Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis

DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious. In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates, named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A modified mini-prep method for economical and rapid extraction of genomic DNA in plants

Molecular markers for map-based cloning, marker-assisted selection in crop breeding, and genetic studies require DNA isolation from a large number of plants in a short span of time. Here we describe a modified DNA extraction method that is economical in terms of cost, time and labour. The method allows DNA extraction from as little as 0.2–0.3 g of leaves that are homogenized in zipper plastic bags, followed by DNA isolation in 1.5-mL Eppendorf tubes. By using the modified method, a DNA yield of 700–800 μg/300 mg leaf tissue was obtained from cotton and wheat samples. The quality of the DNA was quite suitable for PCR-based markers.     

A rapid DNA preparation for PCR fromChlamydomonas reinhardtii andArabidopsis thaliana

A method for preparing DNA for PCR has been adapted from the forensic work of Walsh et al. (Biotechniques 10:506–513) for use withChlamydomonas reinhardtii andArabidopsis thaliana. The method consists of a short incubation of cells or tissue in ethanol, followed by addition of Chelex-100 and heat treatment. Following centrifugation, the supernatant is added directly to the PCR reaction; forChlamydomonas, amplification product is visible over a range of four orders of magnitude of starting cells. Using this method, DNA suitable for PCR template can also be obtained fromArabidopsis leaf tissue without grinding, organic extraction or precipitation steps. This method may prove to be useful for other plant and algal species.     

Single Fish Egg DNA Extraction for PCR Amplification

Modern stock researches on marine biomass are basically genetic and rely increasingly on PCR-based manipulations of informative DNA markers for detecting the genetic diversity. This study developed a simple and rapid single tube method for DNA extraction from a single fish egg. The 15 min protocol was based on the use of Chelex 100 resin and urea to breakdown membrane and connective tissue of eggs. From various sizes of a single egg of walleye pollack (Theragra chalcogramma), the amounts of total nucleic acids were reproducibly obtained to be 18.25 ± 1.92 μg per egg. Using DNA templates diluted ranging 1/10⁰–1/10⁵, PCR amplification for the mitochondrial cytochrome b (Cytb) gene was successfully performed, and the 1/10² diluted template yielded the best result in PCR amplification for three different DNA marker genes. This method is quite simple and economical, and enables to provide the high throughput often demanded by the stock identification of marine biomass, in which large numbers of specimens of single fish eggs must be analyzed.     

An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.     

A protocol for rapid DNA extraction from<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> for PCR analysis

We present a method for instant DNA extraction fromArabidopsis thaliana based on a simple DNA extraction method (Edwards et al., 1991). A piece of rosette leaf (typically 3–5 mg) was ground in a centrifuge tube in extraction solution. Extracted DNA was suitable for PCR analysis, without centrifugation. The feasibility of this method was confirmed by testing 24 primer sets. This method requires less than 1 mg of plant tissue and is useful for genetic mapping, transgene detection, and other experiments.     

Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice

We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube, one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions. The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200–300 individuals in a few hours. These features enabled us to perform rapid largescale seedling genotyping required for marker-assisted selection. We have also examined the applicability of this method for other PCR-based markers: RAPDs, nuclear STS, chloroplast STS and chloroplast microsatellites.     

A rapid and high yielding DNA miniprep for cotton (Gossypium spp.)

A rapid DNA minipreparation method was developed for cotton and yields 500–600 μg DNA from 1.0 g fresh leaf tissue. Cotton DNA extracted using this method is completely digested with restriction enzymes, supports PCR and Southern DNA analyses and was used successfully in these applications. An erratum to this article is available at .     

On visual pigment templates and the spectral shape of invertebrate rhodopsins and metarhodopsins

The absorbance spectra of visual pigments can be approximated with mathematical expressions using as single parameter the absorbance peak wavelength. A comparison of the formulae of Stavenga et al. in Vision Res 33:1011–1017 (1993) and Govardovskii et al. in Vis Neurosci 17:509–528 (2000) applied to a number of invertebrate rhodopsins reveals that both templates well describe the normalized α-band of rhodopsins with peak wavelength > 400 nm; the template spectra are virtually indistinguishable in an absorbance range of about three log units. The template formulae of Govardovskii et al. in Vis Neurosci 17:509–528 (2000) describe the rhodopsin spectra better for absorbances below 10⁻³. The template predicted spectra deviate in the ultraviolet wavelength range from each other as well as from measured spectra, preventing a definite conclusion about the spectral shape in the wavelength range <400 nm. The metarhodopsin spectra of blowfly and fruitfly R1-6 photoreceptors derived from measured data appear to be virtually identical. The established templates describe the spectral shape of fly metarhodopsin reasonably well. However, the best fitting template spectrum slightly deviates from the experimental spectra near the peak and in the long-wavelength tail. Improved formulae for fitting the fly metarhodopsin spectra are proposed.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

Amplification of cox2 (～620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens,Comparing 19 Extraction Methods and 15 Polymerases

During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm² of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; ∼620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.     

An improved method for isolating high-quality DNA from<Emphasis Type="Italic">Vitis vinifera</Emphasis> nuclei

We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A_260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.     

