Laura Natalia Gonzalez-García, Daniela Lozano-Arce, Juan Pablo Londoño, Romain Guyot, Jorge Duitama
Premise
Transposable elements (TEs) make up more than half of the genomes of complex plant species and can modulate the expression of neighboring genes, producing significant variability of agronomically relevant traits. The availability of long-read sequencing technologies allows the building of genome assemblies for plant species with large and complex genomes. Unfortunately, TE annotation currently represents a bottleneck in the annotation of genome assemblies.
Methods and Results
We present a new functionality of the Next-Generation Sequencing Experience Platform (NGSEP) to perform efficient homology-based TE annotation. Sequences in a reference library are treated as long reads and mapped to an input genome assembly. A hierarchical annotation is then assigned by homology using the annotation of the reference library. We tested the performance of our algorithm on genome assemblies of different plant species, including Arabidopsis thaliana, Oryza sativa, Coffea humblotiana, and Triticum aestivum (bread wheat). Our algorithm outperforms traditional homology-based annotation tools in speed by a factor of three to >20, reducing the annotation time of the T. aestivum genome from months to hours, and recovering up to 80% of TEs annotated with RepeatMasker with a precision of up to 0.95.
Conclusions
NGSEP allows rapid analysis of TEs, especially in very large and TE-rich plant genomes.
{"title":"Efficient homology-based annotation of transposable elements using minimizers","authors":"Laura Natalia Gonzalez-García, Daniela Lozano-Arce, Juan Pablo Londoño, Romain Guyot, Jorge Duitama","doi":"10.1002/aps3.11520","DOIUrl":"10.1002/aps3.11520","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Transposable elements (TEs) make up more than half of the genomes of complex plant species and can modulate the expression of neighboring genes, producing significant variability of agronomically relevant traits. The availability of long-read sequencing technologies allows the building of genome assemblies for plant species with large and complex genomes. Unfortunately, TE annotation currently represents a bottleneck in the annotation of genome assemblies.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>We present a new functionality of the Next-Generation Sequencing Experience Platform (NGSEP) to perform efficient homology-based TE annotation. Sequences in a reference library are treated as long reads and mapped to an input genome assembly. A hierarchical annotation is then assigned by homology using the annotation of the reference library. We tested the performance of our algorithm on genome assemblies of different plant species, including <i>Arabidopsis thaliana</i>, <i>Oryza sativa, Coffea humblotiana</i>, and <i>Triticum aestivum</i> (bread wheat). Our algorithm outperforms traditional homology-based annotation tools in speed by a factor of three to >20, reducing the annotation time of the <i>T. aestivum</i> genome from months to hours, and recovering up to 80% of TEs annotated with RepeatMasker with a precision of up to 0.95.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>NGSEP allows rapid analysis of TEs, especially in very large and TE-rich plant genomes.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 4","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11520","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10052135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The isolation of RNA from trees is challenging due to the interference of polyphenols and polysaccharides with downstream processes. Furthermore, many RNA extraction protocols are time consuming and involve hazardous chemicals. To address these issues, we aimed to develop a safe protocol for high-quality RNA extraction from diverse Metrosideros taxa representing a broad range of leaf toughness, pubescence, and secondary metabolites.
Methods and Results
We tested popular RNA isolation kits and protocols that were effective on other recalcitrant trees, including a broad range of optimization and purification steps. We optimized a protocol involving two silica-membrane column-based kits that yielded high-quantity RNA with an RNA integrity number >7 and without DNA contamination. All RNA samples were used successfully in a follow-on RNA-Seq experiment.
Conclusions
We present an optimized high-throughput RNA extraction protocol that yielded high-quality and high-quantity RNA from three contrasting leaf phenotypes within a hyperdiverse woody species complex.
