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Department of Entomology and Plant Pathology, The University of Tennessee, Institute of Agriculture, Knoxville, Tennessee
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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We present a strategy to recover high molecular weight genomic DNA from large numbers of isolates of Phytophthora. Included are steps for generating mycelial mass in 24-well reuseable deep well plates, efficient lyophilization and disruption of the mycelium and genomic DNA extraction with 96-well glass fiber filter plates. The resulting DNA is consistently high molecular weight and is suitable for applications that require high quality DNA such as AFLP analysis and TILLING. A single operator easily can manage mycelium preparation and/or DNA extraction from 384 isolates in a single day and this approach might be useful for other fungi or fungi-like organisms that can be grown in liquid media.
Key words: AFLP, oomycetes, reverse genetics, TILLING
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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, Henikoff and Comai 2003, Perry et al 2003, Till et al 2003, Winkler et al 2005), (ii) large scale surveys of Phytophthora from natural environments (Lamour and Hausbeck 2002, Lamour et al 2003, Lamour and Hausbeck 2003), and (iii) extensive crossing experiments to develop genetic mapping populations and inbred lines (Lamour and Hausbeck 2000). TILLING is accomplished by creating relatively large libraries of chemically mutagenized individuals (2000–10 000 individuals) and then screening a mirror library of DNA to determine which isolates are carrying induced mutations in a specific gene of interest. The result is detection of isolates carrying silent, missense and knock-out mutations within specific genes. For the survey, mapping and inbred line work we measure genetic variation with fluorescently labeled amplified fragment length polymorphism (AFLP) markers. A key factor in the success of AFLP and TILLING is the quality of the DNA (Till et al 2003, Habera et al 2004) which needs to be free of contaminants, similar in concentration and of high molecular weight. Researchers face three main challenges in preparing stocks of high quality DNA from a large number (>1000) of Phytophthora isolates. The first is generating sufficient mycelium from the individual cultures, the second is disrupting the mycelium and the third is extracting the DNA. For the mycelium production and disruption steps isolates of Phytophthora often are grown in Erlenmeyer flasks and the resulting mycelium is either freeze dried or immersed in liquid N and manually disrupted with a mortar and pestle. Once the material has been disrupted DNA is extracted with a variety of techniques including phenol/chloroform and commercially available kits. In our experience there are significant limitations to scaling up DNA preparations based on the currently available methods. These limitations include access to sufficient space and equipment, the costs of labor, exposure to toxic chemicals and the monetary cost of commercial kits.
Over the past 2 y we have developed a strategy useful for large numbers of Phytophthora isolates that lessens the limiting factors and consistently provides high molecular weight genomic DNA from diverse Phytophthora species including P. sojae, P. capsici, P. tropicalis, P. ramorum, P. nicotianae, P. citricola and P. citrophthora. This protocol is based on our need to (i) maximize the use of limited space and monetary resources and (ii) recover consistently high quality DNA that can be stored and used over many years.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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(TABLES I and II provide bulleted summaries of the procedures.)
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). Antibiotics are included to reduce foreign contamination but are not necessary. The broth plates are covered with rayon breathable tape and incubated at room temperature 6 d. The resulting colonies are harvested into a 96-well 2 mL DW plate containing three 3 mm glass balls per well. A Millipore dry dispensing plate is used to dispense the glass balls before loading the biological material. The sample plates then are covered with AeraSeal rayon breathable tape (PGC Scientifics, Frederick, Massachusetts) and frozen to –80 C at least 1 h. The samples are lyophilized for a total of 48 h; 24 h using the STDS with the incubation chamber at 0 C, followed by 24 h with the incubation chamber at 23 C. Immediately after removal of the samples from the chamber a capmat is applied to the deepwell plates with CMA. The samples then are disrupted with the MM300 for a total of 2 min on the highest setting of 30 revolutions per second. After bashing 1 min the 96-well deepwell plate is rotated 180 degrees and bashed an additional minute (TABLE I
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The plates containing pulverized dried mycelium are centrifuged 5 min at 4600 g and the capmats carefully removed. A total of 400 µL of a lysis cocktail (100 mM Tris (pH 8.0), 50 mM EDTA, 500 mM NaCl, 1.33% SDS with 0.8% Fighter F antifoaming agent (Loveland Industries, Greely, Colorado) and 0.2 mg/mL RNase A) is added to each well using the Apricot and a new capmat applied. The plates are agitated vigorously by inverting the plate 5–10 times and the samples incubated in a 65 C chamber 20 min. The plates are centrifuged 2 min at 4600 g and the capmat gently removed. A total of 150 µL of 5 M potassium acetate is added using the Apricot and a new capmat applied. The plates are inverted vigorously 5–10 times and the samples incubated at –20 C 30 min to overnight. Chilled plates then are centrifuged 30 min at 4600 g and 400 µL of the supernatant transferred to a new 2 mL DW plate containing 600 µL of a 0.66 M guanidine hydrochloride and 63.3% ethanol solution using the Apricot. Guanidine hydrochloride is a dangerous irritant and proper protective garments, gloves and eye protection must be worn. Guanidine hydrochloride is highly reactive with bleach and the waste from the following steps must be disposed of properly.
