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1 - Proteins extraction, 2D electrophoresis and silver staining

The megagametophytes and embryos were collected from non germinated seeds and were individually crushed in µl/mg of 3M urea, 4% FSN - 100 (ZONYL Fluorosurfactant, DuPont), 2% ampholytes (Pharmalytes 3 - 10) and 1% dithiothreitol in a 1.5 ml microcentrifuge tube (Anderson et al, 1985; Bahrman and Damerval, 1989). The mixture was briefly sonicated and extracted for 1 h at room temperature. After a brief centrifugation at 15000 g during 2 min, the supernatants were removed and stocked at -20°C until isoelectrofocusing. The megagametophyte from a germinated seed was collected just before the germinant is ready to cast its seed coat. The seed coat still contain the residual megagametophyte inside. They were individually extracted in 6 µl/mg of UKS (9.5M urea, 5mM K2CO3, 1.25% SDS (sodium dodecyl sulphate), 0.5% dithiothreitol, 2% Pharmalyte pH 3-10, and 6 % Triton-100) buffer (Damerval et al, 1986) and the supernatants were stored at -80°C after centrifugation at 15000 g for 2 minutes. Secondary needle and bud proteins were extracted according to Damerval et al (1986). Liquid nitrogen powdered - tissue was homogenised with 10% TCA (trichloroacetic acid) and 0.07% 2-mercaptoethanol in acetone. Proteins were precipitated for 1h at -20°C. After centrifugation at 15000 g for 15 min, the protein pellets were rinsed with acetone containing 0.07% 2-mercaptoethanol for 1h at -20°C. The supernatant was removed and protein pellet vacuum-dried and solubilized in 15µl/mg of UKS buffer. The pollen proteins were extracted directly by UKS buffer, using 30µl of UKS per mg of pollens and the supernatants were saved after centrifugation at 15000 g for 5 minutes. The quantities of extract to be solubilized in UKS buffer was determined on the basis of protein pattern comparisons in these different tissues. Our goal was to obtained similar amount of proteins for 2D gel comparison. The isoelectrofocusing (IEF) rod gels were 24cm long and 1.5mm diameter. The mixture was 4% acrylamide, 9.2M urea, 2% Triton X-100 and 4% ampholytes (3/4 pharmalyte pH 5-8, 1/4 pharmalyte pH 5-6). The IEF was performed at 40000 Vh with 50mM NaOH and 50mM H3PO4. The 2nd dimension electrophoresis was realised on slab gels (200x240x1 mm) bound to Gelbound PAG (Marine colloids) in a Dalt tank. Uniform gel composition was 11% acrylamide, 0.5M Tris-Cl-, 0.15% SDS, and 1% sucrose at pH 8.8 (Bahrman and Damerval 1989). The running buffer was composed of 0.025M Tris, 0.192M glycine and 0.1% SDS. The gels were simultaneously run and silver stained according to Damerval et al. (1987) in the apparatus described by Granier and de Vienne (1986).

2 - DNA extraction

2.1 - from megagametophyte and needles of maritime pine

2.2 - from leaves (eucalyptus)

2.3 - from buds and leaves (oak)

2.1 - Total genomic DNA isolation from megagametophyte and needles (pine)

DNA was extracted using the CTAB method (Doyle and Doyle, 1987; Bousquet et al., 1990) with some modifications. Megagametophyte tissue was ground to a fine powder in 1.5 ml microfuge tube under liquid N2. Preheated 2% CTAB buffer (700 µl) that contained 0.2% 2-mercaptoethanol and 0.5 mg/ml proteinase-K, was added to the powder, then the mixture was homogenized and incubated in a 65°C water bath for 30 min. After the solution cooled to 25°C, 500µl of 24:1 chloroform:isoamyl alcohol (CIA) was added. After centrifugation (12,000 x g ; 5 min ; 25°C), the upper aqueous layer was transferred, extracted with 1/10 volume of 10% CTAB in 0.7 M NaCl, and the CIA step was repeated. After centrifugation, the DNA in the supernatant was precipitated with an equal volume of isopropanol (500 µl). The nucleic acid pellet was collected by centrifugation (12,000 x g, 15 min, 25°C ), washed in 70% and 95% ethanol, dried briefly under vaccum and resuspended in 200 µl of TE buffer (10 mM Tris-HCl, pH 8 ; 1 mM EDTA) containing 10 µg/ml RNAase. The yield of DNA was approximately 2 µg per megagametophyte.

