The combined impact of these factors produces low yields, potentially satisfactory for PCR amplification, but typically insufficient for genomic applications that necessitate large quantities of high-quality DNA. The genus Cycads comprises
Exemplify these predicaments, as this grouping of vegetation is prepared for life in severe, arid landscapes, possessing unusually thick and rigid foliage.
Through the application of a DNA extraction kit, we studied three techniques of mechanical disintegration, examining the variations between stored and directly harvested samples, and mature and aging leaflets. Our findings indicated that the manual pulverization of tissue resulted in the highest DNA concentrations; additionally, both senescing leaflets and leaflets stored for extended periods exhibited sufficient DNA for genomic analysis.
Investigating the extraction of considerable DNA amounts from senescing leaves and/or silica-stored tissue over extended periods is elucidated by these findings. A meticulously crafted DNA extraction protocol, effective on cycads and other plant groups exhibiting hard or stiff leaves, is presented here.
The ability to extract substantial quantities of DNA from senescing leaves and/or silica-stored tissues, retained for considerable durations, is showcased by these findings. An efficient DNA extraction procedure is detailed for cycads and other plant species, capable of dealing with tough or inflexible leaves.
This new protocol for rapid plant DNA extraction, using microneedles, is envisioned to improve botanic surveys, taxonomic classifications, and systematics. This protocol can be carried out in the field, with constraints on laboratory expertise and tools. Sequencing and comparison of results against QIAGEN spin-column DNA extractions, using BLAST analyses, validate the protocol.
Genomic DNA was extracted from 13 species exhibiting a range of leaf anatomical features and phylogenetic classifications using two distinct approaches. Option (i) involved puncturing fresh leaves with custom-designed polymeric microneedle arrays to isolate genomic DNA, while option (ii) utilized standard QIAGEN DNA extraction protocols. Metabolically active, three plastids, each with its specific function, perform their roles with precision.
,
, and
Amplification and sequencing of one nuclear ribosomal (ITS) DNA region, alongside other DNA regions, were performed using Sanger or nanopore technology. The extraction time, as per the proposed method, was reduced to 1 minute, and the DNA sequences obtained matched those from the QIAGEN extractions.
Our method, achieving a dramatic improvement in speed and simplicity, is compatible with nanopore sequencing and is ideally suited for various applications, including the high-throughput identification and monitoring of DNA-based species.
Our method, marked by its considerable speed enhancement and simplicity, is compatible with nanopore sequencing and serves multiple applications, such as high-throughput DNA-based species identifications and monitoring.
Deep dives into the fungi that intertwine with lycophytes and ferns contribute significant knowledge to the early evolution of terrestrial plants. Nonetheless, the majority of previous studies on interactions between ferns and fungi have relied solely on visual examinations of root systems. The present research details a metabarcoding protocol, designed for and evaluated against, fungal communities linked to fern and lycophyte roots.
The general fungal community was screened with two primer pairs for the ITS rRNA region, whereas Glomeromycota (specifically arbuscular mycorrhizal fungi) were targeted by 18S rRNA primers. immune-related adrenal insufficiency In order to verify these approaches, we collected and processed root samples from 12 phylogenetically distant fern and lycophyte species.
A notable divergence in compositional makeup was found between the ITS and 18S datasets. IPI-549 Despite the ITS dataset exhibiting the prominence of the Glomerales (Glomeromycota), Pleosporales, and Helotiales (Ascomycota) orders, the 18S dataset showcased a more expansive diversity within the Glomeromycota phylum. Sample similarities exhibited a substantial geographical pattern, according to the non-metric multidimensional scaling (NMDS) ordination.
A dependable and effective way to examine the fungal communities found in fern and lycophyte roots is the ITS-based approach. The 18S approach is a more suitable method for research specifically targeting detailed identification of arbuscular mycorrhizal fungi.
The fungal communities within fern and lycophyte roots are effectively and reliably assessed employing the ITS-based approach. Studies focusing on a thorough examination of arbuscular mycorrhizal fungi are more suitable for the 18S method.
Preservation of plant tissues through the use of ethanol is commonly perceived as a complex and problematic method. We report that the combined strategy of ethanol preservation and proteinase digestion of leaf material results in high-quality DNA extractions. In addition, employing ethanol as a preliminary treatment can enhance DNA extraction from samples that are resistant to standard procedures.