Quantification and Localization of Bacteria in Plant Tissues Using Quantitative Real-Time PCR and Online Emission Fingerprinting

In order to quantify and localize specific bacterial target genes in plant tissue, this project has generated relevant new insights in the combined application of quantitative real-time PCR in parallel with the in situ PCR + probe-hybridization and online emission fingerprinting using LSM 510 META. After designing an Enterobacter radicincitans species-specific probe, introduced bacterial cells were monitored in growing plant parts and their colonization behaviour was examined in relation to the native bacterial community. For this purpose, the plant growth-promoting rhizobacterial (PGPR) strain Enterobacter radicincitans was applied to Brassica oleracea plants in increasing inoculum concentrations 10⁷, 10⁸ and 10⁹ cells per plant. Inoculation of 10⁹ E. radicincitans cells per plant to Brassica oleracea leaves and roots resulted in significant increases of root, leaf and tuber growth. Total bacterial cell numbers were estimated using quantitative real-time PCR to be between 10⁷ and 10⁹ cells g⁻¹ fresh leaf weight and about 10⁸ cells g⁻¹ fresh root weight of Brassica oleracea plants. Using quantitative real-time PCR, a significant colonization of Brassica oleracea leaves and roots with E. radicincitans cells was measured. Roots were colonized with a density of 10⁷ cells g⁻¹ fresh root weight up to at least 14 days after inoculation. That is equivalent to a proportion of E. radicincitans 16S rDNA-gene copy numbers compared to the total bacterial communities of about 10–16%. Online emission fingerprinting established that the introduced bacteria proliferated on and inside the root and that they colonized the intercellular spaces of the root cortex layer. Hence, E. radicincitans was able to successfully compete with the native bacterial population.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Rapid extraction of genomic DNA from leaves and bracts of dove tree (Davidia involucrata)

Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction. A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and the restriction enzyme digestion needed for Southern blotting.     

Effect of alkali treatment time and extraction time on agar from <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis>

The effects of alkali treatment time and extraction time of native agar and alkali treated agar obtained from Gracilaria vermiculophylla were studied. The response characteristics were mainly agar yield and gel strength. Alkali treatment was carried out at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Agar yield and gel strength decreased with the increase in the time of the alkali treatment. The highest yield (15.3%) and highest gel strength (1,064 g cm⁻²) were obtained at 0.5 h, and therefore this time was used for the next experiment. The extraction of both native and alkali treated agars was carried out at 1.5, 2.0, 2.5, and 3.0 h. The best extraction time for alkali treated agar was 1.5 h, and for native agar 2.5 h. The alkali treated agar obtained with the different alkali treatment and extraction times showed higher melting (92.4–99.7°C) and gelling (35.7–39.6°C) temperatures. Native agar was lower in melting (60.2–64.1°C) and gelling (20.4–23.4°C) temperatures. The 3,6-anhydrogalactose content decreased with increasing alkali treatment time, with the opposite effect during the extraction of native and alkali treated agars.     

Effect of paclobutrazol on wheat salt tolerance at pollination stage

The effects of paclobutrazol (PBZ) (0, 30, 60, and 90 ppm) and NaCl (0, 75, 150, and 225 mM) treatments on a salt-tolerant (Karchia-65) cultivar of wheat (Triticum aestivum L.) at the pollination stage were studied. Salt stress decreased plant height, the length and area of the flag leaf, fresh and dry weights of the shoot, roots, and flag leaf, and water content. On the background of salinity, PBZ treatment further suppressed plant height. Although plants growth was suppressed in PBZ-treated plants, PBZ treatment moderated the negative effect of salinity on some growth parameters. Under PBZ treatments, plants tissues accumulated more watersoluble carbohydrates and reducing sugars than control plants, with the exception of water-soluble carbohydrates in the roots. The Na⁺ content in roots significantly (p ≤ 0.05) increased at 150 and 225 mM NaCl, but PBZ treatment moderated the harmful effect of the highest levels of salinity. Salinity with or without PBZ treatment improved the K⁺, P, and N contents in plants. It is reasonably to suggest that the protection and increasing salt tolerance caused by PBZ was due to the mechanism nearly similar to the salt-tolerant cultivar physiological systems. These observations suggest that PBZ treatment has the potential to increase salt tolerance with a limiting damage caused by salt stress even in salt-tolerant plants. This text was submitted by the authors in English. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 278–284.     

Biological effects of low-energy C ion implantation on sesame ( Sesamum indicum L.)

Field cultivation experiments on white sesame (Sesamum indicum L.) seeds implanted with low-energy C ion showed that different dosages of C ion implantation produce different biological effects. Sesame plants in 6 different dosage groups with C ion density respectively at 1 × 10¹¹, 1 × 10¹², 1 × 10¹⁵, 5 × 10¹⁵, 1 × 10¹⁶, 5 × 10¹⁶ ion/cm² were superior to the control group in plant height, leaf number, stalk diameter and leaf size. Further, sesame plants in these groups flower and seed earlier than those in the control group, and single plant yield also increased. Of all the groups, the 5 × 10¹⁵ ion/cm² dosage group yielded the best effect, whereas the 1 × 10¹⁷/cm² dosage group showed an evident inhibitory effect of ion implantation on the germination and growth of the sesame seeds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2006, 42 (1): 95–97 [译自: 北京师范大学学报 (自然科学版)]