New multi-marker strains and complementing genes for Aspergillus nidulans molecular biology

•We have created four new selectable markers through targeted deletions.•We have determined the sequence changes in three frequently used mutants.•We have created strains of Aspergillus nidulans with up to seven selectable markers.•We have cloned Aspergillus terreus genes that complement each new selectable marker.•These advances will facilitate multiple, sequential transformations. Technical advances in Aspergillus nidulans enable relatively easy deletion of genomic sequences, insertion of sequences into the genome and alteration of genomic sequences. To extend the power of this system we wished to create strains with several selectable markers in a common genetic background to facilitate multiple, sequential transformations. We have developed an approach, using the recycling of the pyrG selectable marker, that has allowed us to create new deletions of the biA, pabaA, choA, and lysB genes. We have deleted these genes in a strain that carries the commonly used pyrG89, riboB2, and pyroA4 mutations as well as a deletion of the sterigmatocystin gene cluster and a deletion of the nkuA gene, which greatly reduces heterologous integration of transforming sequences. The new deletions are fully, easily and cheaply supplementable. We have created a strain that carries seven selectable markers as well as strains that carry subsets of these markers. We have identified the homologous genes from Aspergillus terreus, cloned them and used them as selectable markers to transform our new strains. The newly created strains transform well and the new deletion alleles appear to be complemented fully by the A. terreus genes. In addition, we have used deep sequencing data to determine the sequence alterations of the venerable and frequently used pyrG89, riboB2 and pyroA4 alleles and we have reannotated the choA gene.