DNA from needles was prepared in a similar way. Freeze dried needles (25 to 70 mg) were ground under liquid nitrogen using a prechilled mortar and pestle, and transferred to 1.5 ml microfuge tubes. Samples were incubated for 45 min at 65°C in CTAB buffer that contained 1 mg/ml proteinase-K. DNA was extracted from freeze dried needles (4g) of both grand-parents and the hybrid parent using the same method.

The pine DNA was diluted to a working concentration of approximately 1 ng/µl by comparison with the fluorescence of lambda DNA concentration standards on ethidium bromide stained agarose gel.

2.2 - Total genomic DNA isolation from leaves (Eucalyptus)

The extraction of clean DNA was difficult because of the presence of a variety of secondary metabolism compounds and carbohydrates. Nucleic acids were isolated from dry or fresh leaves using standard methods (Saghai-Maroof et al. 1984) with some modifications. Approximately 300mg of dry tissue was ground under liquid nitrogen with PVP (Polyvinylpolypyrrolidone) and suspended in 9ml of preheated extraction buffer (100mM Tris-HCl pH=8.0, 1.4M NaCl, 20mM EDTA pH=8.0, 2% alkyltrimethylamonium bromide (CTAB), 0.2% b-mercaptoethanol). The homogenate was incubated at 65°C for 90min, cooled and emulsified with 4.5ml of dichloromethane and centrifuged at 1,600g for 10min. The aqueous phase was transferred to a new 15ml tube and emulsified again until a clear aqueous phase was obtained. The aqueous phase was then incubated 1h at 37°C with 30mg RNase A. DNA was precipitaded with the addition of an equal volume of isopropanol, dried and dissolved overnight in 1ml buffer (EDTA 0.1mM, ammonium acetate 10mM). DNA was precipitated again with the adddition of 1/20 volume of 5M sodium acetate and 2.5ml ethanol 95%. After 10min centrifugation at 1,600g, the DNA pellet was washed for 20min in 200mM sodium acetate, ethanol 76% and for 20min in 10mM amonium acetate, ethanol 76%. Finally, the pellet was rinsed with ethanol 76%, vacuum dried and resuspended in distilled water. The above method yielded between 15 µg and 20 µg of clean DNA. DNA concentration was measured with a fluorometer, diluted to a working concentration of 7.5ng/µl in sterile water and stored at -20°C .

2.3 - Total genomic DNA isolation from buds and leaves in microtube (oak)

(adapted from Doyle and Doyle, 1990)


-buds: remove scales from 5 medium-size buds. Cut the larger buds in two pieces.

-leaves: cut 2-3 cm² of preferencially fresh leaves into small pieces.

Roots or cambium can be used when buds are two small or leaves not available.

Put the material in microtubes (1.5 ml).

1) For 36 samples (depending on the capacity of the centrifuge), mix 40 ml of extraction buffer with 80µl of 2-mercaptoethanol (0.2% v/v) and add 250µl of this solution in each of the 36 microtubes.

2) For grinding, we use an electric driller with an home-made glass drill that fits within an Eppendorf tube. Between two samples, rince the drill with dH2O and wipe with paper. Wet the drill and cover it with dry sand. Grind while moving the tube up and down until you get an homogeneous mixture. Add 750 µl of extraction buffer and mix well. Put the tubes horizontally in boxes within the shaking incubator during 1 h at 55°C. Take the tubes out and wait 10 min until they cool.

3) Add 400µl of dichloromethane and mix gently until you get an emulsion. Centrifuge 10 min at 13,000 rpm (4°C). With a pipetman (blue tip), pipet the upper phase (avoid to pipet the interphase) and put it in a new labelled Eppendorf tube. If the upper phase is not clear, repeat this sequence again.

4) Add 400µl (2/3 volume) of isopropanol (let in the freezer since the morning) and mix gently. If the DNA pellet doesn't appear, put the tubes in a freezer 1 h at -20°C. Centrifuge all the tubes (with the pellet or not) 10 min at 13,000rpm (4°C). Remove the supernatant. Dry the tubes by letting them upside down on filter paper during 15 min on the bench.

5) Add 1ml of ethanol 76%, vortex and centrifuge 10 min at 13000 rpm (4°C). Remove the supernatant carefully and dry 15 min in a speed-vac at room temperature (if no speed-vac then dry longer on the bench).

