Protocols for Schizophyllum commune

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Sep 2, 2012 - I generally place plates in a plastic box with a lid that keeps the air humid, but ..... scroll around a bit before considering it not an common A.
Protocols for Schizophyllum commune 2 September 2012

Protocols collected, created and adapted by Bart Nieuwenhuis over the years of my PhD project. The results of the research are published in different papers and in my thesis entitled “Sexual selection in Fungi”. Feel free to use these protocols. Reproduce them, adapt them, republish them. All experiments were performed at the University of Wageningen, the Netherlands. Thesis is available for free from the Wageningen University Library website. Or google it. For any questions comments or whatever, please contact me at

Index Protocols for Schizophyllum commune .............................................................................. 1 Index ................................................................................................................................... 1 Introduction ......................................................................................................................... 3 Media etc. ............................................................................................................................ 4 Minimal medium: Schizophyllum commune SMM ....................................................... 4 Complete medium: Schizophyllum commune SCM ...................................................... 4 Trapping medium: Schizophyllum commune STM ....................................................... 4 Migration complete medium: Schizophyllum commune (MCM) .................................. 5 FeCl3 stock solution ........................................................................................................ 5 SMM + MgSO4 ............................................................................................................... 5 Antibiotics ....................................................................................................................... 6 Trace elements stock solution ......................................................................................... 6 Techniques .......................................................................................................................... 7 Growth and storage of Schizo ......................................................................................... 7 Storage of mycelium at -80oC ......................................................................................... 7 complicated 1 .............................................................................................................. 7 complicated 2 .............................................................................................................. 7 quick and dirty (and GREAT!) ................................................................................... 8 Mushroom production (fructification) ............................................................................ 8 24well plates for cultures ................................................................................................ 8 Different types of selection plates................................................................................... 9 Phleomycin resistance plates: ..................................................................................... 9 Nourseotrycin resistance plates: ................................................................................. 9 Colony Counting plates: ............................................................................................. 9 Colony Counting resistance markers: ......................................................................... 9 Creating liquid culture .................................................................................................. 10 Racetracks ..................................................................................................................... 11 Protoplast generation for isolation of protoclones ........................................................ 12 Materials ................................................................................................................... 12 Procedure .................................................................................................................. 12 Regeneration from fruiting body .................................................................................. 14 Test for mating type ...................................................................................................... 14 mon-mon mating ....................................................................................................... 14 Di-mon mating .......................................................................................................... 15 DNA extraction methods .............................................................................................. 16 Phenol/chlorophorm LETS ....................................................................................... 16 CTAB with nuclei isolation ...................................................................................... 17 Qiagen Mini Plant ..................................................................................................... 18 Chelex ....................................................................................................................... 18 KASP SNP genotyping ................................................................................................. 18 RFLP: mtDNA fragment length polymorphisms .......................................................... 19 Phenotypic characteristics ............................................................................................. 20 Growth speed ............................................................................................................ 20 Nuclear migration speed ........................................................................................... 20 References ......................................................................................................................... 21

Introduction Here I give a short overview of the different protocols as used by me over the last few years during my Phd research. I’ve tried to be as precise as possible when describing the methods and materials used. I’m precise so you can repeat what I’ve done to replicate my results, explain simple things which might seem trivial, but that saved me a lot of time, so why not share, and sometimes these small things can be of importance, or even crucial. See what you can use, and forget the rest. Schizophyllum commune is a fungus that has been used since the mid-20th century by different people as a model basidiomycete fungus. It fructifies easily, grows fast, can be grown on synthetic media and has a fast sexual generation time. The research performed by John Raper and his co-workers that makes up much of the content of the amazing book “Genetics of sexuality in higher fungi” (Raper 1966) is done with Schizo. Many techniques are available for the fungus, such as transformations (van Peer et al. 2009), and protoplasting (de Vries & Wessels 1972). The genome has been sequenced (Ohm et al. 2010) and much research is performed on the proteomics of the fungus. Due to its classification as an emergent pathogen the medical world is spending efforts on investigating the fungus too (Chowdhary et al. 2012). Schizophyllum commune lifecycle. A haploid spore germinates and grows by forming a mycelium - the monokaryon - in which a single type of haploid nuclei is present in all hyphae. Mating occurs by fusion of hyphae of different individuals, which exchange nuclei. The nuclei migrate back from the point of plasmogamy and through the entire mycelium, thereby forming a long-lasting dikaryon. When the conditions are suitable, the dikaryon forms a fruiting body, the mushroom, in which basidia are formed. Resulting haploid air-borne spores disperse at long distances and germinate to form a monokaryon.

