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Enset (Enseteventricosum (Welw.) Cheesman) is a multipurpose, drought-tolerant and a key food security crop, which is the staple food for peoples in the South and Southwestern parts of Ethiopia. Despite its importance, enset is an orphan crop as regards genetic research and breeding. In this study, we characterized genetic diversity, population structure and selection signatures in 226 cultivated and 10 wild enset accessions collected from diverse enset growing regions of Ethiopia using 3505 high-quality SNP markers obtained from ddRAD-sequences. The population structure and cluster analyses clearly distinguished between cultivated and wild enset. AMOVA revealed much higher levels of genetic variation within populations and regions (91.2 and 92.4%, respectively) than between populations and regions (8.8 and 7.6%, respectively). This shows that the region of origin and environmental heterogeneity have little influence on the genetic variation. However, the genetic differentiation between regions was moderate to large (FST = 0.06–0.17). The genetic structure of enset was mainly shaped by eco-geographic factors, mode of propagation and cultivation status. Six genes potentially involved in sexual reproduction and flowering signalling, which are key processes underlying domestication and adaptation, were under positive selection demonstrating that sexual reproduction plays an important role in shaping enset diversity. A lot of unexplored diversity is available for improving enset in Ethiopia, with patterns of diversity consistent with divergent selection on adaptive traits. This diversity also shows potential for introducing enset as a more food secure crop for the food insecure regions in the dry north of Ethiopia.

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.

Conservative flowering behaviors, 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 ten winter-annual Arabidopsis thaliana populations originating along 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 behaviors when grown in common garden and assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod sensitivity inversely correlated with interannual variation in timing of growing season onset (start of favorable spring temperatures). 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 sensitivity and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length perhaps to opportunistically maximize growth.

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.

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.

Microdochium majusandMicrodochium nivalecause serious disease problems in grasses and cereal crops in the temperate regions. Both fungi can infect the plants during winter (causing pink snow mould) as well as under cool humid conditions during spring and fall. We conducted a pathogenicity test of 15 M. nivaleisolates and twoM. majusisolates from Norway at low temperature on four different grass cultivars ofLolium perenneandFestuloliumhybrids. Significant differences betweenM. nivaleisolates in the ability to cause pink snow mould were detected. TheM. nivalestrains originally isolated from grasses were more pathogenic than isolates from cereals. The genetic diversity ofM. nivaleandM. majusisolates was studied by sequencing four genetic regions; Elongation factor-1 alpha (EF-1α), β-tubulin, RNA polymerase II (RPB2)and the Internal Transcribed Spacer (ITS). Phylogenetic trees based on the sequences of these four genetic regions resolvedM. nivaleandM. majusisolates into separate clades. Higher genetic diversity was found amongM. nivaleisolates than amongM. majusisolates.M. nivaleisolates revealed genetic differences related to different host plants (grasses vs. cereals) and different geographic regions (Norway and UK vs. North America). Sequence results from theRPB2and β-tubulin genes were more informative than those from ITS andEF-1α. The genetic and phenotypic differences detected between NorwegianM. nivaleisolates from cereals and grasses support the assumption that host specialization exist withinM. nivaleisolates.

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.