6) Add 100µl dH2O. This is your stock solution. Estimate the DNA concentration and dilute to get a 100ng/l solution. Make a diluted solution at 2ng/l that will be your work solution.

Extraction buffer (1l)

ATMAB (Alkyltrimethylammonium bromide) 20g

EDTA 0.5 M , pH=8 40 ml

Tris HCl 1 M , pH=8 100 ml

NaCl 5 M 280 ml

Heat the solution at 65°C to allow the dissolution of ATMAB.

3 - RAPD in Maritime pine, Eucalyptus and Oak

RAPD assay in Maritime pine

The RAPD reactions were performed following the method described by Williams et al., (1990). Primers were purchased in kits (OP-A through OP-Z) from Operon Technologies Inc. (Alameda, CA). The volume of the reaction mixture was 15 µl and contained 8 mg/ml nonacetylated Bovine Serum Albumin (New England Biolabs), 5 to 10 ng genomic DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton-X, 200 mM of each dATP, dCTP, dGTP, dTTP, 20 ng 10-mer primer and 1 U of Taq DNA Polymerase. The mixture was covered with 50 µl of mineral oil to avoid evaporation and amplifications were carried out in 96-well microtitre plates using MJ Research PT-100 thermal cycler (MJ Research, Inc. Watertown, MA). The reaction conditions were 41 cycles, consisting of 1 min/92°C (denaturation), 1 min/35°C (annealing) and 2 min/72°C (extension) with a ramping time of 1°C /3s. The DNA fragments were separated by standard electrophoretic methods (1X TBE) on 2% horizontal agarose gels, stained with ethidium bromide (0.2 mg/ml) and the gels were photographed.

RAPD assay in Eucalyptus and Oak

Oligonucleotide primers (10-mers) purchased from Operon Technologies Inc. (Alameda, CA) were used as single primers for the amplification of RAPD sequences (Williams et al. 1990). The reaction mixture (25 µl total volume) contained 67mM Tris-HCl pH=7.5, 2mM MgCl2, 1 ng BSA (Bovine Serum Albumin), 0.2% b-mercaptoethanol, 16mM ammonium sulphate, 200mM of each dNTP, 0.55mM of primer, 15ng of genomic DNA and 1U of Taq DNA polymerase (GIBCO BRL), overlaid with mineral oil. Amplification was performed in a thermal cycler model PHC-3 (Techne). The thermocycler programme was : preliminary denaturation (4min, 94°C ), followed by 35 cycles consisting of denaturation (45s, 92°C ), annealing (45s, 40°C ), and extension (1.5 min, 72°C ), and a final extension (10min, 72°C ).

4 - CpDNA and mtDNA primers

5 - PCR - RFLP

The amplification is realised with 5 µl of DNA (~5 ng/l) and 20 µl of a PCR mix. A first denaturation (4 min at 94 °C) is followed by 30 cycles of amplification (the annealing temperature depend of the primers used in the PCR) and a final elongation (10 min at 72°C). The amplification products are stored in the fridge.

PCR Mix for one sample:

2Xbuffer 12.5 µl

Primer 1 (2 µM) 2.4µl

Primer 2 (2 µM) 2.4 µl

dH2O 2.65 µl

Taq polymerase (5 U/µl , Red Goldstar) 0.05 µl (i.e. 0.25 U by sample)

2Xbuffer (1 ml):

10X (provided with the polymerase by Eurogentec) 200 µl

MgCl2 (25 mM, provided with the polymerase by Eurogentec) 144µl

dNTP (5 mM for each dNTP) 40µl

dH2O 616µl

PS: MgCl2 is added in order to have a final concentration in the PCR reaction of 2 mM which was considered to be the optimal concentration for the amplifications.


5µl of the amplification product is added to a digestion mix. At the end of the reaction, 5µl of bromophenol blue is added and the samples are stored in the fridge when they are not immediatly analysed. The migration is realised on 8% polyacrylamide gels, 1.5 mm thick. The electrophoresis apparatus is the model SE600 of HOEFER. We use combs with 28 teeth (a microplate of 96 samples requires 4 gels for one primers-enzyme combination). The migration is made at 300V (the amperage for one gel is comprised between 30 and 50 mA). The gels are cooled at 15°C with a refrigerating system. After migration, the gels are stained with ethidium bromide.