{"title":"An optimized RNA extraction method for diverse leaves of Hawaiian Metrosideros, a hypervariable tree species complex","authors":"Maryam Hadi, Elizabeth A. Stacy","doi":"10.1002/aps3.11518","DOIUrl":"https://doi.org/10.1002/aps3.11518","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>The isolation of RNA from trees is challenging due to the interference of polyphenols and polysaccharides with downstream processes. Furthermore, many RNA extraction protocols are time consuming and involve hazardous chemicals. To address these issues, we aimed to develop a safe protocol for high-quality RNA extraction from diverse <i>Metrosideros</i> taxa representing a broad range of leaf toughness, pubescence, and secondary metabolites.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>We tested popular RNA isolation kits and protocols that were effective on other recalcitrant trees, including a broad range of optimization and purification steps. We optimized a protocol involving two silica-membrane column-based kits that yielded high-quantity RNA with an RNA integrity number >7 and without DNA contamination. All RNA samples were used successfully in a follow-on RNA-Seq experiment.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>We present an optimized high-throughput RNA extraction protocol that yielded high-quality and high-quantity RNA from three contrasting leaf phenotypes within a hyperdiverse woody species complex.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11518","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50154786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Polyploidy has become a central factor in plant evolutionary biological research in recent decades. Methods such as flow cytometry have revealed the widespread occurrence of polyploidy; however, its inference relies on expensive lab equipment and is largely restricted to fresh or recently dried material.
Methods
Here, we assess the applicability of infrared spectroscopy to infer ploidy in two related species of Veronica (Plantaginaceae). Infrared spectroscopy relies on differences in the absorbance of tissues, which could be affected by primary and secondary metabolites related to polyploidy. We sampled 33 living plants from the greenhouse and 74 herbarium specimens with ploidy known through flow cytometrical measurements and analyzed the resulting spectra using discriminant analysis of principal components (DAPC) and neural network (NNET) classifiers.
Results
Living material of both species combined was classified with 70% (DAPC) to 75% (NNET) accuracy, whereas herbarium material was classified with 84% (DAPC) to 85% (NNET) accuracy. Analyzing both species separately resulted in less clear results.
Discussion
Infrared spectroscopy is quite reliable but is not a certain method for assessing intraspecific ploidy level differences in two species of Veronica. More accurate inferences rely on large training data sets and herbarium material. This study demonstrates an important way to expand the field of polyploid research to herbaria.
{"title":"Infrared spectroscopy for ploidy estimation: An example in two species of Veronica using fresh and herbarium specimens","authors":"Daniele Buono, Dirk C. Albach","doi":"10.1002/aps3.11516","DOIUrl":"https://doi.org/10.1002/aps3.11516","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Polyploidy has become a central factor in plant evolutionary biological research in recent decades. Methods such as flow cytometry have revealed the widespread occurrence of polyploidy; however, its inference relies on expensive lab equipment and is largely restricted to fresh or recently dried material.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Here, we assess the applicability of infrared spectroscopy to infer ploidy in two related species of <i>Veronica</i> (Plantaginaceae). Infrared spectroscopy relies on differences in the absorbance of tissues, which could be affected by primary and secondary metabolites related to polyploidy. We sampled 33 living plants from the greenhouse and 74 herbarium specimens with ploidy known through flow cytometrical measurements and analyzed the resulting spectra using discriminant analysis of principal components (DAPC) and neural network (NNET) classifiers.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Living material of both species combined was classified with 70% (DAPC) to 75% (NNET) accuracy, whereas herbarium material was classified with 84% (DAPC) to 85% (NNET) accuracy. Analyzing both species separately resulted in less clear results.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>Infrared spectroscopy is quite reliable but is not a certain method for assessing intraspecific ploidy level differences in two species of <i>Veronica</i>. More accurate inferences rely on large training data sets and herbarium material. This study demonstrates an important way to expand the field of polyploid research to herbaria.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 2","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11516","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50118844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Charlie P. Bailey, Carolyn A. Sonter, Jeremy L. Jones, Sabu Pandey, Simon Haberle, Karen C. B. S. Santos, Maria L. Absy, Romina Rader
Premise
Pollen collected by honey bees from different plant species often differs in color, and this has been used as a basis for plant identification. The objective of this study was to develop a new, low-cost protocol to sort pollen pellets by color using high-energy violet light and visible light to determine whether pollen pellet color is associated with variations in plant species identity.
Methods and Results
We identified 35 distinct colors and found that 52% of pollen subsamples (n = 200) were dominated by a single taxon. Among these near-pure pellets, only one color consistently represented a single pollen taxon (Asteraceae: Cichorioideae). Across the spectrum of colors spanning yellows, oranges, and browns, similarly colored pollen pellets contained pollen from multiple plant families ranging from two to 13 families per color.
