PCR primers for microsatellite loci in the desert tortoise (Gopherus agassizii,Testudinidae)

The desert tortoise, Gopherus agassizii, is a threatened species native to the North American desert southwest and is recognized as having distinct Mojave and Sonoran populations. We identified six polymorphic microsatellite loci in the desert tortoise. All six loci were polymorphic in Sonoran samples. Five of the loci were variable in Mojave samples with varying degrees of amplification success. Two of the loci exhibited low allelic variation (2–3 alleles) while four were highly variable (8–27 alleles).     

Model-based comparisons of phylogeographic scenarios resolve the intraspecific divergence of cactophilic Drosophila mojavensis

The cactophilic fly Drosophila mojavensis exhibits considerable intraspecific genetic structure across allopatric geographic regions and shows associations with different host cactus species across its range. The divergence between these populations has been studied for more than 60years, yet their exact historical relationships have not been resolved. We analysed sequence data from 15 intronic X-linked loci across populations from Baja California, mainland Sonora-Arizona and Mojave Desert regions under an isolation-with-migration model to assess multiple scenarios of divergence. We also compared the results with a pre-existing sequence data set of eight autosomal loci. We derived a population tree with Baja California placed at its base and link their isolation to Pleistocene climatic oscillations. Our estimates suggest the Baja California population diverged from an ancestral Mojave Desert/mainland Sonora-Arizona group around 230,000-270,000years ago, while the split between the Mojave Desert and mainland Sonora-Arizona populations occurred one glacial cycle later, 117,000-135,000years ago. Although we found these three populations to be effectively allopatric, model ranking could not rule out the possibility of a low level of gene flow between two of them. Finally, the Mojave Desert population showed a small effective population size, consistent with a historical population bottleneck. We show that model-based inference from multiple loci can provide accurate information on the historical relationships of closely related groups allowing us to set into historical context a classic system of incipient ecological speciation.     

Microsatellite variation among diverging populations of Drosophila mojavensis

Divergence and speciation may occur by various means, depending on the particular history, selective environments, and genetic composition of populations. In Drosophila mojavensis, a good model of incipient speciation, understanding the population genetic structure within this group facilitates our ability to understand the context in which reproductive isolation among populations is developing. Here we report the genetic structure and relationships of D. mojavensis populations at nuclear loci. We surveyed 29 populations throughout the distribution of D. mojavensis for four microsatellite loci to differentiation among populations of this species. These loci reveal four distinct geographical regions of D. mojavensis populations in the south-western United States and north-western Mexico--(i) Baja California peninsula (Baja), (ii) Sonora, Mexico-southern Arizona, United States (Sonora), (iii) Mojave Desert and Grand Canyon (Mojave), and (iv) Santa Catalina Island (Catalina). While all regions show strong isolation, Mojave and Catalina are highly diverged from other regions. Within any region, populations are largely homogenous over broad geographical distances. Based on the population structure, we find clear geographical barriers to gene flow appear to have a strong effect in isolating populations across regions for this species.     

Genetic characterization and management of the endangered Mohave tui chub

The Mohave tui chub (Siphateles bicolor mohavensis) is the only fish native to the Mojave River, California. The fish were displaced by introduced arroyo chubs (Gila orcutti) throughout most of their range, starting in the 1930s. Two potentially relictual populations and two transplanted populations were genetically characterized using 12 microsatellite DNA loci, along with contemporary cyprinid populations in the Mojave River. We found only un-hybridized Mohave tui chubs in the refuge populations, and only un-hybridized arroyo chubs in the Mojave River. The two largest Mohave tui chub populations (Lake Tuendae and China Lake) exhibit similar, comparatively high genetic variation. Another large population (Camp Cady) with low genetic diversity shows the effect of a bottleneck of ten individuals during the historic founding event. The fourth population (MC Spring) has the fewest alleles, lowest heterozygosity, and is the most divergent, suggesting that genetic drift from a persistently low effective population size has reduced genetic diversity since its apparent isolation in 1934. We recommend instituting artificial gene flow to rebuild genetic variation in Camp Cady from both Lake Tuendae and China Lake, and the establishment of new populations with founders from both Lake Tuendae and China Lake. Additionally, we comment on the infeasibility of restoring populations of Mohave tui chub in their historic habitats.     