Background: Enset (Ensete ventricosum (Welw.) Cheesman) is a staple food for more than 20 million Ethiopians and only cultivated in the native indigenous farming systems of Ethiopia. In contrast to other cultivated species in the Musaceae family, enset has been relatively little studied at the molecular level. Application of advanced molecular genetic techniques requires rapid extraction of DNA of high quality and quantity. Fresh, lyophilized tissues, as well as tissues stored in liquid nitrogen are mainly preferred to avoid DNA degradation, thus most of the DNA extraction protocols recommend these types of tissues as starting material. However, such sample processing techniques are difficult to utilize in many developing countries and at collection sites of many endemic plant species, underutilized or orphan crop species like enset. These situations necessitate the development of alternative protocols for leaf preservation and optimized methods for isolating high-quality DNA from dried or preserved leaf samples. Results: In this study, three different leaf preservation and two DNA extraction methods were compared. Fresh young leaf tissue was preserved using the minor modified saturated NaCl-CTAB solution, silica gel or 96% ethanol at ambient temperature for more than 35 days. Subsequently, DNA was extracted using either the DNeasy Plant Mini Kit or the CTAB method. As compared to silica gel and 96% ethanol, the minor modified saturated NaCl-CTAB solution preserved the quality, quantity, and integrity of enset genomic DNA. This method consistently produced genomic DNA of high-quality and quantity at affordable cost. The DNeasy Plant Mini Kit method was found to be more efficient than the standard CTAB method, being faster and producing genomic DNA of higher quality. Conclusions: Using saturated NaCl-CTAB solution is an accessible, efficient, scalable, and inexpensive way to preserve enset leaves during collection and transportation. The preservation protocol was validated for leaf tissues of all cultivated and wild enset, and Entada landraces. Genomic DNA of high quality and quantity was obtained from preserved enset leaves, which can be used for further downstream applications including PCR and sequencing.

Red clover (Trifolium pratense L.) is the most important forage legume in the Nordic region, but its utilization is limited by poor persistency. The improvement of cultivated red clover can potentially take advantage of the numerous wild populations and landraces conserved in gene banks; however, there is often limited information available on the phenotypic and genetic characteristics of this material. We characterized 48 populations conserved at NordGen for a number of traits and compared them to commercial cultivars. The material was evaluated in field trials at four locations over two years and in an experiment under controlled conditions. Considerable variation was identified, with stem length, growth type and flowering date having the highest broad sense heritabilities. Traits related to plant size were strongly associated with late flowering and upright growth and differed between landraces/cultivars on the one hand and wild populations on the other. There was a large genotype by environment interaction on winter survival, which only partially correlated with freezing tolerance under controlled conditions. A majority of gene bank accessions exceeded the commercial cultivars in winter survival and freezing tolerance and can therefore be a genetic resource for future improvement of these traits. The phenotypic variation among accessions was associated with two main axes of climatic variation at the collection site. Petiole length of young plants under controlled conditions as well as plant size in the field increased with increasing summer temperature and decreasing summer precipitation, while number of leaves and an apparent vernalization requirement, recorded under controlled conditions, increased with decreasing annual and winter temperature. We discuss the implications these results have for collection, conservation and utilization of red clover genetic resources in the Nordic region.

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.

Satisfactory seed yield of red clover (Trifolium pratense L.) cultivars is crucial for the availability of seeds on the market. Many breeders and researchers have used seed yield components to measure, compare, and explain differences in seed yield between diploid and tetraploid red clover cultivars and populations; however, the relative importance of each component varies between studies. In 2011 and 2012, single-plant trials with several tetraploid and one diploid red clover cultivar were established at the Norwegian plant breeding station at Bjorke. The goal was to study the impact of different seed-yield components on the seed yield of tetraploid plants. Seed weight per flower head was the seed-yield component that correlated best with the seed yield plant⁻¹ (r = 0.91 and r = 0.68 in 2011 and 2012, respectively). Path coefficient analysis has also shown that the seed weight per flower head had the highest direct impact on seed yield plant⁻¹ (direct path coefficients were 0.867 and 0.783 in 2011 and 2012, respectively). In comparison, the direct path coefficients for calculated number of flower heads, which was previously highlighted as the most important seed-yield component, were lower and more variable (0.739 and 0.392 in 2011 and 2012, respectively). Since previously seed yield per flower head was also identified as the most important seed-yield component in dense plant canopy, this component might have the potential to select for improved seed yield of new cultivars based on single plants. However, further studies are required to confirm this conclusion.

