Plant Molecular Biology Reporter Volume 11(2) 1993

pages117-121

Commentary

A Procedure for Mini-Preparations of Genomic DNA from Needles of Silver Fir (Abies alba Mill.)

Key Words: DNA, minipreparation, Abies alba, needles

Abstract: We have adapted a procedure for the isolation of genomic DNA from needles of silver fir (Abies alba) to meet the requirements for large-scale analysis of the population genetics of forest trees. Our modifications permit the entire procedure to be carried out in Eppendorf tubes, which greatly minimizes time, plant material, and the amounts of chemicals. DNA is recovered with a mean efficiency of 80 p.g/g needles, is suitable for restriction by the common endonu- cleases, and serves as a substrate for PCR.

T he use of DNA polymorphisms in the study of population genetics involves major problems starting with the isolation of DNA. As population genetics is based on the study of a large number of

genotypes, it is essential to minimize steps that consume substantial amounts of time, plant material, or chemicals. To avoid severe damage to young trees, for example, only small amounts of tissue should be analyzed. In order that the protocol can be followed the year around, the tissue employed for extraction should be continuously available and not be affected by seasonal changes in metabolism. To insure that the polymorphisms detected are genetic and not artefactual, the extracted DNA should be free of either mechanical or enzymatic degradation, and the quality and amount of DNA should be appropriate for PCR and permit comparisons of RFLPs.

Meeting these requirements with woody species has been far more difficult than with annuals or biennials (Couch & Fritz, 1990; Howland et al., 1991). Silver fir (Abies alba) presents especially difficult problems because the needles contain large amounts of polysaccharides and secondary metabolites, such as polyphenols, terpenoids, and resins; the usual procedures for isolating DNA generally fail. Nonetheless, Kreike (1990, 1991 ) working with apices and seeds, and Guillemaut & Mar6chal- Drouard (1992) with mature needles reported success in the isolation of DNA from this plant.

118 Ziegenhagen, Guillemaut, and Scholz

Focusing on needles as the best year-round source, we modified the procedure of Guil lemaut & Mar6chal-Drouard to develop a minipreparation. In contrast to the usual extraction procedure, which requires eight to ten minutes to grind one g of tissue from a single individual, the procedure described here permits the simultaneous extraction of 200 to 300 mg of needles from 10 separate plants in microcentrifuge tubes within an interval of six minutes.

Special equipment required Retsch Schwingrn6hle (shaking mill), type MM2, 220 V, 50 Hz, 2 Teflon racks for five 1.5- to 2.0-mL Eppendorf tubes 5-ram beads of tungsten carbide Haereus Microlitre centrifuge, type Biofuge 15 Hoefer Fluorometer, type TKO 100 Liquid nitrogen

Solutions required 5 M potassium acetate buffer, pH 5.2 5 M sodium acetate buffer, pH 4.8 3 M sodium acetate buffer, pH 5.2 10x TE buffer, pH 8.0 extraction medium:

100 mM sodium acetate, pH 4.8 50 mM EDTA pH 8.0 500 mM NaC1 2% soluble PVP (MW 10,000, Sigma) Adjust the pH to 5.5 and add SDS to 1.4%.

phenol, equilibrated in 1 M Tris-HC1 buffer, pH 8.0 and 0.1% 8- hydroxyquinoline

chloroform RNAse, DNAse-free (Boehringer) Solutions for fluorometric measurements of DNA; as described in Hoefer

protocol

Protocol

Disruption of tissue Pre--cool Teflon tube racks in liquid nitrogen. Weigh 10 200-to 300-mg sets of mature needles (fresh or already

frozen). Transfer needles to pre-cooled Eppendorf tubes, add two beads of

tungsten carbide to each of the ten tubes and pre-cool again in liquid nitrogen for 5 min.

DNA Miniprep from Fir Needles 119

Put the Eppendorf tubes into the Teflon rack and attach the rack to the mill.

Homogenize at maximum speed for 6 min. 1 Remove the Eppendorf tubes with the still-frozen homogenate and

add 1000 ~tL of extraction medium) Gently mix while the mixture thaws. 2-3 Remove the tungsten carbide

beads.

Notes 1. Do not delay. 2. While the homogenate thaws, make sure that any material that sticks to the lid

of the Eppendorf tube remains continuously in contact with the extraction medium.

3. Interfering substances may be removed earlier by adding 200 BL chloroform at this time; a two-phase supernatant will appear after the first centrifugation.

I n c u b a t i o n and p rec ip i t a t i on of D N A The remaining steps are performed on the set of ten Eppendorf tubes:

Incubate 20 min at 65~ and mix from time to time. Centrifuge at 10,000 rpm for 10 min at room temperature. Collect supernatant and add 1/3 vol of 5 M potassium acetate (pH

5.2); mix gently and incubate for 30 min at 0~ Centrifuge at 10,000 rpm for 10 min at 4~ Collect supernatant and add 0.6 vol of isopropanol; mix and incubate

for 30 rain at -20~ Centrifuge at 10,000 rpm for 10 min at 4~ and pour off supernatant. Air-dry DNA pellet for 10 rain. Redissolve DNA pellet in 200 ~L lxTE buffer for 30 rain at room

temperature or overnight at 4~ Add 2 ~tL RNAse and incubate at 37~ for 30 min to I h.