Conclusions
Sorting pollen pellets illuminated under high-energy violet light lit from four directions within a custom-made light box aided in distinguishing pellet composition, especially in pellets within the same color.
{"title":"Does sorting by color using visible and high-energy violet light improve classification of taxa in honey bee pollen pellets?","authors":"Charlie P. Bailey, Carolyn A. Sonter, Jeremy L. Jones, Sabu Pandey, Simon Haberle, Karen C. B. S. Santos, Maria L. Absy, Romina Rader","doi":"10.1002/aps3.11514","DOIUrl":"10.1002/aps3.11514","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Pollen collected by honey bees from different plant species often differs in color, and this has been used as a basis for plant identification. The objective of this study was to develop a new, low-cost protocol to sort pollen pellets by color using high-energy violet light and visible light to determine whether pollen pellet color is associated with variations in plant species identity.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>We identified 35 distinct colors and found that 52% of pollen subsamples (<i>n</i> = 200) were dominated by a single taxon. Among these near-pure pellets, only one color consistently represented a single pollen taxon (Asteraceae: Cichorioideae). Across the spectrum of colors spanning yellows, oranges, and browns, similarly colored pollen pellets contained pollen from multiple plant families ranging from two to 13 families per color.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Sorting pollen pellets illuminated under high-energy violet light lit from four directions within a custom-made light box aided in distinguishing pellet composition, especially in pellets within the same color.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 2","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e5/98/APS3-11-e11514.PMC10083439.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9359451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Christian S. Bowman, Ryan Traband, Xuesong Wang, Sara P. Knowles, Sassoum Lo, Zhenyu Jia, Nicholi Vorsa, Ira A. Herniter
Premise
The measurement of leaf morphometric parameters from digital images can be time-consuming or restrictive when using digital image analysis softwares. The Multiple Leaf Sample Extraction System (MuLES) is a new tool that enables high-throughput leaf shape analysis with minimal user input or prerequisites, such as coding knowledge or image modification.
Methods and Results
MuLES uses contrasting pixel color values to distinguish between leaf objects and their background area, eliminating the need for color threshold–based methods or color correction cards typically required in other software methods. The leaf morphometric parameters measured by this software, especially leaf aspect ratio, were able to distinguish between large populations of different accessions for the same species in a high-throughput manner.
Conclusions
MuLES provides a simple method for the rapid measurement of leaf morphometric parameters in large plant populations from digital images and demonstrates the ability of leaf aspect ratio to distinguish between closely related plant types.
{"title":"Multiple Leaf Sample Extraction System (MuLES): A tool to improve automated morphometric leaf studies","authors":"Christian S. Bowman, Ryan Traband, Xuesong Wang, Sara P. Knowles, Sassoum Lo, Zhenyu Jia, Nicholi Vorsa, Ira A. Herniter","doi":"10.1002/aps3.11513","DOIUrl":"10.1002/aps3.11513","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>The measurement of leaf morphometric parameters from digital images can be time-consuming or restrictive when using digital image analysis softwares. The Multiple Leaf Sample Extraction System (MuLES) is a new tool that enables high-throughput leaf shape analysis with minimal user input or prerequisites, such as coding knowledge or image modification.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>MuLES uses contrasting pixel color values to distinguish between leaf objects and their background area, eliminating the need for color threshold–based methods or color correction cards typically required in other software methods. The leaf morphometric parameters measured by this software, especially leaf aspect ratio, were able to distinguish between large populations of different accessions for the same species in a high-throughput manner.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>MuLES provides a simple method for the rapid measurement of leaf morphometric parameters in large plant populations from digital images and demonstrates the ability of leaf aspect ratio to distinguish between closely related plant types.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 2","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/8d/19/APS3-11-e11513.PMC10083438.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9359453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Denise L. Lindsay, Xin Guan, Nathan E. Harms, James T. Cronin, Laura A. Meyerson, Richard F. Lance
Premise
To genetically discriminate subspecies of the common reed (Phragmites australis), we developed real-time quantitative (qPCR) assays for identifying P. australis subsp. americanus, P. australis subsp. australis, and P. australis subsp. berlandieri.