Four-species mixtures and pure stands of perennial ryegrass, tall fescue, white clover and red clover were grown in three-cut and five-cut systems at As, southern Norway, at a low fertilization rate (100 kg N ha−1 year−1). Over a three-year experiment, we found strong positive effects of species diversity on annual dry-matter yield and yield stability under both cutting frequencies. The overyielding in mixtures relative to pure stands was highest in the five-cut system and in the second year. Among the possible pairwise species interaction effects contributing to the diversity effect, the grass–grass interaction was the strongest, being significant in both cutting systems and in all years. The grass–legume interactions were sometimes significant, but no significant legume–legume interaction could be detected. Competitive relationships between species varied from year to year and also between cutting systems. Estimations based on species identity effects and pair-specific interactions suggested that the optimal proportions of red clover, white clover, perennial ryegrass and tall fescue in seed mixtures would have been around 0·1, 0·2, 0·4 and 0·3 in the three-cut system, and 0·1, 0·3, 0·3 and 0·3 in the five-cut system.

Pink snow mould caused by Microdochium nivale is a serious cereal and grass disease in several temperate regions. In this study, resistance to snow mould was evaluated under controlled conditions i...

In a ×Festulolium population (FuRs0357) of parental origin Lolium perenne × Festuca pratensis, selection of freezing tolerance by freezing tests on whole plants (FT) and chlorophyll a (Chl-a) fluorimetry on frozen detached leaves (CF) was assessed in high and low directions during two cycles of selection. The original population went through two cycles of random mating. All selections and non-selected intercrossed generations of the original population were established in field trials at a coastal site and a continental site in Norway. At the coastal site, analyses of Chl-a fluorimetry parameters and leaf growth on individual plants in autumn and winter hardiness observed in field plots in spring showed that the first-generation selections for high freezing tolerance were associated with winter hardiness and early growth cessation. The second-generation FT-selections for high freezing tolerance were also associated with winter hardiness, whereas the CF-high selections diverged toward high photosynthetic activity. Both low selections were correlated with high photosynthetic activity. There were smaller variations between generations in unselected generations of the original population. Low accumulated leaf growth and early growth cessation were observed in the second-generation FT-selection for high freezing tolerance, whereas high normalized difference vegetation index (NDVI) were seen in Chl-a selections. Both selection methods distinguished diverging selections with significantly different high and low freezing tolerance, but selection efficiency was comparable only for the first selection cycle. Moreover, due to mixed ploidy level in the original population, selection by FT and CF generated diploid and tetraploid plants, respectively, which intensified the response of selection, particularly in the diploid selections. Total dry matter yield (DMY) (mean of three annual cuts for 3 years) of the FT-high selections was lower than for the CF-selections. At coastal sites, selection intensity using freezing tests on whole plants should be adapted to actual climate conditions, to obtain genotypes that balance photosynthetic activity during autumn and good winter hardiness, making them persistent and high yielding.

Perennial ryegrass (Lolium perenne L.) is increasingly important as a forage grass in the Nordic and Baltic region, especially in the light of climate change. However, winter hardiness and persistence need to be improved. This article describes the long-term Nordic/Baltic public-private partnership for pre-breeding in perennial ryegrass, started in 2012. The basis for the project is detailed genotyping and phenotyping of nearly 400 genebank accessions, followed by generation of new broad-based diploid and tetraploid breeding populations, locally adapted and trait-based populations. Preliminary results confirm the need for more robust and stable cultivars with wider adaptations, and genebank accessions need to be introgressed and recombined with adapted materials followed by natural/artificial selection in order to create novel germplasm for these regions.

International audience; Norwegian cultivars and breeding materials of perennial ryegrass and Festulolium were planted at three locations in Denmark, France and Japan for testing of resistance against leaf diseases. In general, all plant materials were susceptible to crown rust. The highest incidence of rust attack occurred at the French site, which due to its climatic conditions might be the most suitable testing site for future scoring of similar plant material. Entries based on introgressed genetic materials from UK were most resistant towards crown rust. Crown rust resistance needs increased focus as a breeding objective in the Nordic region due to climate changes, which will most likely lead to increased infection of leaf diseases.

Mixtures and pure stands of perennial ryegrass, tall fescue, white clover and red clover were grown in a three-cut and a five-cut system in southern Norway, at a low fertilization rate (100 kg N ha−1 year−1). The nutritional quality (annual weighted averages) of the dried forage from the two-first harvesting years was analysed. There was no significant effect of species diversity on crude protein (CP) concentration. In the three-cut system, we found a significant species diversity effect leading to 10% higher concentrations of acid detergent fibre (ADF), 20–22% lower concentrations of water-soluble carbohydrate (WSC) and 4% lower net energy for lactation (NEL) concentrations in mixtures compared with pure stands (averaged across the two-first years). In the five-cut system, similar effects were seen in the first year only. This diversity effect was associated with a reduction in WSC and NEL concentrations and an increase in ADF, NDF and CP concentrations in the grass species, and not in red clover, when grown in mixtures. This is thought to be a combined result of better N availability and more shading in the mixtures. Species diversity reduced the intra-annual variability in nutritional quality in both cutting systems.

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.

Vernalization requirement maintains winter annuals in a cold hardy vegetative state during the winter. Vernalization saturation has been shown to be followed by a loss of freezing tolerance under controlled experimental conditions. The objective of this study was to determine a possible relationship between vernalization saturation and the maintenance of cultivar specific freezing tolerance levels in winter rapeseed (Brassica napus L.) under field conditions. Two cultivars, ‘Banjo’ and ‘Californium’, with differing vernalization requirements were chosen after a screening of 18 cultivars under controlled conditions. Vernalization response and freezing tolerance of the two cultivars grown under field conditions were compared on five different dates throughout the winter of 2010/11 at Apelsvoll Research Station, Norway (60° 42′ N, 10° 52′ E, 250 m a.s.l.). ‘Californium’ reached vernalization saturation on 11 Oct. whereas ‘Banjo’ did 13 days later. Maximum freezing tolerance of both cultivars was attained in early December and it was maintained for 31 days in ‘Californium’ and 67 days in ‘Banjo’. Our results indicate that there is no simple relationship between freezing tolerance and saturation of vernalization. Increasing contents of water, d -glucose and d -fructose and decreasing sucrose and starch levels in the shoot apex from mid to late winter were significantly correlated with a decrease in freezing tolerance (LT50) and shoot regrowth following a −12 °C low temperature treatment and so these parameters may be good predictors of plant survival and vitality in the spring.

Most perennial and winter annual temperate grasses have a vernalization requirement (VR) for flowering, that is, they require a cold period before they can flower in response to long days. From a F1 mapping population of the outbreeding perennial forage grass Festuca pratensis Huds. (meadow fescue) previously used to map several quantitative trait loci (QTLs) for VR, we produced two F2 populations divergently selected for high or low VR. The two populations were characterised for flowering behaviour and gene expression of VRN1 as well as other MADS-box genes with a putative function in the induction of flowering. Expression of FpVRN1 and the VRN1-like genes FpMADS2 and FpMADS3 was associated with flowering but the response of gene expression to vernalization differed between genes and populations. The expression of the SVP-like genes FpMADS10 and FpMADS16 was not affected by vernalization and did not differ between the two F2 populations.

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&nbsp

The establishment of PPP for Pre-breeding, development, present status and the way forward are described in this booklet. The success of the PPP is based on:• Pooled public funding• Participation f ...

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.