P h e n o l - c h l o r o f o r m e x t r a c t i o n Add 200 ~tL phenol, mix thoroughly and centrifuge at 10,000 rpm for

10 min at room temperature. Collect supernatant, add 100 ~L phenol and 100 ~tL chloroform, mix

thoroughly, and centrifuge at 10,000 rpm for 10 min at room temperature.

Collect supernatant, add 200 ~tL chloroform, mix thoroughly, and centrifuge at 10,000 rpm for 5 min at room temperature.

Collect supernatant, adjust to 300 mM sodium-acetate (pH 5.2) and add 2.5 vol of 96% EtOH.

Incubate for 1 h at -80~ or overnight at -20~ Centrifuge at 10,000 rpm for 10 min at 4~ and pour off supernatant. Air-dry DNA pellet for 10 min. Redissolve DNA pellet in lxTE buffer.

120 Ziegenhagen, Guillemaut, and Scholz

Quantitation of DNA We employed the Hoefer protocol for the fluorometric quantitation of DNA in the samples.

R e s u l t s a n d D i s c u s s i o n

We have isolated DNA by the above procedure about 30 times, which corresponds to 300 samples of mature needles. Our evaluation of the procedure and of the DNA obtained is described below.

�9 The DNA obtained is of high molecular weight and is completely restrictable by Eco RI, HinfI, Hae III, and Hind III. The DNA further yielded RAPD fingerprints with decamer primers. We obtained an average of 80 I.lg D N A / g fresh weight of tissue with a maximum yield of 250 ~ g / g . Guillemaut (unpublished) found up to 400 ~g D N A / g of needles from Norway spruce [Picea abies (L.) Karst.]. Kreike et al. (1991) reported 20 ~g DNA from 3 to 5 apices, 5 ~g DNA per embryo, and 20 p.g DNA per endosperm of silver fir. We expect that the large variations in yield in our experiments are due to differences in the age, morphology, and anatomy of the needles, but may also be due to the position of the Eppendorf tube within the Teflon racks (see below). A yield of 80 ~g DNA per g of needles corresponds to about 20 ~g DNA per Eppendorf tube, which should be sufficient for restriction analyses and more than suffi- cient for PCR reactions.

�9 Our slightmodification of the protocol of Guillemaut and Mar6chal- Drouard (1992) simplifies the extraction procedure, and the addi- tion of RNAse treatment and extraction with phenol-chloroform makes further purification unnecessary.

�9 Repeatability depends on the time of harvesting: better repeatabil- ity is obtained during seasons free of frost, and poorer repeatability and lower quality of DNA from samples obtained during freezing weather.

�9 The use of the shaking mill decreases the time required to homog- enize samples by over ninety percent. Because the thawing homogenate is exposed immediately to the extraction buffer, the action of nucleases is minimized.

�9 The procedure also has the advantage that all operations are carried out in Eppendorf tubes, which reduces the consumption of chemicals by at least 10-fold, and makes processing of the samples convenient and rapid. Amounts of DNA suitable for either over- night PCR or restriction analyses can be obtained in a single day.

DNA Miniprep from Fir Needles 121

The p r o c e d u r e can in principle be speeded fur ther by add ing four addi t ional Teflon racks to the shaking mill, which would enable the inves t igator to p e r f o r m 30 s imul taneous homogeniza t ions . We think that repeatabi l i ty can be i m p r o v e d by changing the des ign of the racks to equal ize the pa ths of the beads in the Eppendor f tubes.

In s u m m a r y , the i m p r o v e d protocol mee t s the requ i rements for ana lyz ing D N A p o l y m o r p h i s m s in popu la t ions of w o o d y plants, and to solve some of the p rob l ems associated with plants high in secondary metabol i tes .

Acknowledgments: The molecular genetic investigations on silver fir in the Institute of Forest Genetics are part of a research project funded by the Federal Environmental Agency.Thanks are given to D. Riesner and co-workers (Institute of Biophysics, University of Diisseldorf) who supported the establishment of molecular biological technics in our forest lab. We express our gratitude to M. Schauerte for technical assistance.

B. Ziegenhagen, P. Guillemaut, and F. Scholz (BZ, FS) Bundesforschungsanstalt fCir Forst- und Holzwirtschaft, Institut fCir

Forstgenetik, Sieker Landstr. 2, D-2070 Grot~hansdorf 2, Germany; (PG) Laboratoire de Biologie V6gftale Appliqu6e, Institut Universitaire de

Technologie Louis Pasteur, 3 rue de l'Argonne, F-67000 Strasbourg, France

Refe rences

Couch, J.A., and P.J. Fritz. 1990. Isolation of DNA from plants high in polyphenolics. Plant Mol. Biol. Reptr. 8:8-12.

Howland, D.E., R.P. Oliver, and A.J. Davy. 1991. A method of extraction of DNA from birch. Plant Mol. Biol. Reptr. 9:340-344.

Guillemaut, P., and L. Mar6chal-Drouard. 1992. Isolation of plant DNA: A fast, inexpen- sive, and reliable method. Plant Mol. Biol. Reptr. 10:60-65.

Kreike, J. 1990. Genetic analyses of forest tree populations. Isolation of DNA from spruce and fir apices. Plant Mol. Biol. 14:877-879.

Kreike, J., K. Burg, M. Zechmeister, T. Haider, and J. Gl6ssl. 1991. DNA-fingerprint and RFLP analysis as tools to genetic diversity in populations of fir, spruce and oak. pp. 95- 100 in Genetic Variation in European Populations of Forest Trees, G. MOller-Starck, M. Ziehe, eds. Frankfurt am Main.