Media etc. For none of the media the pH needs to be adjusted.

Minimal medium: Schizophyllum commune SMM (Dons et al. 1979) Dissolve in demi-water: Glucose KH2PO4 K2HPO4. 3 H2O MgSO4. 7 H2O Trace elements solution FeCl3 solution (i.e. 5 mg) L-Asparagin Agar Add after sterilization: Filter sterilized Thiamine 10mg/100ml

Per l 20 g 0.46 g 1.28 g 0.5 g 1 ml 1 ml (=5mg) 1.5 g 20 g (2%) 1.2 ml

Complete medium: Schizophyllum commune SCM (Dons et al. 1979) Dissolve in demi-water Glucose KH2PO4 K2HPO4. 3 H2O MgSO4. 7 H2O Yeast extact Pepton

Per l 20 g 0.46 g 1.28 g 0.5 g 2g 2g

Trapping medium: Schizophyllum commune STM (James & Vilgalys 2001) Dissolve in demi-water Malt extract 1.5% Yeast extract 0.3% Glucose 0.5% Agar 1.5%

Per l 15 g 3g 5g 15 g

Migration complete medium: Schizophyllum commune (MCM) (Snider & Raper 1958) Dissolve in demi-water Bacto peptone Glucose KH2PO4 K2HPO4. 3 H2O MgSO4. 7 H2O Agar 1.5%

Per l 15 g 2g 0.46 g 1.28 g 0.5 g 15 g

FeCl3 stock solution Dissolve in demi-water Per l FeCl2

5g

SMM + MgSO4 (Sonnenberg & Wessels 1987) This medium is used for protoplast recovery. Dissolve in demi-water: Glucose KH2PO4 K2HPO4. 3 H2O MgSO4. 7 H2O Trace elements solution FeCl3 solution (i.e. 5 mg) L-Asparagin Agar Add after sterilization: Filter sterilized Thiamine 10mg/100ml

Per l 20 g 0.46 g 1.28 g 240 g 1 ml 1 ml (=5mg) 1.5 g 20 g (2%) 1.2 ml

Pour at ~60oC. At lower temperatures the medium becomes turbid due to crystallisation of salt. Do not autoclave twice, as the agar will not solidify again. The medium can be melted again, but should be poured at ~65oC because the medium is even more likely to occur than with fresh medium.

Antibiotics Nourseotrycin: Stock: 8 ug/ml Usage: 8 ul/ml (1:1000) Phleomycin: Stock: 25 ug/ml Usage: 10 ul/ml (2:5000) Benomyl: Stock: (standard what we have in fridge) Usage: 1:100 Tetracyclin: Stock: (standard what we have in freezer) Usage: 1:1000 Streptomycin: Stock: (standard what we have in freezer) Usage: 3:1000

Trace elements stock solution (Dons et al. 1979) [i.e. Whitaker 1951] Dissolve in demi-water HBO3 (NH4)6Mo7O24 . 4 H2O CuSO4 . 5 H2O ZnSO4 . 7 H2O MnSO4 . 4 H2O CoCl2. 6 H2O Ca(NO3)2 . 4 H2O

Per l 60 mg 40 mg 0.2 g 2.0 g 0.1 g 0.4 g 1.2 g

Found in MolLab GK FunLab Tr. El. GK kitchen GK kitchen Tr. El. GK kitchen Tr. El. GK kitchen Plofk Kitchen

Techniques Growth and storage of Schizo Strains are generally grown at temperatures of 25-30oC in the dark in climate cabinets. Strains grow about 1 cm per day, but this can vary greatly, depending on strain, medium and temperature. I generally place plates in a plastic box with a lid that keeps the air humid, but doesn’t seal the plates completely. Other labs have climate chambers with humidity control. Dikaryons generally grow faster than monokaryons. Dikaryons grown in the dark grow in a similar fashion to monokaryons, i.e. homogeneous with smooth edges. You can play around with the speed of growth by changing the temperature. For instance, if mating type crosses are not yet perfectly visible, the plates can be left of the bench overnight to get a few more mm growth. Plates can be stored at 4oC for many months. As long as there are no infections of the plates, strains from over a 3 years old can be sub-cultured without obvious phenotypic changes, even when the plate is completely dried out (though I did not test if they really didn’t differ!). Many Schizo strains are capable of growth at 4oC so put parafilm around the plate to avoid cross fertilizations.

