Odd Arne Rognli

Molecular mechanisms underlying frost tolerance in perennial grasses adapted to cold climates

We review recent progress in understanding cold and freezing stress responses in forage grass species, notably Lolium and Festuca species. The chromosomal positions of important frost tolerance and winter survival QTLs on Festuca and Lolium chromosomes 4 and 5 are most likely orthologs of QTLs on Triticeae chromosome 5 which correspond to a cluster of CBF-genes and the major vernalization gene. Gene expression and protein accumulation analyses after cold acclimation shed light on general responses to cold stress. These responses involve modulation of transcription levels of genes encoding proteins involved in cell signalling, cellular transport and proteins associated with the cell membrane. Also, abundance levels of proteins directly involved in photosynthesis were found to be different between genotypes of differing frost tolerance levels, stressing the importance of the link between the function of the photosynthetic apparatus under cold stress and frost tolerance levels. The significance of the ability to undergo photosynthetic acclimation and avoid photoinhibition is also evident from numerous studies in forage grasses. Other interesting candidate mechanisms for freezing tolerance in forage grasses are molecular responses to cold stress which have evolved after the divergence of temperate grasses. This includes metabolic machinery for synthesis of fructans and novel ice-binding proteins.

Overwintering of herbaceous plants in a changing climate. Still more questions than answers.

The increase in surface temperature of the Earth indicates a lower risk of exposure for temperate grassland and crop to extremely low temperatures. However, the risk of low winter survival rate, especially in higher latitudes may not be smaller, due to complex interactions among different environmental factors. For example, the frequency, degree and length of extreme winter warming events, leading to snowmelt during winter increased, affecting the risks of anoxia, ice encasement and freezing of plants not covered with snow. Future climate projections suggest that cold acclimation will occur later in autumn, under shorter photoperiod and lower light intensity, which may affect the energy partitioning between the elongation growth, accumulation of organic reserves and cold acclimation. Rising CO2 levels may also disturb the cold acclimation process. Predicting problems with winter pathogens is also very complex, because climate change may greatly influence the pathogen population and because the plant resistance to these pathogens is increased by cold acclimation. All these factors, often with contradictory effects on winter survival, make plant overwintering viability under future climates an open question. Close cooperation between climatologists, ecologists, plant physiologists, geneticists and plant breeders is strongly required to predict and prevent possible problems.

Development and mapping of DArT markers within the Festuca - Lolium complex.

BackgroundGrasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum.ResultsThe DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins.ConclusionThe resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.

Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses.

BackgroundLittle is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses.ResultsBrachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species.ConclusionsWe conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.

QTL analyses and comparative genetic mapping of frost tolerance, winter survival and drought tolerance in meadow fescue (Festuca pratensis Huds.)

Quantitative trait loci (QTLs) for frost and drought tolerance, and winter survival in the field, were mapped in meadow fescue (Festuca pratensis Huds.) and compared with corresponding traits in Triticeae and rice to study co-location with putatively orthologous QTLs and known abiotic stress tolerance genes. The genomes of grass species are highly macrosyntenic; however, the Festuca/Lolium and Triticeae homoeologous chromosomes 4 and 5 show major structural differences that is especially interesting in comparative genomics of frost tolerance. The locations of two frost tolerance/winter survival QTLs on Festuca chromosome 5F correspond most likely to the Fr-A1 and Fr-A2 loci on wheat homoeologous group 5A chromosomes. A QTL for long-term drought tolerance on chromosome 3F (syntenic with rice 1) support evidence from introgression of Festuca genome segments onto homoeologous Lolium chromosomes (3L) that this genome region is an excellent source of tolerance towards drought stress. The coincident location of several stress tolerance QTL in Festuca with QTL and genes in Triticeae species, notably dehydrins, CBF transcription factors and vernalisation response genes indicate the action of structural or regulatory genes conserved across evolutionarily distant species.

Analysis of genetic diversity in a sweet potato ( Ipomoea batatas L.) germplasm collection from Tanzania as revealed by AFLP

Sweet potato (Ipomoea batatas L.) is the fifth most important crop in the developing countries after rice, wheat, maize and cassava. The amplified fragment length polymorphism (AFLP) method was used to study the genetic diversity and relationships of sweet potato accessions in the germplasm collection of Sokoine University of Agriculture, Morogoro and Sugarcane Research Institute, Kibaha, Tanzania. AFLP analysis of 97 sweet potato accessions using ten primer combinations gave a total of 202 clear polymorphic bands. Each one of the 97 sweet potato accessions could be distinguished based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the un-weight pair-group method using arithmetic average. AFLP-based genetic similarity varied from 0.388 to 0.941, with a mean of 0.709. Cluster analysis using genetic similarity divided the accessions into two main groups suggesting that there are genetic relationships among the accessions. Principal Coordinate analysis confirmed the pattern of the cluster analysis. Analysis of molecular variance revealed greater variation within regions (96.19%) than among regions (3.81%). The results from the AFLP analysis revealed a relatively low genetic diversity among the germplasm accessions and the genetic distances between regions were low. A maximally diverse subset of 13 accessions capturing 97% of the molecular markers diversity was identified. We were able to detect duplicates accessions in the germplasm collection using the highly polymorphic markers obtained by AFLP, which were found to be an efficient tool to characterize the genetic diversity and relationships of sweet potato accessions in the germplasm collection in Tanzania.

Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement.

Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection.

Extremely low genetic variability and highly structured local populations of Arabidopsis thaliana at higher latitudes

The genetic diversity and population structure of Arabidopsis thaliana populations from Norway were studied and compared to a worldwide sample of A. thaliana to investigate the demographic history and elucidate possible colonization routes of populations at the northernmost species limit. We genotyped 282 individuals from 31 local populations using 149 single nucleotide polymorphism markers. A high level of population subdivision (FST = 0.85 ± 0.007) was found indicating that A. thaliana is highly structured at the regional level. Significant relationships between genetic and geographical distances were found, suggesting an isolation by distance mode of evolution. Genetic diversity was much lower, and the level of linkage disequilibrium was higher in populations from the north (65–68°N) compared to populations from the south (59–62°N); this is consistent with a northward expansion pattern. A neighbour‐joining tree showed that populations from northern Norway form a separate cluster, while the remaining populations are distributed over a few minor clusters. Minimal gene flow seems to have occurred between populations in different regions, especially between the geographically distant northern and southern populations. Our data suggest that northern populations represent a homogenous group that may have been established from a few founders during northward expansions, while populations in the central part of Norway constitute an admixed group established by founders of different origins, most probably as a result of human‐mediated gene flow. Moreover, Norwegian populations appeared to be homogenous and isolated compared to a worldwide sample of A. thaliana, but they are still grouped with Swedish populations, which may indicate common colonization histories.

Evidence for adaptive evolution of low‐temperature stress response genes in a Pooideae grass ancestor

Adaptation to temperate environments is common in the grass subfamily Pooideae, suggesting an ancestral origin of cold climate adaptation. Here, we investigated substitution rates of genes involved in low-temperature-induced (LTI) stress responses to test the hypothesis that adaptive molecular evolution of LTI pathway genes was important for Pooideae evolution. Substitution rates and signatures of positive selection were analyzed using 4330 gene trees including three warm climate-adapted species (maize (Zea mays), sorghum (Sorghum bicolor), and rice (Oryza sativa)) and five temperate Pooideae species (Brachypodium distachyon, wheat (Triticum aestivum), barley (Hordeum vulgare), Lolium perenne and Festuca pratensis). Nonsynonymous substitution rate differences between Pooideae and warm habitat-adapted species were elevated in LTI trees compared with all trees. Furthermore, signatures of positive selection were significantly stronger in LTI trees after the rice and Pooideae split but before the Brachypodium divergence (P < 0.05). Genome-wide heterogeneity in substitution rates was also observed, reflecting divergent genome evolution processes within these grasses. Our results provide evidence for a link between adaptation to cold habitats and adaptive evolution of LTI stress responses in early Pooideae evolution and shed light on a poorly understood chapter in the evolutionary history of some of the worlds most important temperate crops.

Molecular Breeding and Functional Genomics for Tolerance to Abiotic Stress

Sustainability is a measure of our ability to produce food with the maximium of efficiency combined with the minimum of damage to the environment. Grasslands represent over 40% of all agricultural land in the European Union, and over 70% in the United Kingdom. Whilst Lolium in Europe is considered to be the ideal source of profitable and safe high quality animal forage, its general poor persistency limits its use to favourable growing areas. Fortunately, genes for abiotic stress resistance are transferred readily from closely related Festuca species by conventional breeding technologies. Introgression mapping allows the assembly of desirable gene combinations and molecular markers to assist with their selection in breeding programmes. Additional new androgenesis techniques have led to novel genotypes rarely observed as outcomes of breeding programmes. Lolium × Festuca hybrids display promiscuous chromosome recombination enabling genes from one species to be transferred readily to homoeologous chromosome regions where they both function normally and remain stable. Despite the close homology between Lolium and Festuca species, repetitive DNA sequences differ sufficiently for their genomes to be distinguished, by genomic in situ hybridisation (GISH). This enables the physical mapping of genes for abiotic stress resistance transferred from Festuca to Lolium. Further chromosome recombination between homoeologous Lolium and Festuca sequences enables Festuca introgressions to be “dissected”, and recombination series created. Knowledge of synteny and gene sequences within model species amongst the Poaceae, combined with the development of sequenced molecular markers, and bacterial artificial chromosomes is enabling the isolation of genes for abiotic stress resistance.