Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

Bressan, Eduardo A.; Rossi, Mônica Lanzoni; Gerald, Lee Tseng Sheng; Figueira, Antônio

doi:10.1186/1756-0500-7-268

Cited by 34 publications

(26 citation statements)

References 16 publications

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“…Of the preservation methods used, silica gel proved consistently reliable, while treatments involving heat or alcohol lead to recovery of less DNA, and of more degraded DNA. This is in line with previous reports (Staats et al, 2011;Särkinen et al, 2012;Bressan et al, 2014;Weiß et al, 2016). Contemporary field collections for molecular work are usually silica gel-dried, or less frequently, collected into high salt buffers or RNAlater; however, it is not always possible to work with recent plant material.…”

Section: Discussionsupporting

confidence: 82%

The Limits of Hyb-Seq for Herbarium Specimens: Impact of Preservation Techniques

et al. 2019

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Over the past 300 years of plant collecting for herbaria, the basic method of preservation has remained remarkably consistent-a plant press with absorbent paper. However, the difficulty of drying plant specimens in the humid tropics has led to a variety of additions to this basic technique, primarily to prevent the specimens succumbing to fungal and bacterial breakdown. These additions include drying gently in tents over low fires, soaking the specimens in alcohol before pressing and, more recently, drying the specimens with forced heat from hair dryers. The process of drying is, however, known to cause breakage and damage to the plant's DNA, as well as providing time for non-plant organisms (bacteria and fungi) to multiply in the tissue, potentially "swamping" the plant specimen's own DNA. Contemporary plant collectors therefore usually collect a separate sample for DNA work, usually rapidly dried in silica gel desiccant; historical collections, however, may have been treated with alcohol and/or heat. We have recently shown that Hyb-Seq provides a reliable method for retrieving robust sequence data from even very old and degraded herbarium specimens. In this study we have used a panel of specimens preserved using a variety of methods to assess whether Hyb-Seq is capable of retrieving informative amounts of sequence data from duplicate specimens preserved using a range of specimen preservation methods. We present data on the amount and quality of DNA and of sequence data retrieved, the variation in error types between preservation techniques and the utility of the data for phylogenetic analysis.

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Section: Discussionsupporting

confidence: 82%

The Limits of Hyb-Seq for Herbarium Specimens: Impact of Preservation Techniques

et al. 2019

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“…Specimens were imaged using an Olympus TG3 camera. For each sample, part of the apical tip of a frond was cleaned of epiphytes, cut into smaller fragments of approximately 0.5 cm × 0.5 cm, preserved in 100% molecular-grade ethanol (Bressan et al 2014), and stored in a -80°C freezer. In total, 25 out of the 44 samples collected were pressed as herbarium vouchers and deposited at the SINU Herbarium.…”

Section: Sampling Sites and Collectionmentioning

confidence: 99%

Diversity and phylogeny of Sargassum (Fucales, Phaeophyceae) in Singapore

et al. 2018

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Sargassum species play key ecological roles on coral reefs, yet their diversity remains poorly known. Precise identification of Sargassum species, however, is improving with molecular genetic tools, though these have yet to be applied rigorously in Singapore. Historical records list 41 species, but no more than ten were verified based on herbarium vouchers, and even fewer (five species) were confirmed in the field based on a single nuclear gene marker in a previous study. Here, we revised the diversity of Sargassum in Singapore by examining all the morphologically distinct forms collected from the local coral reef environment. A total of six morphotypes, Sargassum aquifolium (Turner) C.Argardh (1820), S. cf. granuliferum C.Argardh (1820), S. ilicifolium (Turner) C.Argardh (1820), S. swartzii C.Argardh (1820), S. polycystum C.Argardh (1824), and an undescribed taxon ‘Sargassum sp.’ (Mattio and Payri 2009), were delineated based on morphological characteristics. The morphotypes were placed in five molecular clades based on phylogenetic analyses of the nuclear ITS-2, chloroplastic partial RuBisCO operon rbcLS, and mitochondrial cox3. Sargassum cf. granuliferum, though morphologically distinct from all other species, is not phylogenetically distinct from S. polycystum. Our results provide a species list for Singapore that will be valuable for future studies on macroalgal biogeography and species-specific ecological relationships with other reef organisms, particularly corals.

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“…26 Ethanol was used in our protocol because a previous report indicated that highemolecular weight DNA could be extracted from cells fixed with it and yield DNA of equivalent quality to that retrieved from fresh and frozen tissue. 10 The need for the recovery of high-quality nucleic acids, such as DNA, in the current age of sequencing is 2-fold. High-quality DNA as a template for next-generation sequencing (NGS) technologies means low fragmentation and the availability of large numbers of templates for amplification.…”

Section: Discussionmentioning

confidence: 99%

“…This has proven to result in improved yields of nucleic acids at both the qualitative and quantitative levels. 10 In fact, molecular analysis from a variety of cytology formats (fluids, washes, aspirations, and cell blocks) have been documented to be reliable sources for molecular analysis. [11][12][13][14][15] Looking to further increase the value of cytology specimens, we have developed a microfluidic platform from which the cells in a cytologic specimen can be viewed for diagnostic purposes and later recovered for molecular analysis, all in the context of a formalin-free environment.…”

Section: Introductionmentioning

confidence: 99%