Storage of mycelium at -80oC complicated 1       

Macerate a small piece of mycelium from plate (take as little agar as possible) Grow the macerated mycelium in liquid SMM Filter the mycelium on sterile nylon Resuspend in 15% glycerol solution Store in -20oC for two hours (not ne) Store at -80oC To use: thaw the tube on the bench. Turn over the tube and spread out on plate or plates

complicated 2       

Grow mycelium on SMM covered with cellophane (see also DNA isolation) harvest mycelium into tubes (1 per tube should be enough) add 15% glycerol Store at -80oC To use: thaw tubes, turn over on plate and spread out the mycelium bits. Each bit will start growing. Problem is that each time you use a tube, you use the entire tube, so multiple tubes are needed in this way.

quick and dirty (and GREAT!)    

take a plate with culture and cut it into little blocks put blocks in eppendorf tube and place tube at -80oC To recover: take a block from the tube and place on fresh SMM. Return tube quickly to -80C (always have extra tube in -80oC because of possible freeze/thaw damage) I’ve not tested how long the cubes can be stored in this way, but at least for 3 years.

Mushroom production (fructification) Mushroom production is induced by growth in light:  Inoculate a plate (SMM 5cm or 9cm) with a plug of dikaryotic mycelium in the middle.  Place plate upside down in incubator.  Labstrains (e.g. 4-40x4-39, 4.8AxB) fructify easily. o Place them at 27/28oC in 12h light dark cycle or constant light o Mushrooms within about a week  non-lab strains can be more difficult o Some fructify at 27oC o Some need lower temperatures, especially those collected in West/North Europe.  Don’t use parafilm. Apparently too high CO2 concentrations inhibit fructification (Luis Lugones, personal communications).

24well plates for cultures When isolating single spore isolates or regenerated protoplasts, I found 5cm plates often a bit overkill. Each plate takes 10ml medium, and often will be discarded after a screen as only one or a few isolates are needed. Using 24well plates is often very convenient.  Take a fresh plate and add with a sterile repetition pipet (I used 50ml pipets, sterilized with 70% EtOH) 1ml SMM to each well.  A spore or neohaplont can be transferred and inoculated for 3 or 2 days respectively.  From a culture in a well about 20 mycelium-plugs can be obtained which is most of the time more than enough.  Transfer the strains of interest to a larger plate.  If more is needed, transfer to a larger plate. Pay attention Don’t grow them too long as the isolates will grow out of the wells and can mate with each other! Schizo often continues to grow at 4oC so don’t use them for long term storage!

Different types of selection plates Making resistance plates is easy for round plates. Just add the right amount to an empty plate, add the 55oC SMM on the droplet and then carefully mix the medium on the plate by swirling the plate. For a square plate this does not work. Use a sterile 50ml Greiner tube instead. Add antibiotic, add SMM and carefully mix (careful for bubbles) Pour out in one go. Tubes can be reused. (In case of bubbles, use a red hot needle, move above the bubbles and the heat will pop them, or quickly move a Bunsen-burner over the plate).

Phleomycin resistance plates:   

stock solution: 25mg/ml final concentration: 10ug/ml use: 2:5000 o SMM5  4ul/plate o SMM9  10ul/plate o SMM15 and SMM12sq 20 ul/plate

Nourseotrycin resistance plates:   

stock solution: 8mg/ml final concentration: 8ug/ml use: 1:1000 o SMM5  10ul/plate o SMM9  25ul/plate o SMM15 and SMM12sq  50 ul/plate

Colony Counting plates: use plates with 0.02% Triton. This inhibits spreading of the mycelium and thus results in dense but small colonies.  stock solution 1% or 5% triton  Add 500ul or 100ul per SMM9 plate  make a spore dilution and add the right amount to the plate  spread with glass beads  Dry in flow cabinet until dry  place at 27oC for ~4 days (check daily)  count colonies on colony counter

Colony Counting resistance markers: This is used to count ratios of spores carrying a resistance marker vs. not carrying that marker. For instance after performing competitions, or before competitions to assess actual ratios (used in chapter 6 of thesis).