Plants adapted to cold winters go through annual cycles of gain followed by loss of freezing tolerance (cold acclimation and deacclimation). Warm spells during winter and early spring can cause deacclimation, and if temperatures drop, freezing damage may occur. Many plants are vernalized during winter, a process making them competent to flower in the following summer. In winter cereals, a coincidence in the timing of vernalization saturation, deacclimation, downregulation of cold-induced genes, and reduced ability to reacclimate, occurs under long photoperiods and is under control of the main regulator of vernalization requirement in cereals, VRN1, and/or closely linked gene(s). Thus, the probability of freezing damage after a warm spell may depend on both vernalization saturation and photoperiod. We investigated the role of vernalization and the VRN1-region on freezing tolerance of meadow fescue (Festuca pratensis Huds.), a perennial grass species. Two F2 populations, divergently selected for high and low vernalization requirement, were studied. Each genotype was characterized for the copy number of one of the four parental haplotypes of the VRN1-region. Clonal plants were cold acclimated for 2 weeks or vernalized/cold acclimated for a total of 9 weeks, after which the F2 populations reached different levels of vernalization saturation. Vernalized and cold acclimated plants were deacclimated for 1 week and then reacclimated for 2 weeks. All treatments were given at 8 h photoperiod. Flowering response, freezing tolerance and expression of the cold-induced genes VRN1, MADS3, CBF6, COR14B, CR7 (BLT14), LOS2, and IRI1 was measured. We found that some genotypes can lose some freezing tolerance after vernalization and a deacclimation–reacclimation cycle. The relationship between vernalization and freezing tolerance was complex. We found effects of the VRN1-region on freezing tolerance in plants cold acclimated for 2 weeks, timing of heading after 9 weeks of vernalization, expression of COR14B, CBF6, and LOS2 in vernalized and/or deacclimated treatments, and restoration of freezing tolerance during reacclimation. While expression of VRN1, COR14B, CBF6, LOS2, and IRI1 was correlated, CR7 was associated with vernalization requirement by other mechanisms, and appeared to play a role in freezing tolerance in reacclimated plants.

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar ‘Fagerlin’ based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould.

Flowering time is a major determinant of plant reproductive success and adaptation to local climatic conditions. Vernalization, i.e. a prolonged period of low temperature, is one of the processes controlling flowering time. This study investigates vernalization responses in natural populations of Arabidopsis thaliana from higher latitudes and their significance for local adaptation. Twenty-seven A. thaliana populations were collected across latitudinal (59–68°N) and altitudinal gradients (2–850 m a.s.l.) in Norway, and the populations were screened for responses to 5 different vernalization treatments. These populations represent the northernmost limit of the species distribution worldwide. Variations in vernalization sensitivity and vernalization saturation were tested for relationships with altitude, distance from the ocean and climatic factors associated with the site of origin. The results showed that these traits were associated with both altitude and distance from the ocean. Populations originating from higher altitudes and inland locations were more sensitive and required less time to saturate their vernalization requirement than populations from lower altitudes at coastal locations. Analyses among inland populations revealed higher contribution of distance from the ocean than altitude to the variation in vernalization responses. These results indicate that the continental-oceanic climatic gradient generate clinal variation in adaptive traits.

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., Scientific Reports, 5, ISSN:2045-2322

Small but important differences in winter survival are known to exist between winter canola cultivars. The objective of this study was to compare a short-term (lethal temperature for 50% plant kill [LT 50 ]) and a prolonged (lethal duration time for 50% plant kill [LD 50 ]) freeze test to identify differences in freezing tolerance of winter canola (Brassica napus L. var. oleifera Metzg. and Brassica rapa L. var. oleifera Sink.) species and cultivars. Viability following each freeze test was determined by electrolyte leakage, plant survival, biomass of shoot regrowth, and root regeneration. Plant survival from the LT 50 (cooling rate 3°C h −1 ) and LD 50 (−8°C isothermal for up to 24 d) tests were poorly correlated, and only the LD 50 test was able to identify cultivar differences in freezing tolerance. Electrolyte leakage did not correlate with actual survival measurements in both freeze tests. Shoot regrowth was a more sensitive viability test than plant survival. Brassica rapa plants subjected to the LT 50 test showed greater shoot regrowth than B. napus plants, yet no cultivar differences were found within each species. In the LD 50 freeze test, no difference in shoot regrowth was detected between the species; however, cultivar differences were found within both species. Tolerance to prolonged freezing is critical for winter survival, and the LD 50 freeze test may allow more accurate screening for species and cultivars with improved freezing tolerance than the LT 50 freeze test.

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.

