Rapid Detection of Raffaelea lauricola Directly from Host Plant and Beetle Vector Tissues Using Loop-Mediated Isothermal Amplification.

Since its introduction in 2002, laurel wilt disease has devastated indigenous lauraceous species in the southeastern United States. The causal agent is a fungal pathogen, Raffaelea lauricola, which, after being introduced into the xylem of trees by its vector beetle, Xyleborus glabratus, results in a fatal vascular wilt. Rapid detection and accurate diagnosis of infections is paramount to the successful implementation of disease management strategies. Current management operations to prevent the spread of laurel wilt disease are largely delayed by time-consuming laboratory procedures to confirm the diagnosis. In order to greatly speed up the operations, we developed a loop-mediated isothermal amplification (LAMP) species-specific assay that targets the ß-tubulin gene region of R. lauricola, and allows for the rapid detection of the pathogen directly from host plant and beetle tissues. The assay is capable of amplifying as little as 0.5 pg of fungal DNA and as few as 50 conidia. The assay is also capable of detecting R. lauricola directly from wood tissue of artificially inoculated redbay saplings as early as 10 and 12 days postinoculation, when testing high-quality and crude DNA extracts, respectively. Finally, crude DNA extracts of individual adult female X. glabratus beetles were assayed and the pathogen was detected from all specimens. This assay greatly reduces the time required to confirm a laurel wilt diagnosis and, because LAMP technology is well suited to provide point-of-care testing, it has the potential to expedite and facilitate implementation of management operations in response to disease outbreaks.

Mitogenomic sequences better resolve stock structure of southern Greater Caribbean green turtle rookeries.

Analyses of mitochondrial control region polymorphisms have supported the presence of several demographically independent green turtle (Chelonia mydas) rookeries in the Greater Caribbean region. However, extensive sharing of common haplotypes based on 490-bp control region sequences confounds assessment of the scale of natal homing and population structure among regional rookeries. We screened the majority of the mitochondrial genomes of 20 green turtles carrying the common haplotype CM-A5 and representing the rookeries of Buck Island, St. Croix, United States Virgin Islands (USVI); Aves Island, Venezuela; Galibi, Suriname; and Tortuguero, Costa Rica. Five single-nucleotide polymorphisms (SNPs) were identified that subdivided CM-A5 among regions. Mitogenomic pairwise φ(ST) values of eastern Caribbean rookery comparisons were markedly lower than the respective pairwise F(ST) values. This discrepancy results from the presence of haplotypes representing two divergent lineages in each rookery, highlighting the importance of choosing the appropriate test statistic for addressing the study question. Haplotype frequency differentiation supports demographic independence of Aves Island and Suriname, emphasizing the need to recognize the smaller Aves rookery as a distinct management unit. Aves Island and Buck Island rookeries shared mitogenomic haplotypes; however, frequency divergence suggests that the Buck Island rookery is sufficiently demographically isolated to warrant management unit status for the USVI rookeries. Given that haplotype sharing among rookeries is common in marine turtles with cosmopolitan distributions, mitogenomic sequencing may enhance inferences of population structure and phylogeography, as well as improve the resolution of mixed stock analyses aimed at estimating natal origins of foraging turtles.

Identifying patterns in foraging-area origins in breeding aggregations of migratory species: Loggerhead turtles in the Northwest Atlantic.

Population assessments conducted at reproductive sites of migratory species necessitate understanding the foraging-area origins of breeding individuals. Without this information, efforts to contextualize changes in breeding populations and develop effective management strategies are compromised. We used stable isotope analysis of tissue samples collected from loggerhead sea turtles (Caretta caretta) nesting at seven sites in the Northern Recovery Unit (NRU) of the eastern United States (North Carolina, South Carolina and Georgia) to assign females to three separate foraging areas in the Northwest Atlantic Ocean (NWA). We found that the majority of the females at NRU nesting sites (84.4%) use more northern foraging areas in the Mid-Atlantic Bight, while fewer females use more proximate foraging areas in the South Atlantic Bight (13.4%) and more southerly foraging areas in the Subtropical Northwest Atlantic (2.2%). We did not find significant latitudinal or temporal trends in the proportions of NRU females originating from different foraging areas. Combining these findings with previous data from stable isotope and satellite tracking studies across NWA nesting sites showed that variation in the proportion of adult loggerheads originating from different foraging areas is primarily related differences between recovery units: individuals in the NRU primarily use the Mid-Atlantic Bight foraging area, while individuals from the three Florida recovery units primarily use the Subtropical Northwest Atlantic and Eastern Gulf of Mexico foraging areas. Because each foraging area is associated with its own distinct ecological characteristics, environmental fluctuations and anthropogenic threats that affect the abundance and productivity of individuals at nesting sites, this information is critical for accurately evaluating population trends and developing effective region-specific management strategies.

Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation.

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.

Carbohydrate-related genes and cell wall biosynthesis in vascular tissues of loblolly pine (Pinus taeda).

