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Background  Two reference strains have been sequenced from the mushroom  Coprinopsis cinerea , monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 ( ade8-1 ) and a  para -aminobenzoic acid (PABA)-auxotrophy in AmutBmut ( pab1-1 ) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker  hph , respectively.    Results  Gene  ade8  encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in  ade8-1  rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new  ade8  +  vector p Cc Ade8 complements the auxotrophy of OK130 in transformations. Transformation rates with p Cc Ade8 in single-vector and co-transformations with  ade8  + -selection were similarly high, unlike for  trp1  +  plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected p Cc Ade8 helped in co-transformations of  trp1  strains with a  trp1  + -selection vector to overcome suicidal effects by transferred  trp1  + . Co-transformation rates of p Cc Ade8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26–42% for various laccase genes and up to 67% with  lcc9  silencing vectors. The bacterial gene  hph  can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the  pab1-1  defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase.    Conclusions   ade8-1  and  pab1-1  auxotrophic defects in  C. cinerea  reference strains OK130 and AmutBmut for complementation in transformation are described. p Cc Ade8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene  hph  can also be used as an additional selection marker in OK130, making in combination with  ade8  +  successive rounds of transformation possible.

Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea