Breeding program for studying mitonuclear interactions leading to hybrid breakdown in Pleurotus ostreatus

Mitonuclear interactions play a fundamental role in eukaryotic evolution, fitness, and hybrid viability. Despite extensive studies in animals and plants, their impact on fungi such as Pleurotus ostreatus (P. ostreatus) remains underexplored. In the dN001 (n) strain, long-term subculturing was shown to lead to degeneration, which was associated with mitonuclear incompatibilities, oxidative stress, and reduced growth. This work demonstrates how mitonuclear incompatibilities trigger strain degeneration and influence phenotypic and agronomic performance traits in P. ostreatus. These findings were supported by a divergent breeding programme derived from the commercial hybrid strain dN001 (n), which enabled the generation of fast- and slow-growing parental lines. These were then crossed with the mT1 (t) tester line to produce hybrid strains with identical nuclei, but different mitochondrial types, thereby allowing the molecular basis of mitonuclear interactions in P. ostreatus to be investigated. Strains were evaluated under different temperatures and carbon source conditions, highlighting the importance of mitonuclear–environment interactions in determining performance. RT-qPCR experiments targeting mitochondrial electron transport chain (ETC) components, antioxidant enzymes, and iron homeostasis-related genes revealed that gene expression was closely linked to mitochondrial type and nuclear compatibility. In particular, the (t) mitochondrial type was responsible for the exceptional performance of the d(NS423 × T1, t) strain, which exhibited the highest commercial performance among all strains studied. Overall, the results of this thesis demonstrate the critical role of mitonuclear interactions in growth rate and mushroom production traits. They also suggest that maintaining the two F₁ parental strains of commercial P. ostreatus hybrids in long-term storage, followed by remaking the hybrid shortly before use, may help to preserve strain performance and prevent degeneration.