The level of heterosis can be measured in several different ways. Falconer and MacKay introduced the mid-parent (MP) heterosis as a difference between the trait performance of a hybrid and the mean of the two parental lines . Occasionally, the better-parent (BP) heterosis is used. However, in outbreeding species, populations are often used for the hybrid production. For that, the panmictic mid-parent (PMP) and the panmictic better-parent (PBP) parameters of heterosis were established to reflect the performance of a hybrid population relative to its two parental populations . In this study, we use the MP and BP criteria.
Heterosis in forage grasses is usually studied under space planting and the heterotic effect appears to be more pronounced under this system than under sward conditions [21–23]. However, even when using spaced plants, PMP heterosis for yield is rather modest, from + 1% to + 48% in Lolium perenne compared to -9% to + 8% when plots were used (reviewed in ). Here we decided on the spaced-plant experimental design, for several reasons, including convenience and the ability to gather a wider range of observations than that possible in a sward.
Extent of heterosis for dry matter yield
Very large mid-parent (MP) and better-parent (BP) heterosis values for the hybrids of non-inbred Fp and Fape observed here were far greater than any values reported so far in forage grasses. Posselt  cited 15 studies involving 5 different forage grass species in a range of environments with a maximum MP heterosis for the annual forage yield of + 70% (19% on average), compared to over + 500% in our study. For individual cuts, the maximum MP heterosis reported by Posselt  was + 152%, compared to + 625% here with a comparable experimental design (individual spaced plants). Other studies with sown plots yielded even lower heterosis, usually less than + 20% [8, 25].
Interestingly, the highest reported heterosis values so far were observed in an interspecific hybrid, L. perenne × L. multiflorum . Interspecific hybrids within the Festuca-Lolium complex, commonly referred to as Festulolium, are of interest mainly to combine stress resilience of Festuca species with productivity and quality of Lolium species. Heterosis for yield per se is rather small . Humphreys et al. report yields similar to, at best 10% higher than the respective Lolium parent for the most successful Festulolium candidates . Our triploid Fape × Fp hybrids provide an example of an interspecific hybrid within the Festuca-Lolium complex with a high potential for heterosis for forage yield. This may add a new dimension to the discussion of the potential of interspecific forage grass hybrids. Indeed, exploitation of heterosis is considered a highly promising approach to speed up the supposedly low progress in breeding of forage grasses for yield . Several approaches are followed, such as developing doubled haploids , cytoplasmic male sterility (cms) in hybrid production [29, 30], using the S-Z-self-incompatibility system to develop inbred lines  as well as optimizing the construction of self-incompatible hybrids . However, the use of interspecific hybrids to exploit heterosis for forage yield is not among the prime approaches followed, even though some promising examples exist, such as the energy grass Miscanthus ‘Giganteus’, an interspecific hybrid between Miscanthus sinensis and Miscanthus ogiformis .
It is somewhat surprising that our interspecific hybrid between two closely related species of Festuca show such a large heterosis for biomass yield. F. apennina (Fape) is morphologically so similar to F. pratensis (Fp) that in older literature it was referred to as a subspecies [34, 35] or even a varietas of F. pratensis . However, recent studies clearly show that F. apennina is an allotetraploid originating by hybridization of a progenitor of the current F. pratensis with a so-far unidentified species related to the modern F. glaucescens . One would, nevertheless, expect little heterosis because the Fp genome is present in Fape. However, Fp showed about five times more genetic diversity (estimated by DArT markers) than Fape, suggesting that the Fp genome present in Fape is only distantly related to modern Fp . Generally, heterosis increases as the genetic disparity of the parents or parental populations increases and interspecific hybrids seem to display greater heterosis than intraspecific hybrids .
The triploid Fape × Fp hybrids are almost completely sterile. This can be considered an advantage in nature because it guarantees long term stability of F1 hybridity. Advanced generations of Festulolium hybrids show much less heterosis than would be expected from their F1 hybrids (Marc Ghesquiere, personal communication). Since our Fape × Fp hybrids can efficiently propagate vegetatively through rhizomes , heterosis is conserved and can be expected to contribute significantly to their competitiveness in natural grassland. Highly competitive populations of the triploid hybrids were observed at mid-altitude sites . They dominated the swards in several locations and were the unique cytotype of all 54 Festuca specimens collected in one location at 1350 m a.s.l. However, sterility of triploid hybrids is an obvious obstacle for their further exploitation in grass breeding and agriculture.