As “Colony counter plates” followed by:  when the colonies are still very small, but well visible, count them all  Then add 0.5ml of diluted antibiotic per plate. The dilution should be such that the 0.5ml contains the same amount as would normally be added to the entire plate.  For phleo this is: 0.5 mg/ml, and for nourseo: 0.4 mg/ml  Spread the dilution over the plate by tilting, or when this does not work with beads. If using beads, be careful that no aerial hyphe are present as they might break off and start new colonies elsewhere on the plate!  Dry the plates in flow cabinet  Incubate again at 27oC for ~2 days.  Two types of colonies can be observed now which are in non-overlapping categories: Large and small ones.  Large colonies that continue to grow were resistant, the others not.

Creating liquid culture 

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Prepare in advance: o Sterile Waring blender cup o Sterile Erlenmeyer 250 or 500ml o SMM liquid o Tetracycline (5mg/ml) and streptomycin (10mg/ml) Remove the aluminium foil and keep it sterile! Take a piece (~1 cm2) of agar with culture on it and put in waring blender cup Add 100ml SMM and Add o 300ul tetracycline o 100ul streptomycin o 1 ml benomyl Place Aluminium foil (or green cap) back on blender cup Place cup on blender and blend for 2x 15sec (hold the lid tight with your other hand to avoid liquid all over the place) Transfer the macerate to the Erlenmeyer Place in shaker at 27oC 175rpm Culture is ready after 2 to 3 days Blend in between if yield is low and put macerate in fresh clean sterile Erlenmeyer to avoid contamination. Blend 5 hours before use (1x 15sec) Use pipette tips with cut of tops to prevent clogging.

Racetracks These are the tracks I used to test migration speed of nuclei, or to test competition (described in Chapter 6 of the thesis).  Pour 12 cm square plates with SMM (to increase reproducibility between tracks use a sterile 50ml Greiner tube an pour 50ml per plate, no less or the tracks break when handling them)  Take a sterile container for a blender (in our lab Waring MC3 (50-250 mL)).  Take a fresh 5cm plate (SMM) covered with the monokaryon of choice (When few plates to be made, take half or a quarter plate).  Add 50ml saline and tip in half a plate including the agar medium (of 1 plate and 100ml).  Cover the cup (in our case with the aluminium foil used to sterilize, keep pressed down firmly!)  Blend until the agar is completely gone and no lumps are visible anymore (these will clog your pipet).  Pipet 1ml on a square plate and spread with beads.  Incubate (27oC, dark) until the entire plate is covered in white fluffy mycelium (~4-7 days).  When the plate has formed a homogenous lawn, it is time to cut the plate in tracks.  For this I used a scalpel that I gaffa-taped to a spatula of the same length, spaced with some blocks of eraser so the distance between the spatula and the scalpel was 5mm. The spatula was used as a guide. The first cut made with spatula against the side of the petridish, and then every next lane, by letting the spatula follow the previous cut.  Sterilize the spatula and scalpel by burning with alcohol (don’t light the gaffa!) Use a sharp blade or you’ll damage the mycelium.  Then use a separate scalpel to cut them in the right length.  Racetracks can then be lifted out carefully with a sterile spatula and transferred to other square plates to space them out.  I used 5 tracks per plate, though 6 is still possible.  the tracks dry out faster than plates, so store them at high humidity (e.g. add a tissue sticking out of a beaker with water to the box).

Protoplast generation for isolation of protoclones Based on protocol as described in (de Vries & Wessels 1972)

Materials All per sample 50 plates (SMM + MgSO4 0.5M) Liquid SMM in Erlemeyer Blender Turrax with sterile mixer 500ml MgSO4 1M 500ml MgSO4 0.5M 100ml Sorbitol (1M)

MQ water 4 Büchner funnels Glass funnel with sterile glass wool 100ml MgSO4 0.5M penicillin/streptomycin (100mg/ml)

