We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem
chromosomal duplication was achieved by using strains that had a 5'-deleted pyrG upstream of the region targeted for tandem
chromosomal duplication and a 3'-deleted pyrG downstream of the target region. Consequently,strains bearing a 210-kb targeted tandem
chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem
chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem
chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the
chromosomal duplication did not depend on the introduction of double-stranded breaks(DSBs) by I-SceI. The
chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem
chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased
protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.