Frost survival and gene expression in timothy (Phleum pratense L.) cultivars as affected by age and selection in diverse field environments.

The sustainable production of perennial grasses in Northern Norway is at risk due to the ongoing climate change. The predicted increase in temperatures and variable weather patterns are further expected to create challenges for winter survival of timothy (Phleum pratense L.). Knowledge about the molecular mechanisms underlying freezing tolerance is crucial for developing robust cultivars. The current study is aimed at identifying genes involved in freezing stress response of timothy and studying gene expression differentiation due to field selection in contrasting environments using RNAseq. Four timothy cultivars were field tested for three years in Tromsø and Vesterålen, in Northern Norway. The surviving material from the field tests, along with plants raised from the original seed lots, were subjected to freezing tests. LT50 values varied across cultivars and materials. Many genes coding for transcription factors and proteins known to play an important role in freezing tolerance, like dehydrins, c-repeat binding factors, and late embryogenesis abundant proteins were upregulated with decreasing temperatures. Moreover, genes associated with glycolysis/gluconeogenesis, TCA cycle, glutathione metabolism, proteasome pathways and genes encoding autophagy-related proteins, plasma membrane-associated proteins, sugar and amino acid transporters had elevated expression in field survivors compared to plants raised from the original material. The lower freezing stress tolerance of field survivors despite the elevated expression of several stress-responsive genes might be due to a combination of selection in the field and the age effect. Furthermore, differences in freezing stress response between northern and southern adapted cultivars and surviving material from two field trial locations are discussed.

Responsiveness to long days for flowering is reduced in Arabidopsis by yearly variation in growing season temperatures.

Conservative flowering behaviours, such as flowering during long days in summer or late flowering at a high leaf number, are often proposed to protect against variable winter and spring temperatures which lead to frost damage if premature flowering occurs. Yet, due the many factors in natural environments relative to the number of individuals compared, assessing which climate characteristics drive these flowering traits has been difficult. We applied a multidisciplinary approach to 10 winter-annual Arabidopsis thaliana populations from a wide climactic gradient in Norway. We used a variable reduction strategy to assess which of 100 climate descriptors from their home sites correlated most to their flowering behaviours when tested for responsiveness to photoperiod after saturation of vernalization; then, assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod responsiveness inversely correlated with interannual variation in timing of growing season onset. Time to flowering appeared driven by growing season length, curtailed by cold fall temperatures. The distribution of FLM, TFL2 and HOS1 haplotypes, genes involved in ambient temperature response, correlated with growing-season climate. We show that long-day responsiveness and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length, perhaps to opportunistically maximize growth.

Thermoperiodic Control of Floral Induction Involves Modulation of the Diurnal FLOWERING LOCUS T Expression Pattern.

Thermoperiodism is defined as the ability to discriminate between day temperature (DT) and night temperature (NT). Our aim was to shed light on the mechanistic basis of thermoperiodic floral induction with acceleration under lower DT than NT compared with other DT-NT combinations at the same average daily temperature (ADT), a response exploited in temperate area greenhouses. Arabidopsis thaliana floral pathway mutants and a lhy circadian clock mutant as well as the expression of floral integrators and LHY (LATE ELONGATED HYPOCOTYL) were studied under different DT-NT combinations, all at the same ADT. We show that acceleration of floral induction under lower DT than NT is linked to increased FT expression early during the day and generally increased LFY expression preceding visible flower buds, compared with higher DT than NT or equal DT and NT. Consistent with FLOWERING LOCUS T (FT) action through LEAFY (LFY), time to floral transition in ft-1 and lfy-1 was similar under all treatments, in contrast to the situation for soc1-1, which behaved like the wild type (WT). The lhy-21 mutants did not discriminate between opposite DT-NT combinations, whereas LHY expression in the WT differed in these temperature regimes. This might suggest that LHY plays a role in thermoperiodic control of floral induction. We conclude that thermoperiodic control of floral transition is associated with modulation of the diurnal expression patterns of FT, with timing of temperature alteration being important rather than ADT.

