Search results for: amplicon sequencing

Authors: F. Varga, Z. Liber, J. Jakše, A. Turudić, Z. Šatović, I. Radosavljević, N. Jeran, M. Grdiša

Abstract:

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.; Asteraceae), a source of the commercially dominant plant insecticide pyrethrin, is a species endemic to the eastern Adriatic. Genetic diversity of T. cinerariifolium was previously studied using amplified fragment length polymorphism (AFLP) markers. However, microsatellite markers (single sequence repeats - SSRs) are more informative because they are codominant, highly polymorphic, locus-specific, and more reproducible, and thus are most often used to assess the genetic diversity of plant species. Dalmatian pyrethrum is an outcrossing diploid (2n = 18) whose large genome size and high repeatability have prevented the success of the traditional approach to SSR markers development. The advent of next-generation sequencing combined with the specifically developed method recently enabled the development of, to the author's best knowledge, the first set of SSRs for genomic characterization of Dalmatian pyrethrum, which is essential from the perspective of plant genetic resources conservation. To evaluate the effectiveness of the developed SSR markers in genetic differentiation of Dalmatian pyrethrum populations, a preliminary genetic diversity study was conducted on 30 individuals from three geographically distinct natural populations in Croatia (northern Adriatic island of Mali Lošinj, southern Adriatic island of Čiovo, and Mount Biokovo) based on 12 SSR loci. Analysis of molecular variance (AMOVA) by randomization test with 10,000 permutations was performed in Arlequin 3.5. The average number of alleles per locus, observed and expected heterozygosity, probability of deviations from Hardy-Weinberg equilibrium, and inbreeding coefficient was calculated using GENEPOP 4.4. Genetic distance based on the proportion of common alleles (DPSA) was calculated using MICROSAT. Cluster analysis using the neighbor-joining method with 1,000 bootstraps was performed with PHYLIP to generate a dendrogram. The results of the AMOVA analysis showed that the total SSR diversity was 23% within and 77% between the three populations. A slight deviation from Hardy-Weinberg equilibrium was observed in the Mali Lošinj population. Allele richness ranged from 2.92 to 3.92, with the highest number of private alleles observed in the Mali Lošinj population (17). The average observed DPSA between 30 individuals was 0.557. The highest DPSA (0.875) was observed between several pairs of Dalmatian pyrethrum individuals from the Mali Lošinj and Mt. Biokovo populations, and the lowest between two individuals from the Čiovo population. Neighbor-joining trees, based on DPSA, grouped individuals into clusters according to their population affiliation. The separation of Mt. Biokovo clade was supported (bootstrap value 58%), which is consistent with the previous study on AFLP markers, where isolated populations from Mt. Biokovo differed from the rest of the populations. The developed SSR markers are an effective tool for assessing the genetic diversity and structure of natural Dalmatian pyrethrum populations. These preliminary results are encouraging for a future comprehensive study with a larger sample size across the species' range. Combined with the biochemical data, these highly informative markers could help identify potential genotypes of interest for future development of breeding lines and cultivars that are both resistant to environmental stress and high in pyrethrins. Acknowledgment: This work has been supported by the Croatian Science Foundation under the project ‘Genetic background of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) insecticidal potential’- (PyrDiv) (IP-06-2016-9034) and by project KK.01.1.1.01.0005, Biodiversity and Molecular Plant Breeding, at the Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroP-BioDiv), Zagreb, Croatia.

Keywords: Asteraceae, genetic diversity, genomic SSRs, NGS, pyrethrum, Tanacetum cinerariifolium

Procedia PDF Downloads 87

Authors: David J. Foran, Nhan Do, Samuel Ajjarapu, Wenjin Chen, Tahsin Kurc, Joel H. Saltz

Abstract:

The large-scale data and computational requirements of investigators throughout the clinical and research communities demand an informatics infrastructure that supports both existing and new investigative and translational projects in a robust, secure environment. In some subspecialties of medicine and research, the capacity to generate data has outpaced the methods and technology used to aggregate, organize, access, and reliably retrieve this information. Leading health care centers now recognize the utility of establishing an enterprise-wide, clinical data warehouse. The primary benefits that can be realized through such efforts include cost savings, efficient tracking of outcomes, advanced clinical decision support, improved prognostic accuracy, and more reliable clinical trials matching. The overarching objective of the work presented here is the development and implementation of a flexible Intelligent Retrieval and Interrogation System (IRIS) that exploits the combined use of computational imaging, genomics, and data-mining capabilities to facilitate clinical assessments and translational research in oncology. The proposed System includes a multi-modal, Clinical & Research Data Warehouse (CRDW) that is tightly integrated with a suite of computational and machine-learning tools to provide insight into the underlying tumor characteristics that are not be apparent by human inspection alone. A key distinguishing feature of the System is a configurable Extract, Transform and Load (ETL) interface that enables it to adapt to different clinical and research data environments. This project is motivated by the growing emphasis on establishing Learning Health Systems in which cyclical hypothesis generation and evidence evaluation become integral to improving the quality of patient care. To facilitate iterative prototyping and optimization of the algorithms and workflows for the System, the team has already implemented a fully functional Warehouse that can reliably aggregate information originating from multiple data sources including EHR’s, Clinical Trial Management Systems, Tumor Registries, Biospecimen Repositories, Radiology PAC systems, Digital Pathology archives, Unstructured Clinical Documents, and Next Generation Sequencing services. The System enables physicians to systematically mine and review the molecular, genomic, image-based, and correlated clinical information about patient tumors individually or as part of large cohorts to identify patterns that may influence treatment decisions and outcomes. The CRDW core system has facilitated peer-reviewed publications and funded projects, including an NIH-sponsored collaboration to enhance the cancer registries in Georgia, Kentucky, New Jersey, and New York, with machine-learning based classifications and quantitative pathomics, feature sets. The CRDW has also resulted in a collaboration with the Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC) at the U.S. Department of Veterans Affairs to develop algorithms and workflows to automate the analysis of lung adenocarcinoma. Those studies showed that combining computational nuclear signatures with traditional WHO criteria through the use of deep convolutional neural networks (CNNs) led to improved discrimination among tumor growth patterns. The team has also leveraged the Warehouse to support studies to investigate the potential of utilizing a combination of genomic and computational imaging signatures to characterize prostate cancer. The results of those studies show that integrating image biomarkers with genomic pathway scores is more strongly correlated with disease recurrence than using standard clinical markers.

