Dataset Information

Genetic species identification of ecologically important planthoppers (Prokelisia spp.) of coastal Spartina saltmarshes using High Resolution Melting Analysis (HRMA).

ABSTRACT: Phloem-feeding planthoppers of the genus Prokelisia rank among the most abundant and ecologically important browsers of coastal saltmarsh grasses of eastern North America and the Caribbean. Along the Spartina marshes of the northern Gulf of Mexico, the sympatric species P. marginata and P. dolus are the most abundant, but are difficult to distinguish from each other based solely on morphology. This study seeks to design a molecular assay based on High Resolution Melting Analysis (HRMA) as a fast, cost-effective alternative to differentiate these species. A 450 base pairs (bp) segment of cytochrome c oxidase subunit I (COI) was amplified and sequenced for representative samples of both species, and a short amplicon (SA) HRMA was designed based on the presence of fixed nucleotide differences between species found along a 60?bp segment of COI. The unambiguous identification of individual specimens of P. marginata or P. dolus was possible due to easily discernable differences in the melting temperatures of the two species along this mini barcode. This assay may prove useful for future genetic studies involving these species by preventing the overestimation of genetic diversity via inclusion of conspecifics, and in ecological studies by improving data on the effects of individual species of Prokelisia.

SUBMITTER: Espinoza GJ

PROVIDER: S-EPMC6934748 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Genetic species identification of ecologically important planthoppers (Prokelisia spp.) of coastal Spartina saltmarshes using High Resolution Melting Analysis (HRMA).

Espinoza G Janelle GJ Alvarado Bremer Jaime R JR

Scientific reports 20191227 1

Phloem-feeding planthoppers of the genus Prokelisia rank among the most abundant and ecologically important browsers of coastal saltmarsh grasses of eastern North America and the Caribbean. Along the Spartina marshes of the northern Gulf of Mexico, the sympatric species P. marginata and P. dolus are the most abundant, but are difficult to distinguish from each other based solely on morphology. This study seeks to design a molecular assay based on High Resolution Melting Analysis (HRMA) as a fast ...[more]

PMID: 31882766

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Project description:BackgroundMassively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (P. taeda), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (P. contorta) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies.ResultsWe obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening.ConclusionsThis sequence collection represents a major genomic resource for P. contorta, and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.

Project description:BackgroundThere are various phenotypic methods for identifying class B and class A ?-lactamase enzymes in Pseudomonas aeruginosa. The purpose of this study was to compare the sensitivity and specificity of different phenotypic methods with HRMA assay to detect ?-lactamase-producing P. aeruginosa strains.MethodsEighty-eight of P. aeruginosa isolates were collected from different specimens. Conventional double-disk test (DDT) and EDTA-imipenem microbiological (EIM) were performed to detect ESBL and MBL-producing strains, respectively. Meanwhile, the Modified Hodge test and Carba-NP test were performed on all carbapenem-resistant strains. HRMA method and sensitivity and specificity of primers were determined based on the melt curve temperature range. In all comparisons, PCR was considered as the gold standard.ResultsOf the 402 isolates collected from different clinical specimens, 88 isolates of P. aeruginosa were identified. However, 43 strains were (48.88%) ESBL-producing, and 7 strains (7.95%) were MBL-producing. Also, using the Modified Hodge test and Carba-NP method, 11 (12.5%) and 19 (21.59%) strains were carbapenemase-producing, respectively. The results of the HRMA test revealed that genes coding for blaSHV, blaTEM, blaKPC, blaIMP, blaVIM, and blaGES were detected in 44.31%, 22.72%, 13.63%, 14.7%, 5.6%, and 2.27% of P. aeruginosa isolates. Nonetheless, for blaKPC and blaGES genes, sensitivity and specificity of the Carba-NP test were 90.47%, 94.87%, and 83.36%, 94.80%, respectively. However, sensitivity and specificity of MHT was 91.66%, 98.70%, and 77.77%, 96.42%, respectively. For blaSHV and blaTEM genes, sensitivity and specificity of DDT were 95.55%, 95.55%, and 86%, 83.50%, respectively. However, sensitivity and specificity of EMI were 77.77%, 97.59%, and 91.66%, 97.43% for blaVIM and blaIMP, respectively.ConclusionThe HRMA is a powerful, accurate, closed-tube, rapid method for detecting ?-lactamase genes in P. aeruginosa. The high sensitivity and specificity of this method, along with phenotypic tests, play a useful role in increasing the predictive value of clinical reports.

Dataset Information

Genetic species identification of ecologically important planthoppers (Prokelisia spp.) of coastal Spartina saltmarshes using High Resolution Melting Analysis (HRMA).

Publications

Genetic species identification of ecologically important planthoppers (Prokelisia spp.) of coastal Spartina saltmarshes using High Resolution Melting Analysis (HRMA).

Similar Datasets

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