Cold acclimation in warmer extended autumns impairs freezing tolerance of perennial ryegrass (Lolium perenne) and timothy (Phleum pratense).

The effect of variable autumn temperatures in combination with decreasing irradiance and daylength on photosynthesis, growth cessation and freezing tolerance was investigated in northern- and southern-adapted populations of perennial ryegrass (Lolium perenne) and timothy (Phleum pratense) intended for use in regions at northern high latitudes. Plants were subjected to three different acclimation temperatures; 12, 6 and 9/3°C (day/night) for 4 weeks, followed by 1 week of cold acclimation at 2°C under natural light conditions. This experimental setup was repeated at three different periods during autumn with decreasing sums of irradiance and daylengths. Photoacclimation, leaf elongation and freezing tolerance were studied. The results showed that plants cold acclimated during the period with lowest irradiance and shortest day had lowest freezing tolerance, lowest photosynthetic activity, longest leaves and least biomass production. Higher acclimation temperature (12°C) resulted in lower freezing tolerance, lower photosynthetic activity, faster leaf elongation rate and higher biomass compared with the other temperatures. Photochemical mechanisms were predominant in photoacclimation. The northern-adapted populations had a better freezing tolerance than the southern-adapted except when grown during the late autumn period and at the highest temperature; then there were no differences between the populations. Our results indicate that the projected climate change in the north may reduce freezing tolerance in grasses as acclimation will take place at higher temperatures and shorter daylengths with lower irradiance.

Limited genetic diversity found among genotypes of the Entada landrace (Ensete ventricosum, (Welw.) Chessman) from Ethiopia.

The Entada landrace of enset (Ensete ventricosum (Welw.) Chessman) is probably the most unique indigenous crop in Ethiopia, being maintained and utilized by the Ari people in the South of Ethiopia. Here we describe genetic diversity, selection signatures and relationship of Entada with cultivated and wild enset using 117 Entada genotypes collected from three Entada growing regions in Ethiopia (Sidama, South and North Ari). A total number of 1,617 high-quality SNP markers, obtained from ddRAD-sequences, were used for the diversity studies. Phylogenetic analysis detected a clear distinction between cultivated enset, Entada and wild enset with Entada forming a completely separated clade. However, extremely short branch lengths among the Entada genotypes indicate very little molecular evolution in the Entada lineages. Observed and expected heterozygosities were high, 0.73 and 0.50, respectively. Overall, our results strongly indicate that the Entada genotypes we have studied originated from one or a few clonal lineages that have been propagated and spread among farmers as clones. Prolonged clonal propagation of heterozygous genotypes from a single or few founding lineages has led to populations with very little or no diversity between genotypes, and high heterozygosity within genotypes. Signatures of directional selection were identified at eight loci based on an FST outlier analysis. Four candidate genes detected are involved in axillary shoot growth and might be involved in controlling natural sucker formation in Entada.

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 world's most important temperate crops.

Improving abiotic stress tolerance of forage grasses - prospects of using genome editing.

Due to an increase in the consumption of food, feed, and fuel and to meet global food security needs for the rapidly growing human population, there is a necessity to obtain high-yielding crops that can adapt to future climate changes. Currently, the main feed source used for ruminant livestock production is forage grasses. In temperate climate zones, perennial grasses grown for feed are widely distributed and tend to suffer under unfavorable environmental conditions. Genome editing has been shown to be an effective tool for the development of abiotic stress-resistant plants. The highly versatile CRISPR-Cas system enables increasingly complex modifications in genomes while maintaining precision and low off-target frequency mutations. In this review, we provide an overview of forage grass species that have been subjected to genome editing. We offer a perspective view on the generation of plants resilient to abiotic stresses. Due to the broad factors contributing to these stresses the review focuses on drought, salt, heat, and cold stresses. The application of new genomic techniques (e.g., CRISPR-Cas) allows addressing several challenges caused by climate change and abiotic stresses for developing forage grass cultivars with improved adaptation to the future climatic conditions. Genome editing will contribute towards developing safe and sustainable food systems.

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

BACKGROUND: Little 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. RESULTS: Brachypodium 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. CONCLUSIONS: We 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.

Protein phosphatase 2A B55 and A regulatory subunits interact with nitrate reductase and are essential for nitrate reductase activation.

Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55ß. Interestingly, the homozygous double mutant (b55α × b55ß) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bß and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation.

Genomic Prediction of Complex Traits in Forage Plants Species: Perennial Grasses Case.

The majority of forage grass species are obligate outbreeders. Their breeding classically consists of an initial selection on spaced plants for highly heritable traits such as disease resistances and heading date, followed by familial selection on swards for forage yield and quality traits. The high level of diversity and heterozygosity, and associated decay of linkage disequilibrium (LD) over very short genomic distances, has hampered the implementation of genomic selection (GS) in these species. However, next generation sequencing technologies in combination with the development of genomic resources have recently facilitated implementation of GS in forage grass species such as perennial ryegrass (Lolium perenne L.), switchgrass (Panicum virgatum L.), and timothy (Phleum pratense L.). Experimental work and simulations have shown that GS can increase significantly the genetic gain per unit of time for traits with different levels of heritability. The main reasons are (1) the possibility to select single plants based on their genomic estimated breeding values (GEBV) for traits measured at sward level, (2) a reduction in the duration of selection cycles, and less importantly (3) an increase in the selection intensity associated with an increase in the genetic variance used for selection. Nevertheless, several factors should be taken into account for the successful implementation of GS in forage grasses. For example, it has been shown that the level of relatedness between the training and the selection population is particularly critical when working with highly structured meta-populations consisting of several genetic groups. A sufficient number of markers should be used to estimate properly the kinship between individuals and to reflect the variability of major QTLs. It is also important that the prediction models are trained for relevant environments when dealing with traits with high genotype × environment interaction (G × E). Finally, in these outbreeding species, measures to reduce inbreeding should be used to counterbalance the high selection intensity that can be achieved in GS.

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.

Genetic mapping of DArT markers in the Festuca-Lolium complex and their use in freezing tolerance association analysis.

Species belonging to the Festuca-Lolium complex are important forage and turf species and as such, have been studied intensively. However, their out-crossing nature and limited availability of molecular markers make genetic studies difficult. Here, we report on saturation of F. pratensis and L. multiflorum genetic maps using Diversity Array Technology (DArT) markers and the DArTFest array.The 530 and 149 DArT markers were placed on genetic maps of L. multiflorum and F. pratensis, respectively, with overlap of 20 markers, which mapped in both species. The markers were sequenced and comparative sequence analysis was performed between L. multiflorum, rice and Brachypodium. The utility of the DArTFest array was then tested on a Festulolium population FuRs0357 in an integrated analysis using the DArT marker map positions to study associations between markers and freezing tolerance. Ninety six markers were significantly associated with freezing tolerance and five of these markers were genetically mapped to chromosomes 2, 4 and 7. Three genomic loci associated with freezing tolerance in the FuRs0357 population co-localized with chromosome segments and QTLs previously identified to be associated with freezing tolerance. The present work clearly confirms the potential of the DArTFest array in genetic studies of the Festuca-Lolium complex. The annotated DArTFest array resources could accelerate further studies and improvement of desired traits in Festuca-Lolium species.

Flowering Times of Wild Arabidopsis Accessions From Across Norway Correlate With Expression Levels of FT, CO, and FLC Genes.