Procedure

Lysis

Mycelium preparation

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Isolation

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Grow S. commune mycelium at 25˚C in MM in 100ml Erlenmeyer on a shaker at 200 rpm. Macerate the mycelium the day before harvest for 15 seconds in a Waring blender. Grow the macerate at 25˚C in 100 ml MM in 500ml Erlenmeyer on a shaker at 200 rpm for ~20h. When needed the culture can be diluted with MM after elimination of part of it and grown further. Harvest the mycelium by centrifugation of part of the culture in a sterile 50 ml plastic tube Wash once with 1M MgSO4 and spin down again. Reduce the mycelial pellet to 5 ml. Make a solution of 10 ml 1M MgSO4 adjust to pH5.8 using 30 mM malate buffer and supplement with 20mg lyzing enzymes (Applied Plant Research, Wageningen UR, The Netherlands) Filter sterilize this solution with 0.45µm filter Resuspend the mycelium well in the lysing solution and incubated for 2,5 hours at 30˚C (or at 24oC?) Add one volume sterile water and incubate the tube further for another hour. Centrifuge the tube for 5 minutes at 360 g (1400 rpm in our swing out) and transfer the supernatant to a fresh tube. Discard left over small pieces of mycelium by sieving the suspension trough sterile glass wool. Add one volume sorbitol (1M) and mix well. Put the tube to rest for 15 minutes. Centrifuge the tube for 15 minutes at 2200 g (3500 rpm in our swing out). Discard the supernatant and carefully resuspend the protoplasts in the pellet into 25ml Mg SO4 (0.5M).

Regeneration

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Count protoplasts with a counting chamber Centrifuge again and resuspend in 500µl MgSO4 (0.5M) Make a 6 fold dilution series for 5 times: o 50µl in 250µl MgSO4 + penicillin/streptomycin (1mg/ml) o Repeat this 5 times Plate 20 µl of each dilution on 0.5M MgSO4 + M M plates Incubate the plates at 25oC overnight

Regeneration from fruiting body      

Dissect a small piece from the ‘stem’ of a fruiting body trying not to collect any spores Alternatively, with a sharp blade, cut off a little bit of wood from where the mushroom was broken off. Wash this piece successively in 96% EtOH, 70% EtOH and twice in H2O Put the small piece on a SMM plate with 0.2 ml Augmentin (or other antibiotic which is at hand) Incubate at 28oC Transfer outgrowth to fresh SMM after 2 days of growth

Test for mating type This is based on the paper by Papazian (Papazian 1950). Mating type in Schizophyllum is defined by two unlinked loci (matA and matB). Each mating type is multiallelic. A dikaryon thus carries two alleles of each mating type locus and can produce 4 different types of spores (matA-1+matB-1), (matA-2+matB-1), (matA-1+matB-2) and (matA2+matB-2). To test which mating type a spore has, crosses can be performed.

mon-mon mating     

Use rather thinly poured SMM plates for this. Place two monokaryon mycelium plugs about 5mm from each other. incubate until the two mycelia together are about 3cm in diameter. Check the phenotype. There are four possibilities 1. No mating type shared (Dikaryon): this can be seen by rugged edges and non-homogeneous growth of the mycelium, often an interaction zone



(barrage) between the two mycelia and a the microscopic level clamps. 2. same A, different B (common A): no aerial mycelium is formed. Microscopically the mycelium grows with many bumps on it. 3. different A, same B (common B): like monokaryon but with barrage between the colonies. 4. both A and B same (monokaryon): the two mycelia grow without interaction, and are round and with a smooth edge, with white fluffy mycelium. The microscopic features can be observed by placing the plate upside down under a microscope with 100x to 200x magnification, depending on the microscope (we have one with a 16x objective that has a larger than normal focal distance which can be used with almost all plates with not too much medium). Or use an inverted microscope. o Look at the edge of the colony where the mycelium is not too thick. o Check for clamps at the places where a hypha branches. A clamp is often observable at the opposite side as the branch but in the same plane. o Common A bumps and blips are not always present on all hyphae, so scroll around a bit before considering it not an common A.

Di-mon mating When a dikaryon is mated with a monokaryon, generally, only one nucleus migrates into the monokaryon. A quick mating test can be done on checking the nuclei in a dikaryon. By mating a dikaryon with

DNA extraction methods For all methods use mycelium grown on cellophane covered plates.

