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1. Simple and efficient isolation of plant genomic DNA using magnetic ionic liquids

   https://doi.org/10.1186/s13007-022-00860-8  

   Emaus, Miranda N. ; Cagliero, Cecilia ; Gostel, Morgan R. ; Johnson, Gabriel ; Anderson, Jared L. ( March 2022 , Plant Methods)

   Plant DNA isolation and purification is a time-consuming and laborious process relative to epithelial and viral DNA sample preparation due to the cell wall. The lysis of plant cells to free intracellular DNA normally requires high temperatures, chemical surfactants, and mechanical separation of plant tissue prior to a DNA purification step. Traditional DNA purification methods also do not aid themselves towards fieldwork due to the numerous chemical and bulky equipment requirements.

   In this study, intact plant tissue was coated by hydrophobic magnetic ionic liquids (MILs) and ionic liquids (ILs) and allowed to incubate under static conditions or dispersed in a suspension buffer to facilitate cell disruption and DNA extraction. The DNA-enriched MIL or IL was successfully integrated into the qPCR buffer without inhibiting the reaction. The two aforementioned advantages of ILs and MILs allow plant DNA sample preparation to occur in one minute or less without the aid of elevated temperatures or chemical surfactants that typically inhibit enzymatic amplification methods. MIL or IL-coated plant tissue could be successfully integrated into a qPCR assay without the need for custom enzymes or manual DNA isolation/purification steps that are required for conventional methods.

   The limited amount of equipment, chemicals, and time required to disrupt plant cells while simultaneously extracting DNA using MILs makes the described procedure ideal for fieldwork and lab work in low resource environments.

    

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2. Ethanol preservation and pretreatments facilitate quality DNA extractions in recalcitrant plant species

   https://doi.org/10.1002/aps3.11519  

   Johnson, Gabriel ; Canty, Steven W. ; Lichter‐Marck, Isaac H. ; Wagner, Warren ; Wen, Jun ( May 2023 , Applications in Plant Sciences)

   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.

   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.

   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.

   This study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies.

    

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3. DNA barcoding of herbal supplements on the US commercial market associated with the purported treatment of COVID‐19

   https://doi.org/10.1002/pca.3320  

   Harris, Calin M. ; Kim, Diane Y. ; Jordan, Chevon R. ; Miranda, Miranda I. ; Hellberg, Rosalee S. ( January 2024 , Phytochemical Analysis)

   The COVID‐19 pandemic was associated with an increased global use of traditional medicines, including Ayurvedic herbal preparations. Due to their growing demand, their processed nature, and the complexity of the global supply chain, there is an increased risk of adulteration in these products.

   The objective of this study was to assess the use of DNA barcoding for species identification in herbal supplements on the US market associated with the Ayurvedic treatment of respiratory symptoms.

   A total of 54 commercial products containing Ayurvedic herbs were tested with four DNA barcoding regions (i.e.,rbcL,matK, ITS2, and mini‐ITS2) using two composite samples per product. Nine categories of herbs were targeted: amla, ashwagandha, cinnamon, ginger, guduchi, tribulus, tulsi, turmeric, and vacha.

   At least one species was identified in 64.8% of products and the expected species was detected in 38.9% of products. Undeclared plant species, including other Ayurvedic herbs, rice, and pepper, were detected in 19 products, and fungal species were identified in 12 products. The presence of undeclared plant species may be a result of intentional substitution or contamination during harvest or processing, while fungal DNA was likely associated with the plant material or the growing environment. The greatest sequencing success (42.6–46.3%) was obtained with thematKandrbcLprimers.

   The results of this study indicate that a combination of genetic loci should be used for DNA barcoding of herbal supplements. Due to the limitations of DNA barcoding in identification of these products, future research should incorporate chemical characterization techniques.

    

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4. Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

   https://doi.org/10.7717/peerj.2777  

   Roux, Simon ; Solonenko, Natalie E. ; Dang, Vinh T. ; Poulos, Bonnie T. ; Schwenck, Sarah M. ; Goldsmith, Dawn B. ; Coleman, Maureen L. ; Breitbart, Mya ; Sullivan, Matthew B. ( December 2016 , PeerJ)

   Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).

   Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.

   Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.

   Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

    

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5. A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber

   https://doi.org/10.1038/s41598-020-73958-5  

   Vennapusa, Amaranatha R. ; Somayanda, Impa M. ; Doherty, Colleen J. ; Jagadish, S. V. Krishna ( October 2020 , Scientific Reports)

   Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7–9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.

    

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