A new capmat is applied and the plates inverted vigorously 5–10 times to mix the solutions. One mL of the mixture is added to a Nunc spin column plate (Nalge Nunc Int., Rochester, New York) sitting on a 2 mL DW plate and centrifuged at 4600 g 5 min. The flow-through is discarded and the membrane washed by adding 500 µL wash solution (10 mM Tris [pH 8.0], 1 mM EDTA, 50 mM NaCl, 67% ethanol) and centrifuging at 4600 g 5 min. The membrane is further washed by adding 500 µL of 95% ethanol and centrifuging at 4600 g 5 min. The spin column plate is incubated at 65 C 10 min to dry the membrane. A total of 200 µL of 10 mM Tris (pH 8.0) is added to each well using the Apricot and the plates incubated at room temperature 30 min to 1 h. The DNA is eluted into a clean 1 mL DW plate by centrifugation at 4600 g 2 min. The quantity and quality of the DNA is assessed by separation on a 1% agar gel (FIG. 2, TABLE II).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). DNA isolated with this strategy has been used successfully in standard PCR, fluorescently labeled nested PCR, real time PCR, reverse genetics using the Targeting Induced Local Lesions IN Genomes (TILLING) approach, and fluorescently labeled amplified fragment length polymorphism (AFLP) applications. This approach is attractive because a large number of samples can be processed efficiently in a relatively short period, the total space required is a fraction of that needed for traditional approaches, the cost of performing the DNA extraction is approximately 85% less than currently available commercial kits, and there are no organic solvents such as chloroform or phenol. We have used this same strategy to recover high quality DNA from diverse biological starting materials including soybean (Glycine max), horse-weed (Conyza canadensis), dogwood (Cornus florida), soybean cyst nematode (SCN) (Heterodera glycines) eggs, morel mushroom carpophores (Morchella esculentum) and diverse Pythium species. As the number of genomes with extensive sequence increases, the application of new genetic tools and approaches becomes feasible. For many of these approaches, including mapping and association studies relying on large progeny sets, reverse genetic tests based on random mutagenesis, and large-scale surveys using molecular markers, there is a need for strategies to produce stocks of high quality DNA from large numbers of individuals. The approach outlined above works well for members of the genus Phytophthora and provides a reasonable starting point to develop strategies for other organisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: klamour{at}utk.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Henikoff S, Comai L. 2003. Single-nucleotide mutations for plant functional genomics. Annu Rev Plant Biol 54: 375–401.[CrossRef][Medline]
Lamour KH, Daughtrey ML, Benson DM, Hwang J, Hausbeck MK. 2003. Etiology of Phytophthora drechsleri and P. nicotianae (= P. parasitica) diseases affecting floriculture crops. Plant Dis 87:854–858.[CrossRef]
———, Hausbeck MK. 2000. Mefenoxam insensitivity and the sexual stage of Phytophthora capsici in Michigan cucurbit fields. Phytopathology 90:396–400.[Medline]
———, ———. 2002. The spatiotemporal genetic structure of Phytophthora capsici in Michigan and implications for disease management. Phytopathology 92:681–684.[Medline]
———, ———. 2003. Effect of crop rotation on the survival of Phytophthora capsici and sensitivity to mefenoxam. Plant Dis 87:841–845.[CrossRef]
McCallum CM, Comai L, Greene EA, Henikoff S. 2000. Targeting Induced Local Lesions IN Genomes (TILLING) for plant functional genomics. Plant Phys 123:439–442.
Perry JA, Wang TL, Welham TJ, Gardner S, Pike JM, Yoshida S, Parniske M. 2003. A TILLING reverse genetics tool and web-accessible collection of mutants of the legume Lotus Japonicus. Plant Phys 131:866–871.
Till B, Reynolds S, Greene E, Codomo C, Enns LC, Johnson J, Burtner C, Odden A, Young K, Taylor E, Henikoff J, Comai L, Henikoff S. 2003. Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–530.
Winkler S, Schwabedissen A, Backasch D, Bokel C, Seidel C, Bonisch S, Furthauer M, Kuhrs A, Cobreros L, Brand M, Gonzalez-Gaitan M. 2005. Target-selected mutant screen by TILLING in Drosophila. Genome Res 15:718–723.
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