Phenol/chlorophorm LETS This is just any old DNA extraction method. And it doesn’t work very well for Schizophyllum. When pelleting the DNA from the solution, the solution becomes snotlike (sugars or other stuff in the cells?), and when removing the solution, the DNA is removed with it. Maybe it’s just me, but I suggest using a different method. 1 Add ± 5 glass beads (approx 2-3 mm diameter) to 1.5ml epp-tube that contains the mycelium and throw in liquid Nitrogen canister 2 Take the frozen tube and immediately grind the mycelium for 10 seconds with a bead beater machine (repeat when needed) 3 Add 1 ml of LETS buffer and mix well (vortex) 4 Centrifuge for 5 minutes at 13.000 rpm 5 Transfer 700 μl of the supernatant into a fresh 2 ml eppendorf tube 6 Add 5 μl of proteinase K (stock 20 μg/μl is kept at -20 ºC) and incubate for 1 hour at 37 ºC 7 Add 300 μl phenol and 300 μl SEVAG and mix well (vortex) 8 Centrifuge for 15 minutes at 13.000 rpm 9 Transfer 600 μl of the supernatant to a clean 1.5 ml eppendorf tube 10 Add 300 μl of SEVAG, mix and centrifuge for 15 minutes at 13000 rpm 11 Transfer 500 μl of the supernatant to a clean 1.5 ml eppendorf tube 12 Add 300 μl (0.6 x volume) isopropanol, mix, and store this mixture for 15 minutes at -20 ºC or overnight at 4 ºC 13 Centrifuge nucleic acids out of suspension in an eppendorf centrifuge for 15 minutes at 13.000 rpm 14 Discard the supernatant with a micro-pipet and wash the pellet carefully (pellet must stay in place) using 100 μl of icecold 70% ethanol from the -20 oC freezer 15 Centrifuge 1 minute at 13.000 rpm in an eppendorf centrifuge 16 Carefully remove supernatant with a pipet and dry the pellet under vacuum 17 Dissolve overnight in 50 μl of Milli-Q at 4 oC 18 Mix solution using a pipet and store at 4oC or -20 oC LETS-buffer: o M LiCl  10 mM EDTA pH=8.0  10 mM Tris pH=8.0  0.5 % SDS SEVAG:  isoamylalcohol:chloroform 1:24

CTAB with nuclei isolation I never used this, but this might be useful, as it most likely removes sugars that interfere with the isolation as described above. Especially for larger DNA quantities this method should work very well. The method is derived from Plant Genetics group of the Laboratory of Genetics, Wageningen, where it’s used for DNA isolation in Arabidopsis. CTAB extraction buffer (autoclave before add BME) per liter: [Stock] Add [Final] M Tris pH 7.5 100 ml 100 mM 5M NaCl 140 ml 0.7 M 0.5 M EDTA 20 ml 10 mM BME (2-Mercaptoethanol 10 ml 1% add before use) CTAB 10 g 1% dH2O 730 ml -------Nuclei extraction buffer:  10 mM TRIS-HC1 pH 9.5  10 mM EDTA pH 8.0  100 mM KCl  500 mM sucrose  4 mM spermidine  1 mM spermine  0.1% bata-mercaptoethanol) Lysis Buffer:  10% Triton X-100 in Nuclei extraction buffer 1. Ground 0.5-1 gram mycelium to a fine powder in liquid nitrogen using a mortar and pestle and transfer to a 15-ml polyethylene centrifuge tube containing 10 ml of ice-cold Nuclei extraction buffer. 2. Mix the suspended tissue thoroughly with a wide-bore pipette and filter through (by a brief spin at less than 100g for 5 seconds) two layers of Miracloth (CalBiochem) into an ice-cold 50-ml polyethylene centrifuge tube. 3. Add 2 ml Lysis Buffer to the filtered suspension and mix gently for 2 min on ice. Pellet the nuclei by centrifugation at 2000g for 10 min at 4oC. 4. Add 500 ul CTAB extraction buffer to nuclei pellet, mix well by inverting tube an incubate 30 min at 60oC, let sample cool for 5 min at RT 5. Add 350 ul Chloroform/Iso Amyl Alcohol (24:1) and invert and mix gently for about 5 min and spin for 6k rpm for 10 min 6. Transfer the upper layer (450 ul) to a new 2 ml containing 450 ul Isopropanol, and mix by inverting several times and pellet DNA by spinning13000 rpm for 3 min 7. Wash DNA pellet in 70% EtOH 8. Resuspend DNA in sterile DNase free water (containing RNaseA 10 μg/ml). After re-suspension incubate at 65oC for 20 min to destroy any DNases and RNases and store DNA at 4oC (or -20oC for long term storage).

