Efficient protocols for extracting high-molecular-weight (HMW) DNA from ferns facilitate the long-read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)-based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time.
Methods and Results
We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8).
Conclusions
This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.
{"title":"Modified CTAB protocols for high-molecular-weight DNA extractions from ferns","authors":"Pei-Jun Xie, Ya-Ting Ke, Li-Yaung Kuo","doi":"10.1002/aps3.11526","DOIUrl":"10.1002/aps3.11526","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Efficient protocols for extracting high-molecular-weight (HMW) DNA from ferns facilitate the long-read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)-based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A<sub>260</sub>/A<sub>230</sub> and A<sub>260</sub>/A<sub>280</sub> > 1.8).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/32/c2/APS3-11-e11526.PMC10278929.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9709978","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}
Maia M. Jones, Nathalie S. Nagalingum, Vanessa M. Handley
Premise
Plants with stiff, leathery leaves pose a challenge for standard DNA extraction protocols. These tissues are recalcitrant to mechanical disruption via TissueLyser (or analogous devices) and are often high in secondary metabolites. These compounding factors result in low yields, which may be sufficient for PCR amplification but are generally inadequate for genomic applications that require large quantities of high-quality DNA. Cycads in the genus Encephalartos exemplify these challenges, as this group of plants is fortified for life in harsh, dry habitats with notoriously thick and rigid leaves.
Methods and Results
Using a DNA extraction kit, we tested three methods of mechanical disruption and examined the differences between stored vs. freshly collected samples and mature vs. senescing leaflets. We found that the manual method of pulverizing tissue yields the highest concentrations of DNA, and that both senescing leaflets and leaflet tissue that has been stored for extended periods yield sufficient DNA for genomic analyses.
Conclusions
These findings shed light on the feasibility of using senescing leaves and/or tissue that has been stored on silica for long periods of time when attempting to extract large amounts of DNA. We provide here an optimized DNA extraction protocol that can be applied to cycads and other plant groups with tough or rigid leaves.
{"title":"Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos","authors":"Maia M. Jones, Nathalie S. Nagalingum, Vanessa M. Handley","doi":"10.1002/aps3.11525","DOIUrl":"https://doi.org/10.1002/aps3.11525","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Plants with stiff, leathery leaves pose a challenge for standard DNA extraction protocols. These tissues are recalcitrant to mechanical disruption via TissueLyser (or analogous devices) and are often high in secondary metabolites. These compounding factors result in low yields, which may be sufficient for PCR amplification but are generally inadequate for genomic applications that require large quantities of high-quality DNA. Cycads in the genus <i>Encephalartos</i> exemplify these challenges, as this group of plants is fortified for life in harsh, dry habitats with notoriously thick and rigid leaves.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>Using a DNA extraction kit, we tested three methods of mechanical disruption and examined the differences between stored vs. freshly collected samples and mature vs. senescing leaflets. We found that the manual method of pulverizing tissue yields the highest concentrations of DNA, and that both senescing leaflets and leaflet tissue that has been stored for extended periods yield sufficient DNA for genomic analyses.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>These findings shed light on the feasibility of using senescing leaves and/or tissue that has been stored on silica for long periods of time when attempting to extract large amounts of DNA. We provide here an optimized DNA extraction protocol that can be applied to cycads and other plant groups with tough or rigid leaves.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11525","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50150733","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}
Myoungbo Kang, Andre Chanderbali, Seungyeon Lee, Douglas E. Soltis, Pamela S. Soltis, Sangtae Kim
Premise
Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third-generation sequencing. To efficiently use technologies capable of generating long-read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples.
Methods and Results
We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively.
Conclusions
This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research.
{"title":"High-molecular-weight DNA extraction for long-read sequencing of plant genomes: An optimization of standard methods","authors":"Myoungbo Kang, Andre Chanderbali, Seungyeon Lee, Douglas E. Soltis, Pamela S. Soltis, Sangtae Kim","doi":"10.1002/aps3.11528","DOIUrl":"10.1002/aps3.11528","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third-generation sequencing. To efficiently use technologies capable of generating long-read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11528","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9764041","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}
Thais Guillen-Otero, Soon-Jae Lee, Cheng-Wei Chen, Peter Szoevenyi, Michael Kessler
Premise
Detailed studies of the fungi associated with lycophytes and ferns provide crucial insights into the early evolution of land plants. However, most investigations to date have assessed fern–fungus interactions based only on visual root inspection. In the present research, we establish and evaluate a metabarcoding protocol to analyze the fungal communities associated with fern and lycophyte roots.