As the raven flies: using genetic data to infer the history of invasive common raven (Corvus corax) populations in the Mojave Desert

Common raven (Corvus corax) populations in Mojave Desert regions of southern California and Nevada have increased dramatically over the past five decades. This growth has been attributed to increased human development in the region, as ravens have a commensal relationship with humans and feed extensively at landfills and on road-killed wildlife. Ravens, as a partially subsidized predator, also represent a problem for native desert wildlife, in particular threatened desert tortoises (Gopherus agassizii). However, it is unclear whether the more than 15-fold population increase is due to in situ population growth or to immigration from adjacent regions where ravens have been historically common. Ravens were sampled for genetic analysis at several local sites within five major areas: the West Mojave Desert (California), East Mojave Desert (southern Nevada), southern coastal California, northern coastal California (Bay Area), and northern Nevada (Great Basin). Analyses of mtDNA control region sequences reveal an increased frequency of raven 'Holarctic clade' haplotypes from south to north inland, with 'California clade' haplotypes nearly fixed in the California populations. There was significant structuring among regions for mtDNA, with high F(ST) values among sampling regions, especially between the Nevada and California samples. Analyses of eight microsatellite loci reveal a mostly similar pattern of regional population structure, with considerably smaller, but mostly significant, values. The greater mtDNA divergences may be due to lower female dispersal relative to males, lower N(e), or effects of high mutation rates on maximal values of F(ST). Analyses indicate recent population growth in the West Mojave Desert and a bottleneck in the northern California populations. While we cannot rule out in situ population growth as a factor, patterns of movement inferred from our data suggest that the increase in raven populations in the West Mojave Desert resulted from movements from southern California and the Central Valley. Ravens in the East Mojave Desert are more similar to ones from northern Nevada, indicating movement between those regions. If this interpretation of high gene flow into the Mojave Desert is correct, then efforts to manage raven numbers by local control may not be optimally effective.     

Eleven new highly polymorphic microsatellite loci in the yellow croaker,Pseudosciaena crocea

We developed 11 new microsatellite markers in Pseudosciaena crocea by screening an enriched genomic library using nonradioactive polymerase chain reaction (PCR) techniques. All loci were found to be polymorphic with an average of 14.9 alleles per locus (range four to 30). The mean observed and expected heterozygosities were 0.86 (range 0.57–1.00) and 0.90 (range 0.62–0.98), respectively. Four loci showed significant Hardy–Weinberg disequilibrium. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for population genetic studies of P. crocea.     

PCR‐based markers detect genetic variation at growth and immune function‐related loci in chinook salmon (Oncorhynchus tshawytscha)

We developed seven polymerase chain reaction (PCR) based markers that detect genetic variation at loci related to growth (four) and immune function (three) in chinook salmon. One assay shows a length polymorphism following PCR; the others show restriction fragment length polymorphisms (PCR‐RFLP). Two alleles were detected in each assay, and the common alleles were found at frequencies of 0.67–0.95 in seven wild populations in British Columbia, Canada. These loci also amplified in other salmonid species. These markers, by detecting variation linked to genes with high fitness relevance, are expected to be useful in a range of theoretical and applied studies.     