Qiagen Mini Plant Do according to manufacturer’s protocol. Use 1 colony grown on SMM with cellophane of about 3cm diameter. This method gives clean DNA for e.g. KASP genotyping

Chelex This method results in a dirty DNA solution, but it’s very quick and can be used for standard PCR DNA amplification. Do use extra MgCl in the buffer then.  Use a 5% w/v suspension of Chelex X-100 (mix before using)  Add 200ul to a pulverized colony  Add 10ul proteinaseK (20 mg/ml)  Incubate for 15min at 57oC  Incubate at 95oC for 5 min  Spin down the Chelex before use.

KASP SNP genotyping KASPAR snp detection (see chapter 3 of thesis for primers used). SNPs are identified using KASPar v4.0 SNP Genotyping Systems (KBioscience, Hoddesdon, England). This is a simple and very cheap method to test for snps. Only the primers have to be developed for the right location. The company used to have a program for that, but they figured out they can make more money with designing primers than just supplying consumables, so that program is not available anymore. I might still have a copy, and maybe some other people too (though with our upgrade to Windows7 the program stopped working, so an old computer is needed). The idea is that you do a PCR with a mix of two forward primers and a universal revers. The two fwd differ at the 5’ and 3’side. At the 3’ with one basepair (the SNP of interest) and at the 5’ they have different tails. To the tails a dye can stick (a different flurophore for each tail) which results in a quantitative reading. By looking at the relative expression of the two different florophores can be deduced if the genotype is snp1, snp2 or heterozygous. Primers used can be found on the next page. Primers are very long, because the mtDNA has a very high AT content. I had to change the standard protocol a bit, to make it work for mtDNA DNA used needs to be clean. I used the Qiagen plant mini dnEasy kit for this. Use 10 times diluted DNA Per sample:  5ul DNA  5ul KASP 2X mix  0.08 ul Essay-mix  0.14 ul MgCl2 50mM as provided by KBiosciences to increase concentration to 2.2mM Essay-mix is:  12 ul forward primer 1  12 ul forward primer 2

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30 ul reverse common primer 46 ul ddH2O

I used a CFX96 PCR machine from Bio-Rad Laboratories and the green 96well microtiter plates that come with it. The standard KASPar thermocycling protocol was optimized for low GC content  94oC 15 min  Cycle 9 times o 94oC for 20” o 65oC for 1’ with decrement of 0.7oC per cycle  Cycle 34 times o 94oC for 20” o 57oC for 1’  20oC for 30”  Do measurements for VIC and FAM  50oC 1’ The ‘allelic discrimination’ tool of CFX Manager Software (V2.0, Bio-Rad Laboratories) can be used to determine the SNP allele for each strain.

RFLP: mtDNA fragment length polymorphisms According to (Specht et al. 1983) We tried this for chapter 3. Unfortunately this did not work very well. We did get it working at some point for some strains, which indeed showed some polymorphisms with these two enzymes, though due to difficulties with isolating enough DNA from each strain with standard isolation techniques (see DNA isolation), this was not a feasible approach for the many samples we had planned to do in our experiment. We resorted to SNP identification for the mtDNA. Enzyme Cleavage site(s) Cut frequency HaeIII 1752, 2090, 2265, 4205, 6604, 11190, 26313, 28400, 14 36435, 38047, 40837, 41247, 43527, 43833 MspI 2280, 3007, 3030, 8485, 12148, 14746,16253, 17555, 17 18411, 22201, 22207, 24874,28924, 30398, 34730, 42620, 46859

Phenotypic characteristics Growth speed Measuring growth can be easily done by inoculating a 9cm plate with medium and inoculating it at a certain temperature. Measure after a day and mark it. Remark it after 2 more days and measure the difference. This is much more precise than only measuring once, and gives highly reproducible results.

Nuclear migration speed This is easy to measure, but not very insightful. Speed is generally not different between migrating nuclei of different strains, but depends on the receiving mycelium. See chapter 6 of the thesis and (Ellingboe 1964). One problem is that migration through a racetrack can lead to nuclei further in the mycelium, while the in between bit remains monokaryon. I was able to measure a maximum migration speed.

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