Keywords: clinical data warehouse, decision support, data-mining, intelligent databases, machine-learning.

Procedia PDF Downloads 88

Authors: Maria João Ferreira, Natalia Sierra-Garcia, Javier Cremades, Carla António, Ana M. Rodrigues, Helena Silva, Ângela Cunha

Abstract:

Salicornia ramosissima, a halophyte that grows naturally in coastal areas of the northern hemisphere, is often considered the most promising halophyte candidate for extensive crop cultivation and saline agriculture practices. The expanding interest in this plant surpasses its use as gourmet food and includes their potential application as a source of bioactive compounds for the pharmaceutical industry. Despite growing well in saline soils, sustainable and ecologically friendly techniques to enhance crop production and the nutritional value of this plant are still needed. The root microbiome of S. ramosissima proved to be a source of taxonomically diverse plant growth-promoting bacteria (PGPB). Halotolerant strains of Bacillus, Salinicola, Pseudomonas, and Brevibacterium, among other genera, exhibit a broad spectrum of plant-growth promotion traits [e.g., 3-indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophores, phosphate solubilization, Nitrogen fixation] and express a wide range of extracellular enzyme activities. In this work, three plant growth-promoting bacteria strains (Brevibacterium casei EB3, Pseudomonas oryzihabitans RL18, and Bacillus aryabhattai SP20) isolated from the rhizosphere and the endosphere of S. ramosissima roots from different saltmarshes along the Portuguese coast were inoculated in S. ramosissima seeds. Plants germinated from inoculated seeds were grown for three months in pots filled with a mixture of perlite and estuarine sediment (1:1) in greenhouse conditions and later transferred to a growth chamber, where they were maintained two months with controlled photoperiod, temperature, and humidity. Pots were placed on trays containing the irrigation solution (Hoagland’s solution 20% added with 10‰ marine salt). Before reaching the flowering stage, plants were collected, and the fresh and dry weight of aerial parts was determined. Non-inoculated seeds were used as a negative control. Selected dried stems from the most promising treatments were later analyzed by GC-TOF-MS for primary metabolite composition. The efficiency of inoculation and persistence of the inoculum was assessed by Next Generation Sequencing. Inoculations with single strain EB3 and co-inoculations with EB3+RL18 and EB3+RL18+SP20 (All treatment) resulted in significantly higher biomass production (fresh and dry weight) compared to non-inoculated plants. Considering fresh weight alone, inoculation with isolates SP20 and RL18 also caused a significant positive effect. Combined inoculation with the consortia SP20+EB3 or SP20+RL18 did not significantly improve biomass production. The analysis of the profile of primary metabolites will provide clues on the mechanisms by which the growth-enhancement effect of the inoculants operates in the plants. These results sustain promising prospects for the use of rhizospheric and endophytic PGPB as biofertilizers, reducing environmental impacts and operational costs of agrochemicals and contributing to the sustainability and cost-effectiveness of saline agriculture. Acknowledgments: This work was supported by project Rhizomis PTDC/BIA-MIC/29736/2017 financed by Fundação para a Ciência e Tecnologia (FCT) through the Regional Operational Program of the Center (02/SAICT/2017) with FEDER funds (European Regional Development Fund, FNR, and OE) and by FCT through CESAM (UIDP/50017/2020 + UIDB/50017/2020), LAQV-REQUIMTE (UIDB/50006/2020). We also acknowledge FCT/FSE for the financial support to Maria João Ferreira through a PhD grant (PD/BD/150363/2019). We are grateful to Horta dos Peixinhos for their help and support during sampling and seed collection. We also thank Glória Pinto for her collaboration providing us the use of the growth chambers during the final months of the experiment and Enrique Mateos-Naranjo and Jennifer Mesa-Marín of the Departamento de Biología Vegetal y Ecología, the University of Sevilla for their advice regarding the growth of salicornia plants in greenhouse conditions.

Keywords: halophytes, PGPB, rhizosphere engineering, biofertilizers, primary metabolite profiling, plant inoculation, Salicornia ramosissima

Procedia PDF Downloads 125