Identification of DNA-Microsatellite Markers for the Characterization of Somatic Embryos in Quercus suber

Gómez-Garay, Arancha; Bueno, Ángeles; Pintos, Beatríz

doi:10.1007/978-1-62703-389-3_8

Cited by 2 publications

(5 citation statements)

References 12 publications

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“…We optimized two DNA extraction methods (M1 and M2, Figure 1 ) from commercial kits that are typically inappropriate for leaf samples of woody species rich in secondary metabolites and sugars (such as Quercus palustris ) [ 24 , 25 , 26 , 39 , 40 ]. M1 is based on the DNeasy Plant DNA extraction kit (Qiagen, Hilden, Germany) using cetyl timethylammonium bromide (CTAB) for extraction, and M2 on the Power Plant extraction kit (MoBio, Carlsbad, CA, USA), precipitating contaminants with a phenol solution.…”

Section: Resultsmentioning

confidence: 99%

“…Genomic DNA was extracted from in situ dark- and light-adapted leaves by modifying the initial key grinding, lysis, and few intermediate steps of the PowerPlant and DNeasy DNA isolation procedures developed by MoBio and Qiagen, respectively ( Figure 1 ). We adapted the procedures for efficient, high quality, and quantity DNA extraction of rigid leaf samples that are rich in polysaccharides, phenolics, and other contaminants, conditions reported to lead to low yield in a variety of species with these kits [ 24 , 25 , 26 , 27 , 30 ]. We chose to modify and compare the procedures of two accessible commercial kits, the CTAB-based DNeasy Plant Mini Kit developed by Qiagen and the phenolic separation based MoBio Power Plant Kit developed by MoBio.…”

Section: Methodsmentioning

confidence: 99%

“…North American oaks also synthesize terpenoid molecules, such as isoprene, while some clades of European oaks produce other terpenoids, for example, monoterpenes [ 21 , 22 , 23 ]. Therefore typically soft, non- photosynthesizing tissues (such as young roots, germinating seedlings, buds, flowers or fruits) or young, developing leaves are preferred for genomic DNA isolation from these species as they contain higher amounts of DNA and lower amounts of potential inhibitory metabolites than mature leaves [ 24 , 25 , 26 , 27 ]. However, these tissues are often available only briefly during the lifecycle of many long-lived woody species, unless they are maintained under controlled conditions or in vitro, in tissue, or protoplast cultures, which require time consuming and costly preparation, maintenance, and additional resources that often unavailable at education centered, applied learning institutions [ 28 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

“…The season-long availability of mature leaves is often greater for many woody species of interest for genomic studies in temperate regions, or year-round in some species, such as live oaks ( Quercus virginiana ) in the Southern United States or the Japanese evergreen oaks ( Quercus acuta ) in Eastern Asia. However, their direct applications are limited due to the difficulties in isolating high quality and quantity DNA from the metabolite rich tissue [ 24 , 25 , 26 , 27 ].…”

Section: Introductionmentioning

confidence: 99%

“…Commercial kits are relatively ineffective in extracting DNA from mature leaves of many species of interest [ 10 , 11 , 12 , 13 , 14 , 24 , 25 , 26 , 27 , 30 , 31 ] and require time-consuming and costly pre- and/or post-processing of samples. This may include the digestion of cell walls, isolation of intact protoplasts, enzymatic break down of sugars, isolation of nuclei, mitochondria or chloroplasts, the concentration of the extract, or the separation of DNA from contaminants [ 3 , 5 , 32 , 33 , 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

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In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

Barta

Bolander

Bilby

et al. 2017

Plants

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Mature oak (Quercus spp.) leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS) from pin oak (Quercus palustris), whose full genome has not yet been sequenced.

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