Multi-location trials and population-based genotyping reveal high diversity and adaptation to breeding environments in a large collection of red clover
Abstract:Red clover (Trifolium pratense L.) is an outcrossing forage legume that has adapted to a wide range of climatic and growing conditions across Europe. Red clover is valued for its high yield potential and its forage quality. The high amount of genetic diversity present in red clover provides an invaluable, but often poorly characterized resource to improve key traits such as yield, quality, and resistance to biotic and abiotic stresses. In this study, we examined the genetic and phenotypic diversity within a di… Show more
“…4 Principal component analysis of traits recorded in 110 accessions from the EUCLEG red clover panel, grouped into four regions of origin. Canopy height in late October and clover rot resistance expressed during the following winter was recorded in the Norwegian EUCLEG field experiment [ 23 , 27 ], clover rot resistance data are from Frey et al [ 24 ] and freezing tolerance data from Zanotto et al [ 28 ]. Correlation coefficients between traits are given in Supplementary Table 8A …”
Section: Resultsmentioning
confidence: 99%
“… Fig. 5 Correlation between regrowth of non-acclimated and non-inoculated (but incubated) plants in experiment 1 with canopy height in late October and clover rot resistance expressed during the following winter in the Norwegian EUCLEG field experiment [ 23 , 27 ]. Correlation coefficients between traits are given in Supplementary Table 8B …”
Section: Resultsmentioning
confidence: 99%
“…The populations used in the experiments (Supplementary Table 2) were all diploid and belonged to the set of populations characterized in the EUCLEG project ( www.eucleg.eu ) [ 27 ]. The populations were selected to represent a broad range from susceptible to resistant to clover rot according to observations in the Norwegian EUCLEG field trial.…”
Background
There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth.
Results
Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms.
Conclusions
We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.
“…4 Principal component analysis of traits recorded in 110 accessions from the EUCLEG red clover panel, grouped into four regions of origin. Canopy height in late October and clover rot resistance expressed during the following winter was recorded in the Norwegian EUCLEG field experiment [ 23 , 27 ], clover rot resistance data are from Frey et al [ 24 ] and freezing tolerance data from Zanotto et al [ 28 ]. Correlation coefficients between traits are given in Supplementary Table 8A …”
Section: Resultsmentioning
confidence: 99%
“… Fig. 5 Correlation between regrowth of non-acclimated and non-inoculated (but incubated) plants in experiment 1 with canopy height in late October and clover rot resistance expressed during the following winter in the Norwegian EUCLEG field experiment [ 23 , 27 ]. Correlation coefficients between traits are given in Supplementary Table 8B …”
Section: Resultsmentioning
confidence: 99%
“…The populations used in the experiments (Supplementary Table 2) were all diploid and belonged to the set of populations characterized in the EUCLEG project ( www.eucleg.eu ) [ 27 ]. The populations were selected to represent a broad range from susceptible to resistant to clover rot according to observations in the Norwegian EUCLEG field trial.…”
Background
There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth.
Results
Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms.
Conclusions
We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.
“…Red clover is known to be a genetically and phenotypically diverse crop ( Nay et al., 2023 ). Ergon et al.…”
Section: Discussionmentioning
confidence: 99%
“…A total of 393 red clover accessions were phenotyped for FT under controlled experimental conditions. These accessions are cultivars, breeding material, landraces and ecotypes mostly of European origin ( Table 1 ; Supplementary Table 1 ) collected and characterized within the EUCLEG project 1 ( Nay et al., 2023 ). FT was determined in young plants after a short cold acclimation treatment (two weeks at 3–4°C, 12 h photoperiod and 110 μmol m -2 s −1 photosynthetic photon flux density (PPFD) and expressed as LT50 (temperature required to kill 50% of the plants), in a similar way as described by Zanotto et al.…”
Improvement of persistency is an important breeding goal in red clover (Trifolium pratense L.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.
Symbiotic mutualisms provide critical ecosystem services throughout the world. Anthropogenic stressors, however, may disrupt mutualistic interactions and impact ecosystem health. The plant‐rhizobia symbiosis promotes plant growth and contributes to the nitrogen (N) cycle. While off‐target herbicide exposure is recognized as a significant stressor impacting wild plants, we lack knowledge about how it affects the symbiotic relationship between plants and rhizobia. Moreover, we do not know whether the impact of herbicide exposure on symbiotic traits or plant fitness might be ameliorated by plant or rhizobial genetic variation. To address these gaps, we conducted a greenhouse study where we grew 17 full‐sibling genetic families of red clover (Trifolium pratense) either alone (uninoculated) or in symbiosis with one of two genetic strains of rhizobia (Rhizobium leguminosarum) and exposed them to a concentration of the herbicide dicamba that simulated “drift” (i.e., off‐target atmospheric movement) or a control solution. We recorded responses in immediate vegetative injury, key features of the plant‐rhizobia mutualism (nodule number, nodule size, and N fixation), mutualism outcomes, and plant fitness (biomass). In general, we found that rhizobial variation more than plant variation determined outcomes of mutualism and plant fitness in response to herbicide exposure. Herbicide damage response depended on plant family, but also whether plants were inoculated with rhizobia and if so, with which strain. Rhizobial strain variation determined nodule number and size, but this was herbicide treatment‐dependent. In contrast, strain and herbicide treatment independently impacted symbiotic N fixation. And while herbicide exposure significantly reduced plant fitness, this effect depended on inoculation state. Furthermore, the differential fitness benefits that the two rhizobial strains provided plants seemed to diminish under herbicidal conditions. Altogether, these findings suggest that exposure to low levels of herbicide impact key components of the plant‐rhizobia mutualism as well as plant fitness, but genetic variation in the partners determines the magnitude and/or direction of these effects. In particular, our results highlight a strong role of rhizobial strain identity in driving both symbiotic and plant growth responses to herbicide stress.
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