Methods and Results
Utilizing study-generated chloroplast DNA sequences, we developed three novel qPCR assays. Assays were verified on individuals of each subspecies and against two non-target species, Arundo donax and Phalaris arundinacea. One assay amplifies only P. australis subsp. americanus, one amplifies P. australis subsp. australis and/or P. australis subsp. berlandieri, and one amplifies P. australis subsp. americanus and/or P. australis subsp. australis. This protocol enhances currently available rapid identification methods by providing genetic discrimination of all three subspecies.
Conclusions
The newly developed assays were validated using P. australis samples from across the United States. Application of these assays outside of this geographic range should be preceded by additional testing.
{"title":"DNA assays for genetic discrimination of three Phragmites australis subspecies in the United States","authors":"Denise L. Lindsay, Xin Guan, Nathan E. Harms, James T. Cronin, Laura A. Meyerson, Richard F. Lance","doi":"10.1002/aps3.11512","DOIUrl":"https://doi.org/10.1002/aps3.11512","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>To genetically discriminate subspecies of the common reed (<i>Phragmites australis</i>), we developed real-time quantitative (qPCR) assays for identifying <i>P. australis</i> subsp. <i>americanus</i>, <i>P. australis</i> subsp. <i>australis</i>, and <i>P. australis</i> subsp. <i>berlandieri</i>.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>Utilizing study-generated chloroplast DNA sequences, we developed three novel qPCR assays. Assays were verified on individuals of each subspecies and against two non-target species, <i>Arundo donax</i> and <i>Phalaris arundinacea</i>. One assay amplifies only <i>P. australis</i> subsp. <i>americanus</i>, one amplifies <i>P. australis</i> subsp. <i>australis</i> and/or <i>P. australis</i> subsp. <i>berlandieri</i>, and one amplifies <i>P. australis</i> subsp. <i>americanus</i> and/or <i>P. australis</i> subsp. <i>australis</i>. This protocol enhances currently available rapid identification methods by providing genetic discrimination of all three subspecies.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The newly developed assays were validated using <i>P. australis</i> samples from across the United States. Application of these assays outside of this geographic range should be preceded by additional testing.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 2","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11512","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50140455","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Peggy Martinez, Marcelo Serpe, Rachael Barron, Sven Buerki
Premise
Determining the tolerance of plant populations to climate change requires the development of biotechnological protocols producing genetically identical individuals used for genotype-by-environment experiments. Such protocols are missing for slow-growth, woody plants; to address this gap, this study uses Artemisia tridentata, a western North American keystone shrub, as model.
Methods and Results
The production of individual lines is a two-step process: in vitro propagation under aseptic conditions followed by ex vitro acclimation and hardening. Due to aseptic growth conditions, in vitro plantlets exhibit maladapted phenotypes, and this protocol focuses on presenting an approach promoting morphogenesis for slow-growth, woody species. Survival was used as the main criterion determining successful acclimation and hardening. Phenotypic changes were confirmed by inspecting leaf anatomy, and shoot water potential was used to ensure that plantlets were not water stressed.
Conclusions
Although our protocol has lower survival rates (11–41%) compared to protocols developed for herbaceous, fast-growing species, it provides a benchmark for slow-growth, woody species occurring in dry ecosystems.
{"title":"Acclimation and hardening of a slow-growing woody species emblematic to western North America from in vitro plantlets","authors":"Peggy Martinez, Marcelo Serpe, Rachael Barron, Sven Buerki","doi":"10.1002/aps3.11515","DOIUrl":"10.1002/aps3.11515","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Determining the tolerance of plant populations to climate change requires the development of biotechnological protocols producing genetically identical individuals used for genotype-by-environment experiments. Such protocols are missing for slow-growth, woody plants; to address this gap, this study uses <i>Artemisia tridentata</i>, a western North American keystone shrub, as model.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>The production of individual lines is a two-step process: in vitro propagation under aseptic conditions followed by ex vitro acclimation and hardening. Due to aseptic growth conditions, in vitro plantlets exhibit maladapted phenotypes, and this protocol focuses on presenting an approach promoting morphogenesis for slow-growth, woody species. Survival was used as the main criterion determining successful acclimation and hardening. Phenotypic changes were confirmed by inspecting leaf anatomy, and shoot water potential was used to ensure that plantlets were not water stressed.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Although our protocol has lower survival rates (11–41%) compared to protocols developed for herbaceous, fast-growing species, it provides a benchmark for slow-growth, woody species occurring in dry ecosystems.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 2","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a6/24/APS3-11-e11515.PMC10083460.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9359456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
C. Gary Olds, Jessie W. Berta-Thompson, Justin J. Loucks, Richard A. Levy, Andrew W. Wilson
Premise
Fungaria are an underutilized resource for understanding fungal biodiversity. The effort and cost of producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor-efficient and cost-effective solution for sequencing the fungal DNA barcodes of hundreds of specimens at once.