Digestion mix:

enzyme buffer           1.5 µl

enzyme (10 U/µl)     0.5µl

( i.e. 5U by sample)

dH2O                       13 µl

Acrylamide 40%:

acrylamide (Biowhitaker (Serva) 390 g

bisacrylamide (Sigma)                    10g

8% polyacrylamide gel:

H2O                         26.25 ml

Acrylamide 40%     7.5 ml

TBE 10X                 3.75 µl

APS 10%                180µl

TEMED                  30µl

TBE 10X(for 1 liter):

Tris                                108 g

Boric acid                         55 g

 EDTA pH=8 (0.5M)      40 ml

Final pH=8.3

6 - SSCP

For the SSCP analysis, the digested PCR products (2 µl) are added to a denaturing solution (5 µl) containing 95% formamide, 10mM NaOH, 0.05% of xylene cyanol and 0.05% of bromophenol blue. After 4 minutes at 94°C, to separate the two strands, the solution is immediately cooled on ice. The single-stranded DNA fragments are separated in a 0.75mm x 16cm x 18 cm, non-denaturing Mutation Detection Enhancement (MDE) gel (0.5X MDE, Bioprobe Systems; 0.6X TBE). Electrophoresis lasts 16 to 18 h at 7.5 to 10 V/cm at a constant temperature of 15°C in 0.6X TBE buffer. The gels are then silver-stained with the method of Bassam et al. (1991). After fixation with acetic acid 10% during 20 minutes, the gels are impregnated with silver nitrate and formaldehyde.The gels are then rinced with deionized water (3 times during 2 minutes). Colour development is obtained with sodium carbonate, formaldehyde and sodium thiosulfate and the reaction is stopped with acetic acid 10% during 10 minutes. The gels are finally rinsed with deionized water during 10 minutes and dried between plastic sheets .

colour impregnation (30 min):

silver nitrate 0.1%

formaldehyde 0.056%

colour development (5 to 10 min):

formaldehyde 0.056%

sodium thiosulfate 2 mg/l

7 - Microsatellites

Microsatellites or simple sequence repeats (SSRs) are highly mutable loci which may be present at many sites in a genome. Since the flanking sequences at each of these sites may be unique, if SSR loci are cloned and sequenced, primers to the flanking regions can be designed to produce a sequence-tagged microsatellite (Tautz, 1989).

Microsatellite primers used for the two species Q.petraea and Q.robur were developed by Austrian colleagues (Steinkellner et al., Plant. Mol. Biol., in press.).

Reactions are performed in a total volume of 25µl, included 0.2 µM of each primer, 67 mM. Tris HCL, pH 8, 1 ng. of BSA, 0.2% mercaptoethanol, 16 mM. ammonium acetate, 100µM of dNTP and 0.8 unit of Taq DNA polymerase (Gibco BRL). Amplification proceeded first with one denaturing step of 94°C for 4 min. and then with 30 cycles of 94°C for 45 s., Ta for 30 s. , and 72°C for 30 s..

PCR products are then mixed with a stop solution ( 95% Formamide, 0.05% Bromophenol Blue, 0.05% Xylene cyanol, 10µM NaOH) and denaturated at 94°C for 4min. before being resolved on 6% standard denaturing polyacrylamide gels : 42 g urea, 15 ml 40% acrylamide, 10 ml 1X TBE, 100 ml qsp. deionized water. For the following silver staining, Bind Silane (PHARMACIA) is applied on one of the plates of the sequencing electrophoresis system before pouring the gel.

The run parameters depend on the equipment (length and thickness of the gel). It should be performed at constant Watts to reach and maintain a constant temperature of 60°C during electrophoresis. Time of the run depends on length sizes of the PCR products.

The staining was performed following a fast silver staining protocol.

The gel remains sticked to the plate during the following steps :

- fixation : 30 min. in 10% acetic acid solution,

- 4 baths of 2 min. in deionized water,

- staining : 30 min. in a solution with 0.1% AgNO3, and 0.056 % formaldehyde,

- development in a solution with sodium carbonate 30g/l, sodium thiosulfate 2mg/l, and 0.056 % formaldehyde, until the PCR products are clearly detected,

The reaction is then stopped with chilled 10% acetic acid solution.

The gel can then be scanned and analyzed on an image analysis software.

8 - AFLP (Amplified Fragment Length Polymorphism)

This technique is essentially intermediate between RFLP and PCR (Vos et al. 1995). It involves restriction digestion of the genomic DNA, followed by preamplification and selective amplification of the restricted fragments. The amplified products are separated on a sequencing gel and can be visualised after exposure to X-ray film (when radioactive labels are used).
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