Methods
We used two primer pairs focused on the ITS rRNA region to screen the general fungal communities, and the 18S rRNA to target Glomeromycota fungi (i.e., arbuscular mycorrhizal fungi). To test these approaches, we collected and processed roots from 12 phylogenetically distant fern and lycophyte species.
Results
We found marked compositional differences between the ITS and 18S data sets. While the ITS data set demonstrated the dominance of orders Glomerales (phylum Glomeromycota), Pleosporales, and Helotiales (both in phylum Ascomycota), the 18S data set revealed the greatest diversity of Glomeromycota. Non-metric multidimensional scaling (NMDS) ordination suggested an important geographical effect in sample similarities.
Discussion
The ITS-based approach is a reliable and effective method to analyze the fungal communities associated with fern and lycophyte roots. The 18S approach is more appropriate for studies focused on the detailed screening of arbuscular mycorrhizal fungi.
{"title":"A metabarcoding protocol targeting two DNA regions to analyze root-associated fungal communities in ferns and lycophytes","authors":"Thais Guillen-Otero, Soon-Jae Lee, Cheng-Wei Chen, Peter Szoevenyi, Michael Kessler","doi":"10.1002/aps3.11523","DOIUrl":"10.1002/aps3.11523","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>Detailed studies of the fungi associated with lycophytes and ferns provide crucial insights into the early evolution of land plants. However, most investigations to date have assessed fern–fungus interactions based only on visual root inspection. In the present research, we establish and evaluate a metabarcoding protocol to analyze the fungal communities associated with fern and lycophyte roots.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>We used two primer pairs focused on the ITS rRNA region to screen the general fungal communities, and the 18S rRNA to target Glomeromycota fungi (i.e., arbuscular mycorrhizal fungi). To test these approaches, we collected and processed roots from 12 phylogenetically distant fern and lycophyte species.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>We found marked compositional differences between the ITS and 18S data sets. While the ITS data set demonstrated the dominance of orders Glomerales (phylum Glomeromycota), Pleosporales, and Helotiales (both in phylum Ascomycota), the 18S data set revealed the greatest diversity of Glomeromycota. Non-metric multidimensional scaling (NMDS) ordination suggested an important geographical effect in sample similarities.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>The ITS-based approach is a reliable and effective method to analyze the fungal communities associated with fern and lycophyte roots. The 18S approach is more appropriate for studies focused on the detailed screening of arbuscular mycorrhizal fungi.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/53/3c/APS3-11-e11523.PMC10278937.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9712585","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}
Gisel Y. De La Cerda, Jacob B. Landis, Evan Eifler, Adriana I. Hernandez, Fay-Wei Li, Jing Zhang, Carrie M. Tribble, Nisa Karimi, Patricia Chan, Thomas Givnish, Susan R. Strickler, Chelsea D. Specht
Premise
We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results.
Methods
Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA.
Results
Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced.
Discussion
Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.
{"title":"Balancing read length and sequencing depth: Optimizing Nanopore long-read sequencing for monocots with an emphasis on the Liliales","authors":"Gisel Y. De La Cerda, Jacob B. Landis, Evan Eifler, Adriana I. Hernandez, Fay-Wei Li, Jing Zhang, Carrie M. Tribble, Nisa Karimi, Patricia Chan, Thomas Givnish, Susan R. Strickler, Chelsea D. Specht","doi":"10.1002/aps3.11524","DOIUrl":"10.1002/aps3.11524","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Four species of <i>Calochortus</i> (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11524","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9712582","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}
Edward V. McAssey, Cassidy Downs, Mitsuko Yorkston, Clifford Morden, Karolina Heyduk
Premise
The use of DNA from herbarium specimens is an increasingly important source for evolutionary studies in plant biology, particularly in cases where species are rare or difficult to obtain. Here we compare the utility of DNA from herbarium tissues to their freezer-stored DNA counterparts via the Hawaiian Plant DNA Library.
Methods
Plants collected for the Hawaiian Plant DNA Library were simultaneously accessioned as herbarium specimens at the time of collection, from 1994–2019. Paired samples were sequenced using short-read sequencing and assessed for chloroplast assembly and nuclear gene recovery.
Results
Herbarium specimen–derived DNA was statistically more fragmented than freezer-stored DNA derived from fresh tissue, leading to poorer chloroplast assembly and overall lower coverage. The number of nuclear targets recovered varied mostly by total sequencing reads per library and age of specimen, but not by storage method (herbarium or long-term freezer). Although there was evidence of DNA damage in the samples, there was no evidence that it was related to the length of time in storage, whether frozen or as herbarium specimens.
Discussion
DNA extracted from herbarium tissues will continue to be invaluable, despite being highly fragmented and degraded. Rare floras would benefit from both traditional herbarium storage methods and extracted DNA freezer banks.
