealed 40 new SNPs not previously detected in the species, and our sequence data can be used in future studies of ecotypic diversity in salmonid species.

Alaska

Isolation and characterization of eight novel microsatellite loci in the double-crested cormorant ( Phalacrocorax auritus )

We describe the isolation and characterization of eight microsatellite loci from the double-crested cormorant ( Phalacrocorax auritus ). Genetic variability was assessed using 60 individuals from three populations. All loci were variable with the number of alleles ranging from two to 17 per locus, and observed heterozygosity varying from 0.05 to 0.89. No loci showed signs of linkage disequilibrium and all loci conformed to Hardy–Weinberg equilibrium frequencies. Further, all loci amplified and were polymorphic in two related Phalacrocorax species. These loci should prove useful for population genetic studies of the double-crested cormorant and other pelecaniform species.

Conservation Genetics Resources

Characterization of ten microsatellite loci in midget faded rattlesnake (Crotalus oreganus concolor)

Primers for 10 microsatellite loci were developed for midget faded rattlesnake ( Crotalus oreganus concolor ), a small bodied subspecies of the Western Rattlesnake, which is found in the Colorado Plateau of eastern Utah, western Colorado and southwestern Wyoming. In a screen of 23 individuals from the most northern portion of the subspecies range in southwestern Wyoming, the 10 loci were found to have levels of variability ranging from 4 to 11 alleles. No loci were found to be linked, although one locus revealed significant departures from Hardy–Weinberg equilibrium. These microsatellite loci will be applicable for population genetic analyses, which will ultimately aid in management efforts for this rare subspecies of rattlesnake.

Conservation Genetics Resources

Development and characterization of 17 polymorphic microsatellite loci in the faucet snail, Bithynia tentaculata (Gastroposa: Caenogastropoda; Bithyniidae)

Bithynia tentaculata (Linnaeus, 1758), a snail native to Europe, was introduced into the US Great Lakes in the 1870's and has spread to rivers throughout the Northeastern US and Upper Mississippi River (UMR). Trematode parasites, for which B. tentaculata is a host, have also been introduced and are causing widespread waterfowl mortality in the UMR. Waterfowl mortality is caused by ingestion of trematode-infected B. tentaculata or insects infected with parasites released from the snails. We isolated and characterized 17 microsatellite loci from the invasive faucet snail, B. tentaculata (Gastropoda: Caenogastropoda: Bithyniidae). Loci were screened in 24 individuals of B. tentaculata. The number of alleles per locus ranged from 2 to 6, observed heterozygosity ranged from 0.050 to 0.783, and the probability of identity values ranged from 0.10 to 0.91. These new loci provide tools for examining the origin and spread of invasive populations in the US and management activities to prevent waterfowl mortality.

Conservation Genetics Resources

Tools for assessing kinship, population structure, phylogeography, and interspecific hybridization in Asian carps invasive to the Mississippi River, USA: isolation and characterization of novel tetranucleotide microsatellite DNA loci in silver carp Hypophthalmichthys molitrix

We document the isolation and characterization of novel tetranucleotide microsatellite DNA markers for the invasive silver carp Hypophthalmichthys molitrix and provide the results of cross-species amplification for three additional invasive carp species: bighead (H. nobilis), grass (Ctenopharyngodon idella) and black (Mylopharyngodon piceus). In the target species these markers yielded levels of allelic diversity (average 4.4 alleles/locus) and heterozygosity (average 54.7%) sufficient to: (1) provide unique multilocus genotypes; (2) delineate kinship relationships; (3) differentiate populations/species; (4) estimate effective population sizes; and (5) provide unique demographic perspectives for control or eradication. Currently these markers are being utilized to determine the degree of introgressive hybridization between H. molitrix and H. nobilis, to quantify gene flow between different sub-basins established in the central United States, and to assess the demographic status of sub-basin groups. This information will be critically important in the management/control of these invasive species.

Mississippi River

Characterization of small microsatellite loci isolated in endangered Indiana bat ( Myotis sodalis ) for use in non-invasive sampling

Primers for 10 microsatellite loci were developed specifically to amplify low quantity and quality DNA in the endangered Indiana Bat ( Myotis sodalis ). In a screen of 20 individuals from a population in Missouri, the 10 loci were found to have levels of variability ranging from seven to 18 alleles. No loci were found to be linked, although two loci revealed significant departures from Hardy–Weinberg equilibrium. These microsatellite loci will be applicable for population genetic analyses and for use in mark-recapture studies that utilize DNA collected non-invasively from fecal pellets, which will ultimately aid in management efforts.

Indiana