Winter survival in red clover: experimental evidence for interactions among stresses.

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.

Including marker x environment interactions improves genomic prediction in red clover (Trifolium pratense L.).

Genomic prediction has mostly been used in single environment contexts, largely ignoring genotype x environment interaction, which greatly affects the performance of plants. However, in the last decade, prediction models including marker x environment (MxE) interaction have been developed. We evaluated the potential of genomic prediction in red clover (Trifolium pratense L.) using field trial data from five European locations, obtained in the Horizon 2020 EUCLEG project. Three models were compared: (1) single environment (SingleEnv), (2) across environment (AcrossEnv), (3) marker x environment interaction (MxE). Annual dry matter yield (DMY) gave the highest predictive ability (PA). Joint analyses of DMY from years 1 and 2 from each location varied from 0.87 in Britain and Switzerland in year 1, to 0.40 in Serbia in year 2. Overall, crude protein (CP) was predicted poorly. PAs for date of flowering (DOF), however ranged from 0.87 to 0.67 for Britain and Switzerland, respectively. Across the three traits, the MxE model performed best and the AcrossEnv worst, demonstrating that including marker x environment effects can improve genomic prediction in red clover. Leaving out accessions from specific regions or from specific breeders' material in the cross validation tended to reduce PA, but the magnitude of reduction depended on trait, region and breeders' material, indicating that population structure contributed to the high PAs observed for DMY and DOF. Testing the genomic estimated breeding values on new phenotypic data from Sweden showed that DMY training data from Britain gave high PAs in both years (0.43-0.76), while DMY training data from Switzerland gave high PAs only for year 1 (0.70-0.87). The genomic predictions we report here underline the potential benefits of incorporating MxE interaction in multi-environment trials and could have perspectives for identifying markers with effects that are stable across environments, and markers with environment-specific effects.

Multi-location trials and population-based genotyping reveal high diversity and adaptation to breeding environments in a large collection of red clover.

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 diverse set of 395 diploid red clover accessions via genome wide allele frequency fingerprinting and multi-location field trials across Europe. We found that the genetic structure of accessions mostly reflected their geographic origin and only few cases were detected, where breeders integrated foreign genetic resources into their local breeding pools. The mean dry matter yield of the first main harvesting season ranged from 0.74 kg m-2 in Serbia and Norway to 1.34 kg m-2 in Switzerland. Phenotypic performance of accessions in the multi-location field trials revealed a very strong accession x location interaction. Local adaptation was especially prominent in Nordic red clover accessions that showed a distinct adaptation to the growing conditions and cutting regime of the North. The traits vigor, dry matter yield and plant density were negatively correlated between the trial location in Norway and the locations Great Britain, Switzerland, Czech Republic and Serbia. Notably, breeding material and cultivars generally performed well at the location where they were developed. Our results confirmed that red clover cultivars were bred from regional ecotypes and show a narrow adaptation to regional conditions. Our study can serve as a valuable basis for identifying interesting materials that express the desired characteristics and contribute to the adaptation of red clover to future climatic conditions.

De novo and reference transcriptome assembly of transcripts expressed during flowering provide insight into seed setting in tetraploid red clover.

Red clover (Trifolium pratense L.) is one of the most important legume forage species in temperate livestock agriculture. Tetraploid red clover cultivars are generally producing less seed than diploid cultivars. Improving the seed setting potential of tetraploid cultivars is necessary to utilize the high forage quality and environmentally sustainable nitrogen fixation ability of red clover. In the current study, our aim was to identify candidate genes involved in seed setting. Two genotypes, 'Tripo' with weak seed setting and 'Lasang' with strong seed setting were selected for transcriptome analysis. De novo and reference based analyses of transcriptome assemblies were conducted to study the global transcriptome changes from early to late developmental stages of flower development of the two contrasting red clover genotypes. Transcript profiles, gene ontology enrichment and KEGG pathway analysis indicate that genes related to flower development, pollen pistil interactions, photosynthesis and embryo development are differentially expressed between these two genotypes. A significant number of genes related to pollination were overrepresented in 'Lasang', which might be a reason for its good seed setting ability. The candidate genes detected in this study might be used to develop molecular tools for breeding tetraploid red clover varieties with improved seed yield potentials.