Temperate species often require or flower most rapidly in the long daylengths, or photoperiods, experienced in summer or after prolonged periods of cold temperatures, referred to as vernalization. Yet, even within species, plants vary in the degree of responsiveness to these cues. In Arabidopsis thaliana, CONSTANS (CO) and FLOWERING LOCUS C (FLC) genes are key to photoperiod and vernalization perception and antagonistically regulate FLOWERING LOCUS T (FT) to influence the flowering time of the plants. However, it is still an open question as to how these genes vary in their interactions among wild accessions with different flowering behaviors and adapted to different microclimates, yet this knowledge could improve our ability to predict plant responses in variable natural conditions. To assess the relationships among these genes and to flowering time, we exposed 10 winter-annual Arabidopsis accessions from throughout Norway, ranging from early to late flowering, along with two summer-annual accessions to 14 weeks of vernalization and either 8- or 19-h photoperiods to mimic Norwegian climate conditions, then assessed gene expression levels 3-, 5-, and 8-days post vernalization. CO and FLC explained both FT levels and flowering time (days) but not rosette leaf number at flowering. The correlation between FT and flowering time increased over time. Although vernalization suppresses FLC, FLC was high in the late-flowering accessions. Across accessions, FT was expressed only at low FLC levels and did not respond to CO in the late-flowering accessions. We proposed that FT may only be expressed below a threshold value of FLC and demonstrated that these three genes correlated to flowering times across genetically distinct accessions of Arabidopsis.

ForageGrassBase: molecular resource for the forage grass meadow fescue (Festuca pratensis Huds.).

Meadow fescue (Festuca pratensis Huds.) is one of the most important forage grasses in temperate regions. It is a diploid (2n = 14) outbreeding species that belongs to the genus Festuca. Together with Lolium perenne, they are the most important genera of forage grasses. Meadow fescue has very high quality of yield with good winter survival and persistency. However, extensive genomic resources for meadow fescue have not become available so far. To address this lack of comprehensive publicly available datasets, we have developed functionally annotated draft genome sequences of two meadow fescue genotypes, 'HF7/2' and 'B14/16', and constructed the platform ForageGrassBase, available at http://foragegrass.org/, for data visualization, download and querying. This is the first open-access platform that provides extensive genomic resources related to this forage grass species. The current database provides the most up-to-date draft genome sequence along with structural and functional annotations for genes that can be accessed using Genome Browser (GBrowse), along with comparative genomic alignments to Arabidopsis, L. perenne, barley, rice, Brachypodium and maize genomes. We have integrated homologous search tool BLAST also for the users to analyze their data. Combined, GBrowse, BLAST and downloadable data gives a user-friendly access to meadow fescue genomic resources. To our knowledge, ForageGrassBase is the first genome database dedicated to forage grasses. The current forage grass database provides valuable resources for a range of research fields related to meadow fescue and other forage crop species, as well as for plant research communities in general. The genome database can be accessed at http://foragegrass.org.

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

BACKGROUND: Grasses 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. RESULTS: The 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. CONCLUSION: The 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 x Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.

Allele Frequency Changes Provide Evidence for Selection and Identification of Candidate Loci for Survival in Red Clover (Trifolium pratense L.).

Survivor populations of red clover (Trifolium pratense L.) from plots in a field experiment in southern Norway were genetically characterized using genotyping by sequencing, and compared with the original population and each other. Genetic differentiation between populations was characterized on the basis of allele frequencies of single nucleotide polymorphisms (SNPs), using principal component analysis. SNPs that had been under selection, i.e., SNPs with significantly different allele frequencies in survivor populations relative to the original population, or between survivor populations that had received different treatments, were identified by analysis of F ST values, using BayeScan and a simple and stringent F ST-based test utilizing replicate populations from the field experiment. In addition, we tested the possibility of pooling DNA samples prior to sequencing, and pooling leaf samples prior to DNA extraction and sequencing, followed by allele frequency estimation on the basis of number of variant reads. Overall, survivor populations were more different from each other than from the original population, indicating random changes in allele frequency, selection in response to local variation in conditions between plots in the field experiment, or sampling error. However, some differentiation was observed between plots sown as pure stands or species mixtures, plots sown at different densities, and plots subjected to different harvesting regimes. Allele frequencies could be accurately estimated from pooled DNA, and SNPs under selection could be identified when leaf samples were pooled prior to DNA extraction. However, substantial sampling error required replicate populations and/or a high number of sampled individuals. We identified a number of chromosomal loci that had been under selection in pure stand plots relative to the original sown population, and loci that had been under differential selection in pure stands of red clover vs. red clover grown in species mixtures. These are all candidate loci for establishment success or persistence in red clover.