Methods
We applied a two-step PCR approach using nested, barcoded primers to sequence the fungal nrITS2 region of 766 macrofungal specimens using the Illumina platform. The specimens represent a broad taxonomic sampling of the Dikarya. Of these, 382 Lactarius specimens were analyzed to identify molecular operational taxonomic units (MOTUs) using a phylogenetic approach. The raw sequences were trimmed, filtered, assessed, and analyzed using the DADA2 amplicon de-noising toolkit and Biopython. The sequences were compared to the NCBI and UNITE databases and Sanger nrITS sequences from the same specimens.
Results
The taxonomic identities derived from the nrITS2 sequence data were >90% accurate across all specimens sampled. A phylogenetic analysis of the Lactarius sequences identified 20 MOTUs.
Discussion
The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections to advance fungal diversity identification and documentation.
{"title":"Applying a modified metabarcoding approach for the sequencing of macrofungal specimens from fungarium collections","authors":"C. Gary Olds, Jessie W. Berta-Thompson, Justin J. Loucks, Richard A. Levy, Andrew W. Wilson","doi":"10.1002/aps3.11508","DOIUrl":"10.1002/aps3.11508","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Fungaria are an underutilized resource for understanding fungal biodiversity. The effort and cost of producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor-efficient and cost-effective solution for sequencing the fungal DNA barcodes of hundreds of specimens at once.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>We applied a two-step PCR approach using nested, barcoded primers to sequence the fungal nrITS2 region of 766 macrofungal specimens using the Illumina platform. The specimens represent a broad taxonomic sampling of the Dikarya. Of these, 382 <i>Lactarius</i> specimens were analyzed to identify molecular operational taxonomic units (MOTUs) using a phylogenetic approach. The raw sequences were trimmed, filtered, assessed, and analyzed using the DADA2 amplicon de-noising toolkit and Biopython. The sequences were compared to the NCBI and UNITE databases and Sanger nrITS sequences from the same specimens.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The taxonomic identities derived from the nrITS2 sequence data were >90% accurate across all specimens sampled. A phylogenetic analysis of the <i>Lactarius</i> sequences identified 20 MOTUs.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections to advance fungal diversity identification and documentation.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11508","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10768797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alexia Stettinius, Hal Holmes, Qian Zhang, Isabelle Mehochko, Misa Winters, Ruby Hutchison, Adam Maxwell, Jason Holliday, Eli Vlaisavljevich
Premise
Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis.
Methods
FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana).
Results
We observed that FUSE can extract high quantities of DNA in 9–15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next-generation sequencing.
Discussion
These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field-deployable systems to increase the cataloging of genetic biodiversity, particularly in low-resource biodiversity hotspots.
{"title":"DNA release from plant tissue using focused ultrasound extraction (FUSE)","authors":"Alexia Stettinius, Hal Holmes, Qian Zhang, Isabelle Mehochko, Misa Winters, Ruby Hutchison, Adam Maxwell, Jason Holliday, Eli Vlaisavljevich","doi":"10.1002/aps3.11510","DOIUrl":"10.1002/aps3.11510","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (<i>Castanea dentata</i>), tulip poplar (<i>Liriodendron tulipifera</i>), red maple (<i>Acer rubrum</i>), and chestnut oak (<i>Quercus montana</i>).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>We observed that FUSE can extract high quantities of DNA in 9–15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next-generation sequencing.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field-deployable systems to increase the cataloging of genetic biodiversity, particularly in low-resource biodiversity hotspots.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/df/34/APS3-11-e11510.PMC9934592.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10768800","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号