Loblolly pine (Pinus taeda L.), the most widely planted tree species in the United States, is an important source of wood and wood fibers for a multitude of consumer products. Wood fibers are primarily composed of secondary cell walls, and cellulose, hemicelluloses and lignin are major components of wood. Fiber morphology and cell wall composition are important determinants of wood properties. We used comparative genomics to identify putative genes for cellulose and hemicellulose synthesis in loblolly pine that are homologous to genes implicated in cell wall synthesis in angiosperms. Sequences encoding putative secondary cell wall cellulose synthase genes, cellulose synthase-like genes, a membrane-bound endoglucanase gene, a sucrose synthase gene, a UDP-glucose pyrophosphorylase gene and GDP-mannose pyrophosphorylase genes were identified in expressed sequence tag (EST) collections from loblolly pine. Full-length coding sequences were obtained from cDNA clones isolated from a library constructed from developing xylem. Phylogenetic relationships between the genes from loblolly pine and angiosperm taxa were examined and transcriptional profiling in vascular tissues was conducted by real-time quantitative, reverse transcriptase-polymerase chain reaction. The putative cell wall synthesis genes were expressed at high levels in vascular tissues and a subset was differentially regulated in xylem and phloem tissues. Inferred phylogenetic relationships and expression patterns for the genes from loblolly pine were consistent with roles in synthesis of complex carbohydrates of the cell wall. These studies suggest functional conservation of homologous wood formation genes in gymnosperm and angiosperm taxa.

Exploring the loblolly pine (Pinus taeda L.) genome by BAC sequencing and Cot analysis.

Loblolly pine (LP; Pinus taeda L.) is an economically and ecologically important tree in the southeastern U.S. To advance understanding of the loblolly pine (LP; Pinus taeda L.) genome, we sequenced and analyzed 100 BAC clones and performed a Cot analysis. The Cot analysis indicates that the genome is composed of 57, 24, and 10% highly-repetitive, moderately-repetitive, and single/low-copy sequences, respectively (the remaining 9% of the genome is a combination of fold back and damaged DNA). Although single/low-copy DNA only accounts for 10% of the LP genome, the amount of single/low-copy DNA in LP is still 14 times the size of the Arabidopsis genome. Since gene numbers in LP are similar to those in Arabidopsis, much of the single/low-copy DNA of LP would appear to be composed of DNA that is both gene- and repeat-poor. Macroarrays prepared from a LP bacterial artificial chromosome (BAC) library were hybridized with probes designed from cell wall synthesis/wood development cDNAs, and 50 of the "targeted" clones were selected for further analysis. An additional 25 clones were selected because they contained few repeats, while 25 more clones were selected at random. The 100 BAC clones were Sanger sequenced and assembled. Of the targeted BACs, 80% contained all or part of the cDNA used to target them. One targeted BAC was found to contain fungal DNA and was eliminated from further analysis. Combinations of similarity-based and ab initio gene prediction approaches were utilized to identify and characterize potential coding regions in the 99 BACs containing LP DNA. From this analysis, we identified 154 gene models (GMs) representing both putative protein-coding genes and likely pseudogenes. Ten of the GMs (all of which were specifically targeted) had enough support to be classified as intact genes. Interestingly, the 154 GMs had statistically indistinguishable (α = 0.05) distributions in the targeted and random BAC clones (15.18 and 12.61 GM/Mb, respectively), whereas the low-repeat BACs contained significantly fewer GMs (7.08 GM/Mb). However, when GM length was considered, the targeted BACs had a significantly greater percentage of their length in GMs (3.26%) when compared to random (1.63%) and low-repeat (0.62%) BACs. The results of our study provide insight into LP evolution and inform ongoing efforts to produce a reference genome sequence for LP, while characterization of genes involved in cell wall production highlights carbon metabolism pathways that can be leveraged for increasing wood production.

Microsatellite markers for population studies of the salt marsh species Juncus roemerianus (Juncaceae).

PREMISE OF THE STUDY: Juncus roemerianus (Juncaceae) is a foundational species and ecosystem engineer of salt marshes in the Gulf of Mexico. These ecosystems provide coastal flood attenuation, nurseries for important species, and other ecosystem services, but are experiencing significant decline. Nuclear microsatellite markers were developed for J. roemerianus to study genetic diversity and population structure for conservation and restoration efforts. METHODS AND RESULTS: Illumina NextSeq high-throughput sequencing was used to develop a panel of 19 polymorphic microsatellite markers that were tested across individuals from three populations on the Gulf Coast. All markers were polymorphic, with observed and expected heterozygosities ranging from 0.212 to 0.828 and from 0.362 to 0.873, respectively. Allelic richness ranged from two to 13 alleles per locus with an average of 5.737. CONCLUSIONS: The 19 microsatellite markers are useful for population studies throughout the range of J. roemerianus. Three loci cross-amplified in the related taxon J. effusus.

Characterization of a subtropical hawksbill sea turtle (Eretmocheyles imbricata) assemblage utilizing shallow water natural and artificial habitats in the Florida Keys.