Novel high-throughput genotyping technologies have facilitated rapid genotyping of single nucleotide polymorphisms in non-model organisms. Most plant species have complex genomes with a large proportion of their genes having one or more paralogous copies due to single gene duplications and ancient or recent polyploidization events. These paralogous gene copies are potential sources of genotyping errors, and hence genotyping of plant genomes is inherently difficult. Here we present a case study that exemplifies paralog-related problems in high-throughput genotyping of plant genomes. We used the MassARRAY genotyping platform to genotype the LpIRI locus in L. perenne populations; this gene is thought to be involved in low-temperature stress tolerance. The dissection of the molecular genetics underlying the genotyping results provides a good example of how unknown paralogs can mask the true genotype of the locus, instructive to the non-specialist plant researcher and breeder.

In Tanzania sweet potato ranks as the third most important crop after cassava and potato. We studied the phenotypic diversity of morphological plant and root descriptor traits in accessions of the sweet potato germplasm collection of Sokoine University of Agriculture, Morogoro and Sugarcane Research Institute, Kibaha, Tanzania, using phenotypic characters. A total number of 105 sweet potato accessions of different geographic origins were studied in field trials of The Sugarcane Research Institute at Kibaha Tanzania, and data were recorded for 27 phenotypic characters. Estimates of pair-wise phenotypic similarities using the Manhattan coefficient varied from 0.023 to 0.814, with a mean of 0.285. Cluster analysis was conducted using the unweighted pair group method with arithmetic mean (UPGMA) and Principal Coordinate Analysis (PCO). The clustering of phenotypic data resulted in a dendrogram which was discordant with geographic origin and AFLP data. The analysis of variance (ANOVA) revealed highly significant variation among the accessions for 21 out of the 27 characters studied. Phenotypic analyses revealed a wider range of variability than AFLP analyses. Comparison of molecular and phenotypic data using the Mantel test showed a very low correlation (r2 = 0.0007). Molecular and phenotypic classifications are discordant, and both are necessary to classify the germplasm correctly and to clarify genetic relationships among sweet potato accessions.

Argyranthemum frutescens is a commercially important ornamental species with extensive breeding programmes in several countries. Since it is vegetatively propagated there is a great need for tools for identification of cultivars. Vegetatively propagated species require clean-up from diseases, often performed through meristem-tip cultures. Forty-three cultivars of A. frutescens propagated by meristem-tip culture and traditional cuttings were analyzed for genetic relatedness and possible somaclonal variation using AFLP. Five primer combinations resulted in a relatively high degree of polymorphism. Ten molecular markers generated by one primer combination could distinguish between all 43 cultivars. Differences in fingerprints between meristem-tip culture and cuttings from the same cultivars were found, but the proportion of fragments being specific for either tissue culture or cuttings was relatively low. Some cultivars that did not display somaclonal variation as judged by the AFLP-fingerprints may still be genetically unstable since some morphological variation was observed in the true to type test.

The genetic diversity within and among 81 local populations from Norway and Lithuania and 26 cultivars of meadow fescue (Festuca pratensis Huds.) from Norway, Sweden, Finland, Denmark, Lithuania, Latvia, and Estonia was investigated using amplifi ed fragment length polymorphism markers. Genetic diversity was high, with nearly 70% of the molecular variation within populations. Principal coordinates (PCO) analyses indicate that local populations from Norway and Lithuania consist of both older natural populations and naturalized populations established recently by migration from sown meadows. This is based on the fact that naturalized populations cluster with cultivars from the respective countries. The PCO analysis separates the natural local populations from Norway and Lithuania. They belong to different gene pools and refl ect dispersal from different glacial refugia after the last glaciation. The Nordic and Baltic cultivars are quite similar (6.25% of variation between groups), and the Baltic cultivars are highly homogenous, with 91% of variation within the cultivars, indicating narrow breeding populations of this species in the Nordic–Baltic region. All cultivars are most closely related to local populations from the Baltic. The results can be used to improve management and utilization of gene bank resources of meadow fescue. Conservation of gene bank accessions should focus on those local populations least infl uenced by gene fl ow from cultivated meadow fescue since they probably represent pure natural populations.