Effect of altitude on relative performance of triploid hybrids and their parents
The performance of triploid hybrids relative to their parents was affected by the altitude of the trial site, and this effect increased over time. In the year A0, the MP heterosis ranged among the sites from + 64 to + 111% without a clear altitudinal pattern (Fig. 1). In H1, it was lower, at about + 45% at Reckenholz (440 m a.s.l.) but increased to about + 180% at Fruehbuehl (1000 m a.s.l.) and about + 140% at Maran (1850 m a.s.l.) (Fig. 2). At Olomouc (200 m a.s.l.), the relative performance of hybrids was better than at Reckenholz, but not as good as at Maran. In H2, a clear altitudinal pattern was observed (Fig. 3), with negative values for the MP heterosis at Olomouc, low values at Reckenholz, and an overarching maximum at Fruehbuehl of about + 500%. In H2, the MP heterosis was also high in Maran, with over + 350%. These patterns were highly consistent for both crosses (A and B) investigated. The highest levels of precipitation combined with a deep, well-drained soil at the mid-altitude site, Fruehbuehl (Additional file 2), probably contributed to good performance of Fape relative to Fp, and to the maximum expression of heterosis in the hybrids. The shallow soil at Maran with poorer water holding capacity and nutrient delivery potential was probably responsible for the poorer relative performance of Fape, compared to Fruehbuehl, and the somewhat lesser expression of heterosis. The altitudinal and temporal patterns of the MP heterosis were related to the relative performance of the two parental species, Fape and Fp. When Fape performed very poorly compared to Fp, such as at low altitude in H2, the MP heterosis of triploid hybrids was also low or even negative. When Fape performed moderately well, higher values for the MP heterosis were observed. In H2, a consistent altitudinal pattern of the relative performance of Fp and Fape was reached, with performance of Fape rising steadily with altitude.
Impact of biotic and abiotic stresses
Gradual dying of Fape plants to extinction during the last year of the experiment at low altitudes was responsible for their overall low performance at Olomouc and Reckenholz. At Reckenholz, bacterial wilt was the most damaging factor. Some plants died already during the spring 2018 when the disease was scored, and a dramatic loss of plants occurred after the first cut in H1. Yield losses and lack of regrowth after cutting is a common feature of heavy infestation with Xanthomonas [38, 39]. Therefore, relatively good Xanthomonas resistance of the hybrids contributed to the longer persistence of positive heterosis for yield at the Reckenholz site. At Olomouc, summer drought in 2018 was the likely cause of Fape death. While most F ape plants survived until the second cut, a significant loss of plants occurred in July and August when the drought was the most severe. The hybrids responded differently to these stresses. At Reckenholz, the hybrids showed considerable variation in the susceptibility to Xanthomonas. Resistant plants survived without visible symptoms and kept producing biomass, while the susceptible ones reduced growth, and 25% died during 2018. This produced a very strong negative correlation between the Xanthomonas susceptibility and yields of hybrids (Additional file 1). This correlation was also highly significant for Fape, but low and insignificant for the largely resistant Fp. Over all genotypes in the study, Xanthomonas susceptibility was also highly significantly and negatively correlated with subsequent yield, reflecting the higher susceptibility of Fape and, to a lesser extent, the hybrids, compared to resistant Fp. Resistance to Xanthomas appears to be controlled by few major genes . The two elite Fp plants which were used to produce our triploid hybrids were from the Agroscope breeding program after several cycles of recurrent selection for Xanthomonas resistance, and likely carried such genes. The marked segregation among the triploids from crosses with a highly susceptible Fape plant suggests heterozygosity for resistance genes in the Fp parents. The response of triploid to the dry conditions at Olomouc was more gradual. Similar numbers of Fp plants and the hybrids survived the drought in H1 (Fig. 5), but yields of the hybrids relative to Fp (the better parent) declined significantly during H1 (2018) (see Fig. 4). Attrition of the hybrids continued in H2 and yield decreased further. Among the Fape × Fp hybrids, a higher drought tolerance of the Fp parent was apparently not sufficient to adequately reduce the high water requirement of the Fape parent. Probably, the difficult soil conditions at Olomouc contributed to the poor performance of Fape and the hybrids after the drought of summer 2018.
At low altitude, Fape left winter with apparent damage (brown leaves). This dormancy response can be viewed as a survival mechanism, however it results in slow spring growth and lower first cut yields, compared to the winter green Fp with hardly any signs of winter damage. The hybrids were intermediate between Fape and Fp. However, at the highest altitude (Maran), Fape and the hybrids showed significantly less true winter damage by snow mold caused by Microdochium nivale) than Fp. A peculiar situation was observed at Fruehbuehl. Here, the hybrids showed negative heterosis for winter damage (positive for tolerance to winter conditions). Apparently, hybrids were less winter dormant than Fape and less susceptible to snow mold than Fp and thus they showed strong heterosis.
The hybrids derived from the two crosses A and B differed markedly in their response to crown rust infection. Hybrids from cross A showed a strong positive heterosis for susceptibility (negative for resistance), while those from cross B were intermediate between susceptible Fape and resistant Fp, not significantly more susceptible than Fp in two out of three scorings, and thus showed negative heterosis for susceptibility (positive for resistance). Indeed, rust susceptibility/resistance was the only case of negative heterosis for one, and positive for the other cross. This differential response points to a different architecture of the rust resistance in the two parent Fp plants. The crown rust resistance in the Festuca-Lolium complex is polygenic, as evidenced by a successful genomic prediction model to select for rust resistance in perennial ryegrass . Two major and a number of minor QTLs for rust resistance in a segregating pseudo-testcross population of Italian ryegrass were identified . Schubiger and Boller demonstrated the prevalence of several independent race-specific major genes, each of which showed dominant, Mendelian segregation in cross progenies of perennial ryegrass . Likely, the Fp parent of cross B possesses some dominant, partly homozygous major resistance genes, while the resistance of the Fp parent of cross A relies mostly on minor resistance genes overridden by susceptibility genes of the Fape parent.