Dear Gülsüm,

complementation of genes randomly mutated by UV mutagenesis requires that you master genetic transformation of your mold and that you have access to a library of rather large fragments of the genome of your fungus (at least cosmids). These fragments should be cloned in a vector bearing a selectable marker for filamentous fungi. Even though, you must know that transformation of filamentous fungi with DNA pieces as large as cosmids is not always possible, and that the yield of genetic transformation of your fungus must be quite high in order to even think of success.

Otherwise, you may think of a candidate gene approach, if you already have a rather good idea of the kind of genes that could be involved in the phenotype that you are looking for. As you have access to the complete genome of the fungus, you may select putative genes which could lead to the phenotype(s) observed after mutagenesis, and use them to make complementation assays of your UV mutants. But, here again, mastering genetic transformation of your mold is an absolute requirement, and there is no guarantee of success.

If you have any questions, please let me know.

With best regards,

Pierre-Henri

Dear Gülsüm,

in addition to my previous message: you do not say which "mold" you are working on, but as its genome sequence is available, I guess it could be one of the few filamentous fungi which have been used as model genetic systems. Sexual stage of this fungus might have been described and several strains that you can cross could be available. Genetic markers might even have been identified, which would facilitate greatly your work, as you could then use a classical genetic approach to map mutations induced by UV exposure. Reading the literature about your organism will tell you. Still, if you manage to narrow down this way the localization of your mutation to a rather small piece of DNA, you will still need a genetic transformation protocol to make complementation assays and confirm the identity of the genes mutated.

I hope this will help,

Pierre-Henri

Dear Gülsüm,

to be fully comprehensive on the matter, I must also mention TILLING (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC514099/ AND http://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx), even though I am not really familiar with the technology. I am not sure that it has ever been applied to filamentous fungi, but you could probably start your own literature search on the subject. Please note that TILLING is useful only when using chemical mutagenesis with EMS (which specifically induces base pair changes G/C to A/T) and most likely not UV mutagenesis (which can lead to other kinds of point mutations).

Good luck,

Pierre-Henri

Dear Pierre-Henri and Gülsüm,

Research Gate sent me a link to your conversation. With regards to TILLING, the methods typically used for mutation discovery are PCR of a candidate gene region of ~1-1.5 kb followed by enzymatic mismatch cleavage and gel electrophoresis. This approach can be used to discover SNP and small indel (up to ~50bp) mutations. The sequence doesn't matter. While not published yet, my colleagues have used the approach for polymorphism discovery in the fugus C. sativus. TILLING is a reverse-genetic approach and what you are doing is forward genetics, but you could use enzymatic mismatch cleavage to find induced mutations. 

If you followed this approach you would be following a candidate gene approach mentioned by Pierre-Henri.  Instead of sequencing the PCR products you would perform enzymatic mismatch cleavage.  It could save a bit of money if you have many candidate genes to screen.  My assumption is that UV would be causing SNP and small indel mutations in your mold.  If you are interested my lab can send you protocols and kits to try this approach (positive controls - DNA, enzyme, primers). Just let me know your shipping address by emailing me at [email protected]

The genome size of your mold is most likely much smaller than plant genomes. An alternative approach would be to sequence the entire genome to identify all of the induced variation. I am not an expert on fungal genetics, but you would have to verify the putative causative mutations by some method (complementation). You can do this in diploid plants with recessive mutations without transgenic rescue by looking at segregation of mutations with phenotypes in the F2 generation (there are several published examples of this, see http://www.nature.com/nbt/journal/v30/n2/abs/nbt.2095.html for example).  

I hope this information is useful.

Warm regards,

Brad Till

Please see the papers for your reference

https://www.researchgate.net/publication/311668087_Improvement_of_chitinase_Pachi_with_nematicidal_activities_by_random_mutagenesis

https://www.researchgate.net/publication/289367579_Characterization_and_directed_evolution_of_BliGO_a_novel_glycine_oxidase_from_Bacillus_licheniformis

https://www.researchgate.net/publication/282153514_Enhanced_nematicidal_potential_of_the_chitinase_pachi_from_Pseudomonas_aeruginosa_in_association_with_Cry21Aa

https://www.researchgate.net/publication/277326677_Improvement_of_glycine_oxidase_by_DNA_shuffling_and_site-saturation_mutagenesis_of_F247_residue

Regards

Article Improvement of chitinase Pachi with nematicidal activities b...

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