Tracking the evolution of a cold stress associated gene family in cold tolerant grasses.

BACKGROUND: Grasses are adapted to a wide range of climatic conditions. Species of the subfamily Pooideae, which includes wheat, barley and important forage grasses, have evolved extreme frost tolerance. A class of ice binding proteins that inhibit ice re-crystallisation, specific to the Pooideae subfamily lineage, have been identified in perennial ryegrass and wheat, and these proteins are thought to have evolved from a leucine-rich repeat phytosulfokine receptor kinase (LRR-PSR)-like ancestor gene. Even though the ice re-crystallisation inhibition function of these proteins has been studied extensively in vitro, little is known about the evolution of these genes on the molecular level. RESULTS: We identified 15 putative novel ice re-crystallisation inhibition (IRI)-like protein coding genes in perennial ryegrass, barley, and wheat. Using synonymous divergence estimates we reconstructed the evolution of the IRI-like gene family. We also explored the hypothesis that the IRI-domain has evolved through repeated motif expansion and investigated the evolutionary relationship between a LRR-domain containing IRI coding gene in carrot and the Pooideae IRI-like genes. Our analysis showed that the main expansion of the IRI-gene family happened ~36 million years ago (Mya). In addition to IRI-like paralogs, wheat contained several sequences that likely were products of polyploidisation events (homoeologs). Through sequence analysis we identified two short motifs in the rice LRR-PSR gene highly similar to the repeat motifs of the IRI-domain in cold tolerant grasses. Finally we show that the LRR-domain of carrot and grass IRI proteins both share homology to an Arabidopsis thaliana LRR-trans membrane protein kinase (LRR-TPK). CONCLUSION: The diverse IRI-like genes identified in this study tell a tale of a complex evolutionary history including birth of an ice binding domain, a burst of gene duplication events after cold tolerant grasses radiated from rice, protein domain structure differentiation between paralogs, and sub- and/or neofunctionalisation of IRI-like proteins. From our sequence analysis we provide evidence for IRI-domain evolution probably occurring through increased copy number of a repeated motif. Finally, we discuss the possibility of parallel evolution of LRR domain containing IRI proteins in carrot and grasses through two completely different molecular adaptations.

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.

Freezing tolerance revisited-effects of variable temperatures on gene regulation in temperate grasses and legumes.

Climate change creates new patterns of seasonal climate variation with higher temperatures, longer growth seasons and more variable winter climates. This is challenging the winter survival of perennial herbaceous plants. In this review, we focus on the effects of variable temperatures during autumn/winter/spring, and its interactions with light, on the development and maintenance of freezing tolerance. Cold temperatures induce changes at several organizational levels in the plant (cold acclimation), leading to the development of freezing tolerance, which can be reduced/lost during warm spells (deacclimation) in winters, and attained again during cold spells (reacclimation). We summarize how temperature interacts with components of the light regime (photoperiod, PSII excitation pressure, irradiance, and light quality) in determining changes in the transcriptome, proteome and metabolome.

Using a Candidate Gene-Based Genetic Linkage Map to Identify QTL for Winter Survival in Perennial Ryegrass.

Important agronomical traits in perennial ryegrass (Lolium perenne) breeding programs such as winter survival and heading date, are quantitative traits that are generally controlled by multiple loci. Individually, these loci have relatively small effects. The aim of this study was to develop a candidate gene based Illumina GoldenGate 1,536-plex assay, containing single nucleotide polymorphism markers designed from transcripts involved in response to cold acclimation, vernalization, and induction of flowering. The assay was used to genotype a mapping population that we have also phenotyped for winter survival to complement the heading date trait previously mapped in this population. A positive correlation was observed between strong vernalization requirement and winter survival, and some QTL for winter survival and heading date overlapped on the genetic map. Candidate genes were located in clusters along the genetic map, some of which co-localized with QTL for winter survival and heading date. These clusters of candidate genes may be used in candidate gene based association studies to identify alleles associated with winter survival and heading date.