Rhodiola rosea is widely distributed in Norway, but so far limited knowledge exists on the level of genetic diversity. To initiate a selective breeding program, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the Norwegian R. rosea germplasm collection. AFLP analysis of 97 R. rosea clones using five primer combinations gave a total of 109 polymorphic bands. We detected high percentage of polymorphic bands (PPB) with a mean of 82.3% among the clones of R. rosea . Each of the 97 R. rosea clones could be unambiguously identified based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA). Genetic similarity based on the AFLP data ranged from 0.440 to 0.950 with a mean of 0.631. This genetic analysis showed that there was no close genetic similarity among clones related to their original growing county. No gender-specific markers were found in the R. rosea clones. Analysis of molecular variance (AMOVA) revealed a significantly greater variation within regions (92.03%) than among regions (7.97%). A low level of genetic differentiation ( F ST = 0.043) was observed, indicating a high level of gene flow, which had a strong influence on the genetic structure at different counties. Our results indicate high gene flow among R. rosea clones that might be a result of seed dispersal rather than cross-pollination. Further world-wide studies are required to compare the level of genetic diversity and more studies in R. rosea detailing the consequences of different patterns of gene flow (pollen spread and dispersal of seeds and clonal plants) will be useful for characterization of roseroot.

Rognli, O. A. 1987. Genetic variation in Arctic populations of timothy (Phleum prafenseL.). I. Seed production characters. —Hereditus 107:27–54. Lund, Sweden. ISSN 0018–0661. Received October 10, 1986 Genotypic variation for seed production characters within four North Norwegian timothy populations (69–70°N) was assessed over two years in replicated clonal experiments at 60°N. 63.5°N and 69°N latitude in Norway. Seed yield range was 1.2–147.6 gramlplot of 4 clonal ramets. Genotypic and genotype × environment (GE) interacticn variances were highly significant for seed yield. plant height, culm score, and plant size, while variation in earliness was essentially genotypic. Genotypic variances were homogeneous among populations for all characters, while GE-interactions were highly heterogeneous. Besides genotypic and error variances, genotype × location interactions were most important. Seed yield heritability (h2b) was 0.64. Genotypic variability and expected selection response were largest for seed yield. Genotypic and environmental correlations between seed yield and other characters, except ear emergence, were consistently positive, indicating pleiotropy. Correlations involving ear emergence varied substantially among populations and locations, indicating linkage and/or coadaptation. Population differences in correlation pattern are discussed in the context of genetic origin, local adaptation, and agricultural management. Genotypic and environmental variances were largest at the intermediate, high yielding location. Genotypic coefficients of variation (GCV%) were highest at the southern and arctic location. Genotypic correlations showed that the arctic and intermediate location gave the most similar genotypic expressions of the character seed yield. Seed production at 60“N was low, indicating physiological constraints. Adaptation to hay-production at higher latitudes results in high seed production potential. Possible effects for breeding high latitude timothy are discussed.

Rognli, O.A. 1987. Genetic variation in arctic populations of timothy (Phleum pratense L.). II. Phenotypic stability of seed production. —Hereditas 207: 75–94. Lund, Sweden. ISSN 0018–0661. Received November 10, 1986 The present paper is concerned with phenotypic stability of seed production of four North Norwegian timothy populations. In clonal experiments conducted at 69°N. 63.S°N, and 60°N latitude in Norway. the genotype × location interaction was the most important interaction effect. Individual genotypic stability reactions were studied by ecovalence (or stability variance), and mean rank difference. Pattern analysis was used to investigate the form of the individual genotypic reactions. Large differences in stability were found among genotypes. Only 11.5% of the genotypes contributed significantly to the interaction sum of squares. Elimination of very unstable genotypes could therefore be a strategy of population improvement. rather than selecting stable genotypes. Correlation between ecovalence and mean rank difference depended upon the yield level, being low for high yielding genotypes. Ecovalence and average seed yield were positively correlated, but high yielding and stable genotypes could be found. Stability rankings were sufficiently consistent over years to justify a single year test of stability. Pattern analysis (FASTCLUS) was performed on the individual genotypic within-location residuals. Structure of cluster groups revealed specific genotypic and population adaptations, assessed according to the ecovalence concept. Canonical discriminant analysis (CANDISC) was used to aid visual interpretation of group differences.