The investigation grew out of concern about the performance of the commercial hybrid strain dN001, which had been maintained in subculture in the researchers’ laboratory for about 20 years. Its descendants divided into fast- and slow-growing phenotypes. Earlier work associated the slow descendants with degeneration, oxidative stress and altered expression of genes involved in mitochondrial respiration, hypoxia responses, antioxidant defence and cell death.

The new study tested the possibility that repeated mating and subculture had separated nuclear and mitochondrial components that had previously evolved together, producing incompatibilities associated with hybrid breakdown. To create parental lines with contrasting growth rates, the team intercrossed full-sib monokaryons descended from dN001 and sharing its mitochondrial type. Intercrossing fast descendants and, separately, slow descendants generated fourth-generation fast and slow lines.

The resulting lines had mean radial growth rates of 4.07 and 1.12 millimetres per day. The fast line increased the growth rate by about 50% relative to the earlier generation, while the slow line declined by only 1.8%. Nuclear and mitochondrial molecular markers were then used to confirm the construction of eight hybrids: crosses involving two fast parental lines and two slow parental lines, each represented with either the “n” mitochondrial genome inherited from dN001 or the “t” mitochondrial genome inherited from the compatible wild-derived tester strain mT1.

Each pair shared the same nuclear background but differed in mitochondrial type. Mitochondrial inheritance was uniparental, as each hybrid carried only one detected mitochondrial genotype. Under standard laboratory conditions of 24 degrees Celsius, the mitochondrial background made little consistent difference among hybrids made with fast-growing parents.

The clearest contrast instead appeared within the slow-parent set. Under the tested temperatures of 15, 24 and 32 degrees Celsius, d(NS423 × T1, t) consistently grew faster than d(NS423 × T1, n). Relative to the parental average, the “t” hybrid exhibited mid-parent heterosis ranging from 40% to 76% across the temperature conditions.

The second slow-line cross, d(NS426 × T1), showed no detected mitochondrial effect on growth rate, although both versions grew more slowly at 15 degrees Celsius. These divergent outcomes show that a mitochondrial genome did not have a uniform effect across nuclear backgrounds; its influence depended on which nuclear genome accompanied it. Carbon sources altered the picture again.

The fast and slow parental lines generally grew well with glucose, sucrose or glycerol, although glycerol reduced growth in most strains and by about 60% in the tester mT1. The d(NS423 × T1, t) hybrid grew faster than its “n” counterpart across the tested carbon sources, with mid-parent heterosis values from 32% to 100%, depending on the condition and mitochondrial background. By contrast, mitochondrial type did not consistently affect growth in the fast hybrids or the d(NS426 × T1) pair, apart from some differences involving glycerol.

One slow parental strain, mNS423, maintained similar growth across all three carbon sources, indicating that its nuclear background also contributed to metabolic flexibility. The measurements therefore supported genotype-by-environment interactions rather than a simple rule that hybridisation always improves growth. Mitochondrial differences were accompanied by distinct patterns of gene expression and reactive oxygen species, but these observations did not by themselves establish a causal molecular mechanism.

Using reverse-transcription quantitative PCR, the researchers measured the mitochondrial nd1 gene and nuclear bcs1, rip1, cox4 and cox5b genes associated with electron transport chain complexes I, III and IV. They also examined sod1, cat and gpx, encoding superoxide dismutase, catalase and glutathione peroxidase. Several hybrids expressed genes beyond the range of their parents, a pattern the researchers termed transgressive inheritance and classified as overdominance, underdominance or partial dominance depending on the gene and cross.

Slow-growing lines generally showed higher expression of genes connected with respiration, hypoxia and oxidative-stress responses, although the pattern was not identical for every gene. Fluorescent probes supplied a parallel physiological measurement. Slow lines and many of their hybrids accumulated more superoxide and hydrogen peroxide than fast lines.

In d(NS423 × T1, t), however, reactive oxygen levels fell to levels comparable to those of fast hybrids. This strain also showed elevated expression of nd1, bcs1 and rip1, while cox4 and cox5b expression was reduced to levels resembling those in fast-growing hybrids. The authors interpreted these associated patterns as consistent with improved mitochondrial function and lower oxidative stress in the successful hybrid.