{"title":"A comparison of freezer-stored DNA and herbarium tissue samples for chloroplast assembly and genome skimming","authors":"Edward V. McAssey, Cassidy Downs, Mitsuko Yorkston, Clifford Morden, Karolina Heyduk","doi":"10.1002/aps3.11527","DOIUrl":"10.1002/aps3.11527","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>The use of DNA from herbarium specimens is an increasingly important source for evolutionary studies in plant biology, particularly in cases where species are rare or difficult to obtain. Here we compare the utility of DNA from herbarium tissues to their freezer-stored DNA counterparts via the Hawaiian Plant DNA Library.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Plants collected for the Hawaiian Plant DNA Library were simultaneously accessioned as herbarium specimens at the time of collection, from 1994–2019. Paired samples were sequenced using short-read sequencing and assessed for chloroplast assembly and nuclear gene recovery.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Herbarium specimen–derived DNA was statistically more fragmented than freezer-stored DNA derived from fresh tissue, leading to poorer chloroplast assembly and overall lower coverage. The number of nuclear targets recovered varied mostly by total sequencing reads per library and age of specimen, but not by storage method (herbarium or long-term freezer). Although there was evidence of DNA damage in the samples, there was no evidence that it was related to the length of time in storage, whether frozen or as herbarium specimens.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>DNA extracted from herbarium tissues will continue to be invaluable, despite being highly fragmented and degraded. Rare floras would benefit from both traditional herbarium storage methods and extracted DNA freezer banks.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11527","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10032366","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}
John J. Schenk, L. Ellie Becklund, S. James Carey, Paige P. Fabre
Abstract Cetyltrimethylammonium bromide (CTAB)–based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among plant species has necessitated optimization. Research articles often cite a “modified” CTAB protocol without explicitly stating how the protocol had been altered, creating non‐reproducible studies. Furthermore, the various modifications that have been applied to the CTAB protocol have not been rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed the literature for modified CTAB protocols used for the isolation of plant DNA. We found that every stage of the CTAB protocol has been modified, and we summarized those modifications to provide recommendations for extraction optimization. Future genomic studies will rely on optimized CTAB protocols. Our review of the modifications that have been used, as well as the protocols we provide here, could better standardize DNA extractions, allowing for repeatable and transparent studies.
{"title":"What is the “modified” CTAB protocol? Characterizing modifications to the CTAB DNA extraction protocol","authors":"John J. Schenk, L. Ellie Becklund, S. James Carey, Paige P. Fabre","doi":"10.1002/aps3.11517","DOIUrl":"10.1002/aps3.11517","url":null,"abstract":"Abstract Cetyltrimethylammonium bromide (CTAB)–based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among plant species has necessitated optimization. Research articles often cite a “modified” CTAB protocol without explicitly stating how the protocol had been altered, creating non‐reproducible studies. Furthermore, the various modifications that have been applied to the CTAB protocol have not been rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed the literature for modified CTAB protocols used for the isolation of plant DNA. We found that every stage of the CTAB protocol has been modified, and we summarized those modifications to provide recommendations for extraction optimization. Future genomic studies will rely on optimized CTAB protocols. Our review of the modifications that have been used, as well as the protocols we provide here, could better standardize DNA extractions, allowing for repeatable and transparent studies.","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11517","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9709974","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}
Gabriel Johnson, Steven W. J. Canty, Isaac H. Lichter-Marck, Warren Wagner, Jun Wen
Premise
The preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high-quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples.
Methods
DNA was isolated from leaves preserved with 96% ethanol or from silica-desiccated leaf samples and herbarium fragments that were pretreated with ethanol. DNA was extracted from herbarium tissues using a special ethanol pretreatment protocol, and these extracts were compared with those obtained using the standard cetyltrimethylammonium bromide (CTAB) method.
Results
DNA extracted from tissue preserved in, or pretreated with, ethanol was less fragmented than DNA from tissues without pretreatment. Adding proteinase digestion to the lysis step increased the amount of DNA obtained from the ethanol-pretreated tissues. The combination of the ethanol pretreatment with liquid nitrogen freezing and a sorbitol wash prior to cell lysis greatly improved the quality and yield of DNA from the herbarium tissue samples.
Discussion
This study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies.