Mycoplasmosis in free-ranging desert tortoises in Utah and Arizona

Upper respiratory tract disease (URTD) has been associated with major losses of free-ranging desert tortoises (Gopherus agassizii) in the southwestern United States. This prompted a clinical examination of 63 free-ranging desert tortoises for signs of URTD and sampling for Mycoplasma agassizii, the causative agent of URTD. Tortoises were sampled from three sites in the eastern Mojave Desert (1992-93), and from three sites in the Sonoran Desert (1992-94). Plasma samples were tested for antibodies to M. agassizii using an enzyme-linked immunosorbent assay (ELISA). Nasal aspirate samples from 12 Sonoran tortoises were tested using polymerase chain reaction (PCR) test directed at the 16S rRNA gene of M. agassizii. Nasal aspirate samples from all tortoises were cultured for M. agassizii. In the Mojave Desert, nine tortoises had clinical signs of URTD and eight were seropositive for M. agassizii. In the Sonoran Desert, there were no clinical signs of URTD, but two tortoises were seropositive, and two tortoises had positive PCR results.     

Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR

Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.     

Isolation and characterization of microsatellite markers in Fraser fir (Abies fraseri)

We describe the isolation and characterization of 14 microsatellite loci from Fraser fir (Abies fraseri). These markers originated from cloned inserts enriched for DNA sequences containing tandem di‐ and tri‐nucleotide repeats. In total, 36 clones were selected, sequenced and evaluated. Polymerase chain reaction (PCR) primers for 14 of these sequences consistently produced simple PCR profiles and were found to be polymorphic among 13 Fraser fir samples. In addition, more than half of these loci were found to amplify a wide range of samples from several Abies taxa.     

Tetranucleotide markers from the loggerhead sea turtle (Caretta caretta) and their cross-amplification in other marine turtle species

The loggerhead sea turtle (Caretta caretta) is a federally threatened species and listed as endangered by the World Conservation Union (IUCN). We describe primers and polymerase chain reaction (PCR) conditions to amplify 11 novel tetranucleotide microsatellite loci from the loggerhead sea turtle. We tested primers using samples from 22 females that nested at Melbourne Beach, Florida (USA). Primer pairs yielded an average of 11.2 alleles per locus (range of 4–24), an average observed heterozygosity of 0.83 (range 0.59–0.96), and an average polymorphic information content of 0.80 (range 0.62–0.94). We also demonstrate the utility of these primers, in addition to primers for 15 loci previously described, for amplifying microsatellite loci in four additional species representing the two extant marine turtle families: olive ridley (Lepidochelys olivacea), hawksbill (Eretmochelys imbricata), green turtle (Chelonia mydas), and leatherback (Dermochelys coriacea).     

Tetranucleotide and dinucleotide microsatellite loci from the northern bobwhite (Colinus virginianus)

We describe polymerase chain reaction (PCR) primers and conditions to amplify eight dinucleotide, one trinucleotide and 14 tetranucleotide microsatellite DNA loci isolated from the northern bobwhite (Colinus virginianus). The PCR primers were tested on 16 individuals collected from a population located within the Red Hills region of south Georgia and north Florida. The 23 primer pairs developed in this study yielded an average of 6.5 alleles per locus (range 2–11), an average observed heterozygosity of 0.47 (range 0.06–0.94) and average polymorphic information content of 0.60 (range 0.06–0.85).     

Phylogeny, divergence times and species limits of spiny lizards (Sceloporus magister species group) in western North American deserts and Baja California