They also noted two possible explanations for the hybrid vigour: masking of harmful parental mutations and disruption of regulatory safeguards that normally limit growth. The experiments did not distinguish these alternatives conclusively. The mitochondrial contrast persisted under cultivation designed to resemble mushroom production.

Spawn made from each hybrid was mixed with pasteurised wheat straw at a 2% weight-to-weight ratio and packed into 2-kilogram micro-perforated bags before fruiting at 18 degrees Celsius and 90% relative humidity. Spawn colonisation did not differ significantly between mitochondrial versions of the fast hybrids or the d(NS426 × T1) pair. The d(NS423 × T1, t) hybrid, however, colonised grain at a rate comparable to the fast hybrids, and its substrate weight loss matched that group.

Its mitochondrial background was associated with greater straw degradation in this cross, while the other slow hybrid pair remained low-performing regardless of mitochondrial type. The “n” hybrid produced a total yield of 29.2 ± 5.0 grams, fruited after 38 ± 3 days and formed one flush, with values reported as means of three replicates plus or minus standard deviation. The “t” hybrid produced 148.2 ± 11.6 grams, fruited after 26 ± 1 days and formed three flushes.

Total yields among the fast hybrids ranged from 199.7 ± 15.1 to 299.2 ± 77.2 grams, so the improved slow-line hybrid remained below that group in yield even though it behaved more like them in several other traits. Its earlier production and greater number of flushes nevertheless represented a marked improvement over its mitochondrial counterpart. Expression patterns for genes connected with substrate colonisation, iron handling and mitochondrial structure were consistent with additional physiological differences, without proving their role in the yield advantage.

The fast lines expressed more lacc4, whereas slow lines generally showed higher fet3 expression. The successful “t” hybrid stood out through increased fet3 expression and a yfh1 pattern that the authors interpreted as partial restoration of iron availability. It also showed increased expression of fzo1, associated with mitochondrial fusion, and cld1, involved in cardiolipin and mitochondrial membrane functions.

Antioxidant compounds detected by thin-layer chromatography also varied among hybrids, including a DPPH-reactive band at an Rf value of 0.80 restricted to fast hybrids carrying the “n” mitochondrial type. These findings broaden the phenotypic differences associated with mitonuclear background, but their individual contributions to growth or yield remain unresolved. The findings suggest two practical implications for breeding P. ostreatus, both of which remain proposals for further testing rather than outcomes established by this study.

The researchers advocate retaining compatible parental strains in long-term storage and remaking hybrids shortly before commercial use instead of repeatedly subculturing a hybrid, because degeneration may emerge across generations. They are also screening potential bred lines for new heterotic combinations. Candidate parents would need genomic and transcriptomic information, evidence of general and specific combining ability, and tolerance across substrates and temperatures.

For now, the study’s bounded conclusion is clear: in the tested oyster-mushroom crosses, mitochondrial type could reshape transgressive gene expression, reactive oxygen levels, growth, substrate degradation and fruiting performance, with the size and direction of the effect depending on the nuclear background and environment.

Subject of Research: How mitochondrial background affects hybrid growth, oxidative stress and mushroom yield in Pleurotus ostreatus

Article Title: Asymmetric mitonuclear interactions trigger transgressive inheritance and mitochondria-dependent heterosis in hybrids of the model system Pleurotus ostreatus

Article References: Garde, E., Pérez, G., Jiménez, I., Calvo, M. I., Pisabarro, A. G., & Ramírez, L. (2025). Asymmetric mitonuclear interactions trigger transgressive inheritance and mitochondria-dependent heterosis in hybrids of the model system Pleurotus ostreatus. IMA Fungus, 16, Article e165520. https://doi.org/10.3897/imafungus.16.165520

Image Credits: AI Generated

DOI: 10.3897/imafungus.16.165520

Keywords: Pleurotus ostreatus, mitochondrial inheritance, mitonuclear compatibility, hybrid vigour, heterosis, oxidative stress, mushroom breeding, fungal genomics, electron transport chain, mushroom yield, Asymmetric, mitonuclear