{"title":"Ethanol preservation and pretreatments facilitate quality DNA extractions in recalcitrant plant species","authors":"Gabriel Johnson, Steven W. J. Canty, Isaac H. Lichter-Marck, Warren Wagner, Jun Wen","doi":"10.1002/aps3.11519","DOIUrl":"10.1002/aps3.11519","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>The preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high-quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>DNA was isolated from leaves preserved with 96% ethanol or from silica-desiccated leaf samples and herbarium fragments that were pretreated with ethanol. DNA was extracted from herbarium tissues using a special ethanol pretreatment protocol, and these extracts were compared with those obtained using the standard cetyltrimethylammonium bromide (CTAB) method.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>DNA extracted from tissue preserved in, or pretreated with, ethanol was less fragmented than DNA from tissues without pretreatment. Adding proteinase digestion to the lysis step increased the amount of DNA obtained from the ethanol-pretreated tissues. The combination of the ethanol pretreatment with liquid nitrogen freezing and a sorbitol wash prior to cell lysis greatly improved the quality and yield of DNA from the herbarium tissue samples.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Discussion</h3>\u0000 \u0000 <p>This study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://bsapubs.onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11519","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9709975","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}
S. James Carey, L. Ellie Becklund, Paige P. Fabre, John J. Schenk
Premise
The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality.
Methods and Results
We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel–preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium-preserved tissues.
Conclusions
We recommend DNA extractions of silica gel–preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time.
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Fred E. Gouker, Yonghong Guo, Harlan T. Svoboda, Margaret R. Pooler
Premise
The objective of this study was to optimize an existing DNA extraction protocol for recalcitrant plant taxa to obtain high-quality DNA from preserved herbarium tissue suitable for downstream PCR applications.
Methods and Results
Leaf tissue from 30 diverse plant species was obtained from the U.S. National Arboretum Herbarium. Our previous DNA extraction protocol (Gouker et al., 2020, Applications in Plant Sciences 8: e11403) was improved by use of 10X Tris-EDTA buffer, addition of polyvinylpolypyrrolidone, and omission of sample heating during homogenization, and resulted in total DNA yields ranging from 60–2460 ng. Optimized PCR amplification using universal plant primers for the ITS-p3/u4 region and the P6 loop of the trnL (UAA) chloroplast intron was successful for most specimens.
Conclusions
This protocol, which is simple, fast, and uses standard laboratory-grade chemicals, yields DNA from herbarium specimens that is comparable in quality to that from commercially available kits, and is of sufficient quality and quantity for other applications.
本研究的目的是优化现有的顽固植物类群DNA提取方案，以从保存的植物标本组织中获得适合下游PCR应用的高质量DNA。方法与结果从美国国家植物标本室获得30种不同植物的叶片组织。我们之前的DNA提取方案(Gouker et al.， 2020, Applications in Plant Sciences 8: e11403)通过使用10X Tris-EDTA缓冲液，添加聚乙烯聚吡咯烷酮，以及在均质化过程中省略样品加热来改进，并导致总DNA产量在60-2460 ng之间。使用通用植物引物优化的PCR扩增对trnL (UAA)叶绿体内含子的ts -p3/u4区和P6环在大多数标本中都是成功的。该方法简单、快速，使用标准的实验室级化学品，从植物标本馆标本中获得的DNA质量与市售试剂盒相当，并且具有足够的质量和数量用于其他应用。
{"title":"Optimizing efficient PCR-amplifiable DNA extraction from herbarium specimens","authors":"Fred E. Gouker, Yonghong Guo, Harlan T. Svoboda, Margaret R. Pooler","doi":"10.1002/aps3.11521","DOIUrl":"10.1002/aps3.11521","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Premise</h3>\u0000 \u0000 <p>The objective of this study was to optimize an existing DNA extraction protocol for recalcitrant plant taxa to obtain high-quality DNA from preserved herbarium tissue suitable for downstream PCR applications.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods and Results</h3>\u0000 \u0000 <p>Leaf tissue from 30 diverse plant species was obtained from the U.S. National Arboretum Herbarium. Our previous DNA extraction protocol (Gouker et al., 2020, <i>Applications in Plant Sciences</i> 8: e11403) was improved by use of 10X Tris-EDTA buffer, addition of polyvinylpolypyrrolidone, and omission of sample heating during homogenization, and resulted in total DNA yields ranging from 60–2460 ng. Optimized PCR amplification using universal plant primers for the ITS-p3/u4 region and the P6 loop of the <i>trnL</i> (UAA) chloroplast intron was successful for most specimens.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>This protocol, which is simple, fast, and uses standard laboratory-grade chemicals, yields DNA from herbarium specimens that is comparable in quality to that from commercially available kits, and is of sufficient quality and quantity for other applications.</p>\u0000 </section>\u0000 </div>","PeriodicalId":8022,"journal":{"name":"Applications in Plant Sciences","volume":"11 3","pages":""},"PeriodicalIF":3.6,"publicationDate":"2023-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/aps3.11521","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44694339","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}
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