To isolate DNA, samples were taken from leaves preserved in 96% ethanol, as well as from silica-desiccated leaf samples and herbarium fragments that had been pretreated with ethanol. DNA extraction from herbarium tissues was achieved using an ethanol-based pretreatment, and the resulting extracts were juxtaposed with those derived from the standard cetyltrimethylammonium bromide (CTAB) technique.
Ethanol-based pretreatment or preservation of tissue resulted in less fragmented DNA than that extracted from untreated tissue. The addition of proteinase digestion to the lysis stage proved crucial in increasing the DNA yield from the ethanol-treated plant materials. A protocol involving ethanol pretreatment, liquid nitrogen freezing, a sorbitol wash, and subsequent cell lysis demonstrably improved the quality and yield of DNA extracted from herbarium tissue samples.
A critical reevaluation of ethanol's impact on plant tissue preservation, along with an expansion of pretreatment utility for molecular and phylogenomic studies, is the focus of this investigation.
This study re-examines, in a critical way, the repercussions of ethanol on plant tissue preservation and broadens the applications of pretreatment procedures for molecular and phylogenomic analyses.
The inherent difficulty in isolating RNA from trees stems from the obstruction caused by polyphenols and polysaccharides, affecting downstream research applications. Cadmium phytoremediation Furthermore, the protocols for RNA extraction are frequently time-intensive and involve the use of potentially dangerous chemicals. With the goal of addressing these issues, we designed a secure protocol for extracting high-quality RNA from varied sources.
A group of taxa representing a wide variety of leaf textures, including toughness and pubescence, and secondary metabolites.
We analyzed popular RNA isolation kits and protocols, proven successful in other challenging tree samples, along with a broad range of optimization and purification steps to validate their efficiency. A protocol utilizing two silica-membrane column-based kits was optimized, yielding a high quantity of RNA with an RNA integrity number greater than 7, while ensuring the absence of DNA contamination. All RNA samples underwent a successful subsequent RNA sequencing process.
We developed a high-throughput RNA extraction method that effectively yielded high-quality and high-quantity RNA samples from three distinct leaf phenotypes across a remarkably diverse woody species complex.
This optimized RNA extraction method, characterized by high throughput, produced high-quality, high-quantity RNA from three contrasted leaf morphologies in a hyperdiverse woody plant species complex.
Protocols designed for the extraction of high-molecular-weight DNA from ferns are crucial for the sequencing of their extensive and intricate genomes using long-read technologies. We are introducing two distinct cetyltrimethylammonium bromide (CTAB)-based methods to isolate HMW DNA and examine their suitability across a variety of fern taxa for the first time.
We outline two altered CTAB techniques, with key adjustments specifically designed to lessen mechanical disruption during lysis to prevent DNA breakage. This protocol leverages a small portion of fresh tissue to provide a high-efficiency extraction of a substantial quantity of high-molecular-weight DNA. Large quantities of input tissue are processed using a method that starts with the isolation of nuclei, ensuring a high output within a short period. Both methods consistently yielded robust and effective extraction of high-molecular-weight (HMW) DNA from 33 fern species, spanning 19 families. High purity (A) was observed in the majority of DNA extractions, coupled with high DNA integrity and average fragment sizes significantly larger than 50 kilobases.
/A
and A
/A
>18).
Fern DNA extraction methodologies are detailed in this study, with the goal of fostering future genomic sequencing, thus expanding our knowledge of land plant evolution.
High-molecular-weight DNA extraction protocols for ferns are described in this study, in the hope of encouraging further genomic sequencing, which ultimately will enrich our comprehension of land plant diversity.
An economical and effective means of isolating plant DNA is the utilization of cetyltrimethylammonium bromide (CTAB). While the CTAB protocol is frequently adapted for improved DNA extraction, experimental modifications often fail to isolate and systematically assess the impact of individual variables on DNA yield and quality.
This study investigated the relationship between chemical additives, incubation temperature variations, and lysis time on the measured DNA quantity and quality metrics. Modifications to these parameters impacted DNA concentrations and fragment lengths; however, only the purity of the extractant was considerably affected. CTAB and CTAB mixed with polyvinylpyrrolidone solutions ensured the best DNA quality and yield metrics. Compared to herbarium-preserved tissues, silica gel-preserved tissues offered significantly higher DNA yield, longer DNA fragments, and purer extractants.