To construct a set of isogenic auxotrophic strains in Aspergillus niger suited for creating multiple gene deletion mutants and executing parasexual crossings, we have combined mutations in genes involved in colour pigmentation (fwnA and olvA) with well-selectable auxotrophic markers (pyrG, nicB, argB, and adeA). All markers, except for the pyrG marker, were introduced by targeted deletion, omitting UV mutagenesis of the strains. Aspergillus oryzae orthologous genes of the argB, nicB, and adeA markers were used as heterologous selection markers, and all markers were shown to complement to respective auxotrophic A. niger mutants. A quadruple auxotrophic marker was further constructed suitable for multiple gene deletions. Genome sequencing of two auxotrophic colour mutants JN3.2 (olvA::pyrG, argB::hygB) and JN6.2 (olvA::pyrG, nicB::hygB) revealed four SNPs between them in non-coding regions, indicating a high level of isogenicity between both strains. The availability of near-isogenic complementary auxotrophic colour mutants facilitates the selection of diploids and the isolation of haploid segregants from the diploid using the parasexual cycle.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-016-1240-6) contains supplementary material, which is available to authorized users.

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