The broad distribution of the Sceloporus magister species group (squamata: phrynosomatidae) throughout western North America provides an appropriate model for testing biogeographical hypotheses explaining the timing and origins of diversity across mainland deserts and the Baja California Peninsula. We inferred concordant phylogenetic trees describing the higher-level relationships within the magister group using 1.6 kb of mitochondrial DNA (mtDNA) and 1.7 kb of nuclear DNA data. These data provide strong support for the parallel divergence of lineages endemic to the Baja California Peninsula (S. zosteromus and the orcutti complex) in the form of two sequential divergence events at the base of the magister group phylogeny. A relaxed phylogenetic analysis of the mtDNA data using one fossil and one biogeographical constraint provides a chronology of these divergence events and evidence that further diversification within the Baja California clades occurred simultaneously, although patterns of geographical variation and speciation between clades differ. We resolved four major phylogeographical clades within S. magister that (i) provide a novel phylogenetic placement of the Chihuahuan Desert populations sister to the Mojave Desert; (ii) illustrate a mixed history for the Colorado Plateau that includes Mojave and Sonoran Desert components; and (iii) identify an area of overlap between the Mojave and Sonoran Desert clades near Yuma, Arizona. Estimates of bidirectional migration rates among populations of S. magister using four nuclear loci support strong asymmetries in gene flow among the major mtDNA clades. Based on the nonexclusivity of mtDNA haplotypes, nuclear gene flow among populations and wide zones of phenotypic intergradation, S. magister appears to represent a single geographically variable and widespread species.     

Highly polymorphic microsatellite loci for Speyeria idalia (Lepidoptera: Nymphalidae)

Four polymorphic microsatellite loci were identified in the butterfly Speyeria idalia. We constructed a phagemid library and screened approximately 120 000 inserts. Probing with GT15, we identified 36 positives and designed polymerase chain reaction (PCR) primers for 12 potential loci. Of those loci, only four consistently produced polymorphic, diallelic PCR products in the expected size range. These results are consistent with previous studies concerning the low frequency of microsatellite loci in the lepidoptera, although these four loci are highly polymorphic and therefore likely to provide information on the fine scale genetic structure among populations in this species.     

Sexing the Sciuridae: a simple and accurate set of molecular methods to determine sex in tree squirrels, ground squirrels and marmots

We determined the sequence of the male-specific minor histocompatibility complex antigen (Smcy) from the Y chromosome of seven squirrel species (Sciuridae, Rodentia). Based on conserved regions inside the Smcy intron sequence, we designed PCR primers for sex determination in these species that can be co-amplified with nuclear loci as controls. PCR co-amplification yields two products for males and one for females that are easily visualized as bands by agarose gel electrophoresis. Our method provides simple and reliable sex determination across a wide range of squirrel species.     

Defining population structure for the Mojave desert tortoise

We used highly variable microsatellite markers to identify population structure, movement, and biological boundaries for populations of the desert tortoise, Gopherus agassizii, in the Mojave and Colorado Deserts of the southwestern United States. The Mojave desert tortoise (listed as “threatened” by the U.S. Fish and Wildlife Service) has a large geographic range, long generation time, low population densities, and little above-ground activity. Additionally, the dispersal patterns of individual tortoises are virtually unknown, making indirect methods to assess movement among populations valuable. Using Bayesian assignment tests, we detected hierarchical structuring within the Mojave desert tortoise. Three basal groups were identified, and these corresponded to the mitochondrial DNA haplotypes reported in 1989. Additional population structure was evident within each basal unit, and this structure corresponds with major geographic barriers. Our analyses suggest that gene flow among populations was historically high because levels of population differentiation were low across the range. Geographic distance explained a large proportion of variation in genetic distance (68%), which pinpoints that dispersal is limited only on a regional scale. In light of these new analyses of the genetic population structure of the Mojave desert tortoise, we make new recommendations for the number and locations of recovery units for conservation of this species.     

Seven polymorphic microsatellite DNA loci from the red‐spotted newt (Notophthalmus viridescens)

We describe polymerase chain reaction (PCR) primers and amplification conditions for seven microsatellite DNA loci isolated from the red‐spotted newt (Notophthalmus viridescens). Primers were tested on 16 individuals from two populations on the Savannah River Site in Aiken County, South Carolina. We detected six to 10 alleles per locus and an overall observed heterozygosity range of 0.31–0.81. Despite low heterozygosity at two of the seven loci, the high polymorphic information contents (from 0.54 to 0.85) of these markers render them useful for future studies of the behavioural and population ecology of this common salamander.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.