Incorporation of sodium sulfite into extraction protocol minimizes degradation of Acacia DNA

University of Western Australia, Perth City, Western Australia, Australia
BioTechniques (Impact Factor: 2.95). 05/2001; 30(4):742-4, 748.
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Available from: Margaret Byrne, Sep 06, 2014
    • "Samples were also collected from single populations of Acacia aestivalis and Acacia jennerae for use as outgroups. DNA extractions were carried out as per Byrne et al. (1998), with sodium sulphite added to the extraction buffer (Byrne et al. 2001), and these samples were analysed with restriction fragment length polymorphism (RFLP) methodology. Although this is an older technology, it has been proven to be a reliable co-dominant marker system that is valuable for the investigation of species complexes (e.g. "
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    ABSTRACT: Species complexes are often taxonomically challenging because the evolutionary relationships among closely related taxa may not be well reflected by morphological divergence. Molecular data can be a powerful tool in resolving these ambiguities. Using nuclear restriction fragment length polymorphisms (RFLPs), we examined genetic variation among 22 populations of the Acacia microbotrya complex and assessed its congruence with five taxa previously identified using morphological criteria. Four distinct genetic clusters were consistently detected across multiple analytical approaches (Bayesian, UPGMA, maximum likelihood, and principle coordinate). These genetic clusters were strongly congruent with each of four morphologically defined taxa, Acacia amblyophylla, Acacia daphnifolia, A. microbotrya and A. microbotrya (Dandaragan variant). From this, we suggest that the Dandaragan variant warrants formal recognition at the same taxonomic level as the other three taxa. Individuals of the fifth morphologically defined species, Acacia splendens, were not consistently assigned to any one genetic cluster having affinities with A. microbotrya (Dandaragan variant) and A. amblyophylla, although at the population level it showed closest affinity to A. microbotrya (Dandaragan variant). Despite strong genetic and morphological differentiation of these taxa in allopatric regions, we detected a hybrid zone of genetic admixture between A. microbotrya and A. daphnifolia where their geographic ranges overlap. Together, these results highlight a complex evolutionary history of fragmentation, divergence and hybridisation in the A. microbotrya complex. While further work is required to resolve the phylogenetic status of A. splendens, this study provides compelling evidence for the recognition of these four genetic clusters as distinct evolutionary entities in the A. microbotrya complex that should be treated as separate units for conservation, commercial or management purposes.
    No preview · Article · Aug 2015 · Tree Genetics & Genomes
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    • "The aridity index of each provenance location (ratio between annual rainfall and annual potential evapotranspiration) was calculated, as it is regarded as a useful proxy for potential water availability (Gao and Giorgi, 2008). Genomic DNA was extracted from 10–15 g of leaf material (Byrne et al., 1993) with 0Á1 M sodium sulphate (Byrne et al., 2001) and 0Á05 % bovine serum albumin (BSA) added to the extraction buffer. DNA of each sample was digested with six restriction enzymes (BclI, BglII, DraI, EcoRI, EcoRV and XbaI) and hybridized with heterologous probes covering most of the chloroplast genome, according to the protocol described in Byrne et al. (1993). "
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    ABSTRACT: A worldwide increase in tree decline and mortality has been linked to climate change and, where these represent foundation species, this can have important implications for ecosystem functions. This study tests a combined approach of phylogeographic analysis and species distribution modelling to provide a climate change context for an observed decline in crown health and an increase in mortality in Eucalyptus wandoo, an endemic tree of south-western Australia. Phylogeographic analyses were undertaken using restriction fragment length polymorphism analysis of chloroplast DNA in 26 populations across the species distribution. Parsimony analysis of haplotype relationships was conducted, a haplotype network was prepared, and haplotype and nucleotide diversity were calculated. Species distribution modelling was undertaken using Maxent models based on extant species occurrences and projected to climate models of the last glacial maximum (LGM). A structured pattern of diversity was identified, with the presence of two groups that followed a climatic gradient from mesic to semi-arid regions. Most populations were represented by a single haplotype, but many haplotypes were shared among populations, with some having widespread distributions. A putative refugial area with high haplotype diversity was identified at the centre of the species distribution. Species distribution modelling showed high climatic suitability at the LGM and high climatic stability in the central region where higher genetic diversity was found, and low suitability elsewhere, consistent with a pattern of range contraction. Combination of phylogeography and paleo-distribution modelling can provide an evolutionary context for climate-driven tree decline, as both can be used to cross-validate evidence for refugia and contraction under harsh climatic conditions. This approach identified a central refugial area in the test species E. wandoo, with more recent expansion into peripheral areas from where it had contracted at the LGM. This signature of contraction from lower rainfall areas is consistent with current observations of decline on the semi-arid margin of the range, and indicates low capacity to tolerate forecast climatic change. Identification of a paleo-historical context for current tree decline enables conservation interventions to focus on maintaining genetic diversity, which provides the evolutionary potential for adaptation to climate change. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email:
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    • "We have also included the addition of Proteinase K with sufficient concentration to breakdown the cell walls and dissolving the cell membranes. Sodium sulfite was used as a reducing agent for polyphenol oxidase which acts by preventing the production of polyphenolic compounds that will cause the degradation of DNA [13]. According the gel image of DNA extracted using our modified protocol, it produces favorable yield which is >500 ng/µL and high purity that falls within the range of 1.8-2.0. "
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    ABSTRACT: —The isolation of high quality DNA and RNA from plant species harboring high levels of polysaccharides and secondary metabolites are typically problematic, especially those in cactus. These compounds often co-precipitate with DNA and RNA thus causes low recovery and quality of the nucleic acids. Six DNA extraction protocols were tested on the sample of Hylocereus spp. of which the results were compared and analyzed. For comparison, three manufacturer's protocols from different commercial kits and another three conventional DNA extraction protocols were compared. It was found that conventional method generally produces consistent and higher yield. Among the conventional protocols itself, each has their pros and cons. Therefore, a modified protocol which is concise, quick and simple was developed for Hylocereus spp. which is beneficial for further molecular work. This method was proven to be reliable in generating a good quality of DNA from these particular genera. Similarly for RNA extraction, four different extraction protocols were tested on the same sample. The results were analyzed and a modified protocol was developed to obtain a higher quality and yield of RNA for further downstream investigations. The extraction buffer from our modified DNA extraction protocol was tested on the RNA extraction and shows a good yield.
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