In order to provide information to better inform management decisions and direct further research, vessel-based visual transects, snorkel transects, and in-water capture techniques were used to characterize hawksbill sea turtles in the shallow marine habitats of a Marine Protected Area (MPA), the Key West National Wildlife Refuge in the Florida Keys. Hawksbills were found in hardbottom and seagrass dominated habitats throughout the Refuge, and on man-made rubble structures in the Northwest Channel near Cottrell Key. Hawksbills captured (N = 82) were exclusively juveniles and subadults with a straight standard carapace length (SSCL) ranging from 21.4 to 69.0cm with a mean of 44.1 cm (SD = 10.8). Somatic growth rates were calculated from 15 recaptured turtles with periods at large ranging from 51 to 1188 days. Mean SSCL growth rate was 7.7 cm/year (SD = 4.6). Juvenile hawksbills (<50 cm SSCL) showed a significantly higher growth rate (9.2 cm/year, SD = 4.5, N = 11) than subadult hawksbills (50-70 cm SSCL, 3.6 cm/year, SD = 0.9, N = 4). Analysis of 740 base pair mitochondrial control region sequences from 50 sampled turtles yielded 12 haplotypes. Haplotype frequencies were significantly different compared to four other Caribbean juvenile foraging aggregations, including one off the Atlantic coast of Florida. Many-to-one mixed stock analysis indicated Mexico as the primary source of juveniles in the region and also suggested that the Refuge may serve as important developmental habitat for the Cuban nesting aggregation. Serum testosterone radioimmunoassay results from 33 individuals indicated a female biased sex ratio of 3.3 females: 1 male for hawksbills in the Refuge. This assemblage of hawksbills is near the northern limit of the species range, and is one of only two such assemblages described in the waters of the continental United States. Since this assemblage resides in an MPA with intensive human use, basic information on the assemblage is vital to resource managers charged with conservation and species protection in the MPA.

Geographic patterns of genetic variation in a broadly distributed marine vertebrate: new insights into loggerhead turtle stock structure from expanded mitochondrial DNA sequences.

Previous genetic studies have demonstrated that natal homing shapes the stock structure of marine turtle nesting populations. However, widespread sharing of common haplotypes based on short segments of the mitochondrial control region often limits resolution of the demographic connectivity of populations. Recent studies employing longer control region sequences to resolve haplotype sharing have focused on regional assessments of genetic structure and phylogeography. Here we synthesize available control region sequences for loggerhead turtles from the Mediterranean Sea, Atlantic, and western Indian Ocean basins. These data represent six of the nine globally significant regional management units (RMUs) for the species and include novel sequence data from Brazil, Cape Verde, South Africa and Oman. Genetic tests of differentiation among 42 rookeries represented by short sequences (380 bp haplotypes from 3,486 samples) and 40 rookeries represented by long sequences (∼800 bp haplotypes from 3,434 samples) supported the distinction of the six RMUs analyzed as well as recognition of at least 18 demographically independent management units (MUs) with respect to female natal homing. A total of 59 haplotypes were resolved. These haplotypes belonged to two highly divergent global lineages, with haplogroup I represented primarily by CC-A1, CC-A4, and CC-A11 variants and haplogroup II represented by CC-A2 and derived variants. Geographic distribution patterns of haplogroup II haplotypes and the nested position of CC-A11.6 from Oman among the Atlantic haplotypes invoke recent colonization of the Indian Ocean from the Atlantic for both global lineages. The haplotypes we confirmed for western Indian Ocean RMUs allow reinterpretation of previous mixed stock analysis and further suggest that contemporary migratory connectivity between the Indian and Atlantic Oceans occurs on a broader scale than previously hypothesized. This study represents a valuable model for conducting comprehensive international cooperative data management and research in marine ecology.

Loggerhead turtle eggshells as a source of maternal nuclear genomic DNA for population genetic studies.

Tagging studies on nesting beaches are commonly used to estimate nesting frequency, remigration interval and nesting population size for marine turtle rookeries. Estimates of these demographic parameters from tagging projects may be biased because of the small scale of tagging efforts relative to female nest site fidelity and the logistical difficulty of intercepting all nesting females. Therefore, alternative and supplemental means of individual identification of nesting females are required. We demonstrate that maternal nuclear microsatellite DNA can be isolated from unincubated eggshells of the loggerhead sea turtle (Caretta caretta) through comparison of DNA extracted from 59 eggs collected within 15 h of oviposition and DNA derived from skin samples from respective nesting females. Scorable microsatellite genotypes were produced in 897 of 994 (90.2%) single-locus egg amplifications attempted. Among eggs from known females, 730 of 748 (97.6%) single-locus, egg-derived genotypes matched the respective skin-derived genotypes. Allelic dropout was the most common type of error, followed by the presence of nonmaternal, presumably paternal, alleles. Genotypes derived from unincubated eggshells permit individual assignment of nests and therefore demographic parameter estimates for loggerhead turtle nesting populations, despite genotyping errors that require further optimization. Although sampling unincubated eggs is destructive, this technique is noninvasive to nesting females and is applicable in marine turtle population genetics studies when individual resolution is required but direct interception of nesting females is undesirable or logistically infeasible.

Microsatellite markers for eastern hemlock (Tsuga canadensis).

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2-14), an average observed heterozygosity of 0.45 (range 0.14-0.73), and an average polymorphic information content of 0.54 (range 0.28-0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga.