Evolution of drought and frost responses in cool season grasses (Pooideae): was drought tolerance a precursor to frost tolerance?

Frost tolerance has evolved many times independently across flowering plants. However, conservation of several frost tolerance mechanisms among distant relatives suggests that apparently independent entries into freezing climates may have been facilitated by repeated modification of existing traits ('precursor traits'). One possible precursor trait for freezing tolerance is drought tolerance, because palaeoclimatic data suggest plants were exposed to drought before frost and several studies have demonstrated shared physiological and genetic responses to drought and frost stress. Here, we combine ecophysiological experiments and comparative analyses to test the hypothesis that drought tolerance acted as a precursor to frost tolerance in cool-season grasses (Pooideae). Contrary to our predictions, we measured the highest levels of frost tolerance in species with the lowest ancestral drought tolerance, suggesting that the two stress responses evolved independently in different lineages. We further show that drought tolerance is more evolutionarily labile than frost tolerance. This could limit our ability to reconstruct the order in which drought and frost responses evolved relative to each other. Further research is needed to determine whether our results are unique to Pooideae or general for flowering plants.

Melica as an emerging model system for comparative studies in temperate Pooideae grasses.

BACKGROUND AND AIMS: Pooideae grasses contain some of the world's most important crop and forage species. Although much work has been conducted on understanding the genetic basis of trait diversification within a few annual Pooideae, comparative studies at the subfamily level are limited by a lack of perennial models outside 'core' Pooideae. We argue for development of the perennial non-core genus Melica as an additional model for Pooideae, and provide foundational data regarding the group's biogeography and history of character evolution. METHODS: Supplementing available ITS and ndhF sequence data, we built a preliminary Bayesian-based Melica phylogeny, and used it to understand how the genus has diversified in relation to geography, climate and trait variation surveyed from various floras. We also determine biomass accumulation under controlled conditions for Melica species collected across different latitudes and compare inflorescence development across two taxa for which whole genome data are forthcoming. KEY RESULTS: Our phylogenetic analyses reveal three strongly supported geographically structured Melica clades that are distinct from previously hypothesized subtribes. Despite less geographical affinity between clades, the two sister 'Ciliata' and 'Imperfecta' clades segregate from the more phylogenetically distant 'Nutans' clade in thermal climate variables and precipitation seasonality, with the 'Imperfecta' clade showing the highest levels of trait variation. Growth rates across Melica are positively correlated with latitude of origin. Variation in inflorescence morphology appears to be explained largely through differences in secondary branch distance, phyllotaxy and number of spikelets per secondary branch. CONCLUSIONS: The data presented here and in previous studies suggest that Melica possesses many of the necessary features to be developed as an additional model for Pooideae grasses, including a relatively fast generation time, perenniality, and interesting variation in physiology and morphology. The next step will be to generate a genome-based phylogeny and transformation tools for functional analyses.

The Original Form of C4-Photosynthetic Phosphoenolpyruvate Carboxylase Is Retained in Pooids but Lost in Rice.

Poaceae is the most prominent monocot family that contains the primary cereal crops wheat, rice, and maize. These cereal species exhibit physiological diversity, such as different photosynthetic systems and environmental stress tolerance. Phosphoenolpyruvate carboxylase (PEPC) in Poaceae is encoded by a small multigene family and plays a central role in C4-photosynthesis and dicarboxylic acid metabolism. Here, to better understand the molecular basis of the cereal species diversity, we analyzed the PEPC gene family in wheat together with other grass species. We could designate seven plant-type and one bacterial-type grass PEPC groups, ppc1a, ppc1b, ppc2a, ppc2b, ppc3, ppc4, ppcC4, and ppc-b, respectively, among which ppc1b is an uncharacterized type of PEPC. Evolutionary inference revealed that these PEPCs were derived from five types of ancient PEPCs (ppc1, ppc2, ppc3, ppc4, and ppc-b) in three chromosomal blocks of the ancestral Poaceae genome. C4-photosynthetic PEPC (ppcC4 ) had evolved from ppc1b, which seemed to be arisen by a chromosomal duplication event. We observed that ppc1b was lost in many Oryza species but preserved in Pooideae after natural selection. In silico analysis of cereal RNA-Seq data highlighted the preferential expression of ppc1b in upper ground organs, selective up-regulation of ppc1b under osmotic stress conditions, and nitrogen response of ppc1b. Characterization of wheat ppc1b showed high levels of gene expression in young leaves, transcriptional responses under nitrogen and abiotic stress, and the presence of a Dof1 binding site, similar to ppcC4 in maize. Our results indicate the evolving status of Poaceae PEPCs and suggest the functional association of ppc1-derivatives with adaptation to environmental changes.

Indole-diterpenes alkaloid profiles of native grasses involved in tremorgenic syndromes in the Argentine Patagonia.

One of the main intoxications to livestock in the Patagonia region of Argentina is the tremorgenic disease "Mal de Huecú", attributed to the consumption of the native grasses Poa huecu and/or Festuca argentina. In this report, five outbreaks of spontaneous intoxications were investigated. Several indole-diterpene alkaloids were identified in Poa huecu and Festuca argentina including the known tremorgen terpendole C and are likely the cause of "Mal de Huecú" disease.

Development of a Sequence Searchable Database of Celiac Disease-Associated Peptides and Proteins for Risk Assessment of Novel Food Proteins.

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

Major niche transitions in Pooideae correlate with variation in photoperiodic flowering and evolution of CCT domain genes.

The external cues that trigger timely flowering vary greatly across tropical and temperate plant taxa, the latter relying on predictable seasonal fluctuations in temperature and photoperiod. In the grass family (Poaceae) for example, species of the subfamily Pooideae have become specialists of the northern temperate hemisphere, generating the hypothesis that their progenitor evolved a flowering response to long days from a short-day or day-neutral ancestor. Sampling across the Pooideae, we found support for this hypothesis, and identified several secondary shifts to day-neutral flowering and one to short-day flowering in a tropical highland clade. To explain the proximate mechanisms for the secondary transition back to short-day-regulated flowering, we investigated the expression of CCT domain genes, some of which are known to repress flowering in cereal grasses under specific photoperiods. We found a shift in CONSTANS 1 and CONSTANS 9 expression that coincides with the derived short-day photoperiodism of our exemplar species Nassella pubiflora. This sets up the testable hypothesis that trans- or cis-regulatory elements of these CCT domain genes were the targets of selection for major niche shifts in Pooideae grasses.

Phylotranscriptomics Resolves the Phylogeny of Pooideae and Uncovers Factors for Their Adaptive Evolution.

Adaptation to cool climates has occurred several times in different angiosperm groups. Among them, Pooideae, the largest grass subfamily with ∼3,900 species including wheat and barley, have successfully occupied many temperate regions and play a prominent role in temperate ecosystems. To investigate possible factors contributing to Pooideae adaptive evolution to cooling climates, we performed phylogenetic reconstruction using five gene sets (with 1,234 nuclear genes and their subsets) from 157 transcriptomes/genomes representing all 15 tribes and 24 of 26 subtribes. Our phylogeny supports the monophyly of all tribes (except Diarrheneae) and all subtribes with at least two species, with strongly supported resolution of their relationships. Molecular dating suggests that Pooideae originated in the late Cretaceous, with subsequent divergences under cooling conditions first among many tribes from the early middle to late Eocene and again among genera in the middle Miocene and later periods. We identified a cluster of gene duplications (CGD5) shared by the core Pooideae (with 80% Pooideae species) near the Eocene-Oligocene transition, coinciding with the transition from closed to open habitat and an upshift of diversification rate. Molecular evolutionary analyses homologs of CBF for cold resistance uncovered tandem duplications during the core Pooideae history, dramatically increasing their copy number and possibly promoting adaptation to cold habitats. Moreover, duplication of AP1/FUL-like genes before the Pooideae origin might have facilitated the regulation of the vernalization pathway under cold environments. These and other results provide new insights into factors that likely have contributed to the successful adaptation of Pooideae members to temperate regions.

Phylogenetic, ecological and intraindividual variability patterns in grass phytolith shape.

BACKGROUND AND AIMS: Grass silica short cell (GSSC) phytoliths appear to be the most reliable source of fossil evidence for tracking the evolutionary history and paleoecology of grasses. In recent years, modern techniques that quantitatively assess phytolith shape variation have widened opportunities for the classification of grass fossil phytoliths. However, phylogenetic, ecological and intraindividual variability patterns in phytolith shape remain largely unexplored. METHODS: The full range of intraindividual phytolith shape variation [3650 two-dimensional (2-D) outlines] from 73 extant grass species, 48 genera, 18 tribes and eight subfamilies (particularly Pooideae) was analysed using geometric morphometric analysis based on semi-landmarks spanning phytolith outlines. KEY RESULTS: The 2-D phytolith shape is mainly driven by deep-time diversification of grass subfamilies. There is distinct phytolith shape variation in early-diverging lineages of Pooideae (Meliceae, Stipeae). The amount of intraindividual variation in phytolith shape varies among species, resulting in a remarkable pattern across grass phylogeny. CONCLUSIONS: The phylogenetic pattern in phytolith shape was successfully revealed by applying geometric morphometrics to 2-D phytolith shape outlines, strengthening the potential of phytoliths to track the evolutionary history and paleoecology of grasses. Geometric morphometrics of 2-D phytolith shape is an excellent tool for analysis requiring large numbers of phytolith outlines, making it useful for quantitative palaeoecological reconstruction.

Antagonism to Plant Pathogens by Epichloë Fungal Endophytes-A Review.

Epichloë is a genus of filamentous fungal endophytes that has co-evolved with cool-season grasses with which they form long-term, symbiotic associations. The most agriculturally important associations for pasture persistence for grazing livestock are those between asexual vertically transmitted Epichloë strains and the pasture species, perennial ryegrass, and tall fescue. The fungus confers additional traits to their host grasses including invertebrate pest deterrence and drought tolerance. Selected strains of these mutualistic endophytes have been developed into highly efficacious biocontrol products and are widely utilized within the Americas, Australia, and New Zealand for pasture persistence. Less publicized is the antagonism Epichloë endophytes display towards multiple species of saprophytic and pathogenic microbes. This opinion piece will review the current literature on antimicrobial properties exhibited by this genus of endophyte and discuss the reasons why this trait has historically remained a research curiosity rather than a trait of commercial significance.

Variation in climatic tolerance, but not stomatal traits, partially explains Pooideae grass species distributions.

BACKGROUND AND AIMS: Grasses in subfamily Pooideae live in some of the world's harshest terrestrial environments, from frigid boreal zones to the arid windswept steppe. It is hypothesized that the climate distribution of species within this group is driven by differences in climatic tolerance, and that tolerance can be partially explained by variation in stomatal traits. METHODS: We determined the aridity index (AI) and minimum temperature of the coldest month (MTCM) for 22 diverse Pooideae accessions and one outgroup, and used comparative methods to assess predicted relationships for climate traits versus fitness traits, stomatal diffusive conductance to water (gw) and speed of stomatal closure following drought and/or cold. KEY RESULTS: Results demonstrate that AI and MTCM predict variation in survival/regreening following drought/cold, and gw under drought/cold is positively correlated with δ 13C-measured water use efficiency (WUE). However, the relationship between climate traits and fitness under drought/cold was not explained by gw or speed of stomatal closure. CONCLUSIONS: These findings suggest that Pooideae distributions are at least partly determined by tolerance to aridity and above-freezing cold, but that variation in tolerance is not uniformly explained by variation in stomatal traits.

A 313 plastome phylogenomic analysis of Pooideae: Exploring relationships among the largest subfamily of grasses.

In this study, we analyzed 313 plastid genomes (plastomes) of Poaceae with a focus on expanding our current knowledge of relationships among the subfamily Pooideae, which represented over half the dataset (164 representatives). In total, 47 plastomes were sequenced and assembled for this study. This is the largest study of its kind to include plastome-level data, to not only increase sampling at both the taxonomic and molecular levels with the aim of resolving complex and reticulate relationships, but also to analyze the effects of alignment gaps in large-scale analyses, as well as explore divergences in the subfamily with an expanded set of 14 accepted grass fossils for more accurate calibrations and dating. Incorporating broad systematic assessments of Pooideae taxa conducted by authors within the last five years, we produced a robust phylogenomic reconstruction for the subfamily, which included all but two supergeneric taxa (Calothecinae and Duthieeae). We further explored how including alignment gaps in plastome analyses oftentimes can produce incorrect or misinterpretations of complex or reticulate relationships among taxa of Pooideae. This presented itself as consistently changing relationships at specific nodes for different stripping thresholds (percentage-based removal of gaps per alignment column). Our summary recommendation for large-scale genomic plastome datasets is to strip alignment columns of all gaps to increase pairwise identity and reduce errant signal from poly A/T bias. To do this we used the "mask alignment" tool in Geneious software. Finally, we determined an overall divergence age for Pooideae of roughly 84.8 Mya, which is in line with, but slightly older than most recent estimates.

Cross-reactivity in Skin Prick Test Results of Members Within Pooideae Subfamily.

OBJECTIVE: Molecular similarities of grass pollen antigens have led to the view that cross-reactivity exists within members of the Pooideae subfamily of grasses. This has resulted in testing for only the most antigenically representative member of Pooideae, Timothy grass (Phleum pratense), despite little literature to support the claim that Phleum is the most representative member or that in vitro cross-reactivity correlates with in vivo cross-reactivity. The aim of the study was to determine if patients with allergic rhinitis symptoms and positive skin prick test results to meadow fescue (Festuca pratensis) also have positive results to Timothy grass. STUDY DESIGN: Retrospective cross-sectional study. SETTING: Tertiary care center in middle Missouri. METHODS: A retrospective chart review identified patients ≥12 years old with a diagnosis of allergic rhinitis who underwent skin prick testing between March 2016 and July 2018, by using a search with CPT code 95004 (Current Procedural Terminology). Positive skin prick test results were based on wheal produced ≥3 mm than the negative control. RESULTS: After review of 2182 charts, 1587 patients met criteria to test for Phleum and Festuca. In total, 1239 patients had a positive result for Phleum or Festuca. Of these, 479 (38.6%) tested positive for Festuca alone, while 342 (27.6%) and 418 (33.7%) tested positive for Phleum alone and Phleum+Festuca, respectively. CONCLUSION: Clinical cross-reactivity among Pooideae members may not be as complete as traditionally thought. P pratense may not be the most antigenically representative subfamily member, and other grasses may need to be included in skin prick testing.

EARLY FLOWERING 3 and Photoperiod Sensing in Brachypodium distachyon.

The proper timing of flowering, which is key to maximize reproductive success and yield, relies in many plant species on the coordination between environmental cues and endogenous developmental programs. The perception of changes in day length is one of the most reliable cues of seasonal change, and this involves the interplay between the sensing of light signals and the circadian clock. Here, we describe a Brachypodium distachyon mutant allele of the evening complex protein EARLY FLOWERING 3 (ELF3). We show that the elf3 mutant flowers more rapidly than wild type plants in short days as well as under longer photoperiods but, in very long (20 h) days, flowering is equally rapid in elf3 and wild type. Furthermore, flowering in the elf3 mutant is still sensitive to vernalization, but not to ambient temperature changes. Molecular analyses revealed that the expression of a short-day marker gene is suppressed in elf3 grown in short days, and the expression patterns of clock genes and flowering time regulators are altered. We also explored the mechanisms of photoperiodic perception in temperate grasses by exposing B. distachyon plants grown under a 12 h photoperiod to a daily night break consisting of a mixture of red and far-red light. We showed that 2 h breaks are sufficient to accelerate flowering in B. distachyon under non-inductive photoperiods and that this acceleration of flowering is mediated by red light. Finally, we discuss advances and perspectives for research on the perception of photoperiod in temperate grasses.

The transferability of microsatellite loci from a homoploid to a polyploid hybrid complex: an example from fine-leaved Festuca species (Poaceae).

BACKGROUND: Microsatellite loci, or single sequence repeats (SSR), are widely used as powerful markers in population genetics. They represent an attractive tool for studying plants such as grasses, whose evolution is driven by hybridisation and polyploidization. However, the development of microsatellite markers has been challenging and time-consuming, especially for non-model organisms lacking available genome-wide sequence data. One straightforward and low-cost approach is to transfer the SSR loci developed for one species, or complex, to another closely-related one. This work evaluates the transferability of microsatellite loci from homoploid to allopolyploid complexes of fine-leaved Festuca species and to assess their use in two new species. The studied complex (F. amethystina-F. tatrae) is a useful model for research on the local adaptability of grasses with different ploidy levels. Since both species can be considered as rare or threatened (F. tatrae-as a mountain and narrow endemic species and F. amethystina-a mountain species with relict lowland populations), any tool enabling studies on genetic diversity and population genetics, such as SSR markers, could also be very useful in a conservation context. METHODS: The ploidy level within populations was estimated using flow cytometry. One diploid and one tetraploid population of F. amethystina and a diploid population of F. tatrae were chosen to test the transferability of SSR loci. Because our work describes the transfer of SSR nuclear markers designed originally for F. gautieri, a phylogenetic tree was prepared based on the ITS marker to assess the genetic distance between the studied complexes. The PCR products were separated on a high-resolution agarose gel, intended for SSR marker analysis. Appropriate solutions for the allotetraploid population and whole mixed-ploidy complex were implemented. RESULTS: Flow cytometry confirmed earlier data regarding DNA content in the investigated species and cytotypes. The phylogenetic ITS tree indicated a small genetic distance between F. gautieri complexes and the studied species. Ten microsatellite markers were successfully transferred. All markers were polymorphic. In total, 163 different alleles were scored from the 10 SSR loci. PCoA of accessions revealed well-separated groups corresponding to studied populations. Over 60% of the total variance is explained by differentiation within populations and one third among them. CONCLUSIONS: The transferred markers are valid tools for the study of population genetics and inheritance relationships within cytotypes and species and between them. The presented markers can be used to study inbreeding depression in the Festuca species, and variations in the degrees of genetic diversity between different cytotypes in mountain and lowland areas. Our findings can also be applied to study conservation strategies for ensuring biodiversity at the genetic level in polyploid complexes.

Increased above-ground resource allocation is a likely precursor for independent evolutionary origins of annuality in the Pooideae grass subfamily.

Semelparous annual plants flower a single time during their 1-yr life cycle, investing much of their energy into rapid reproduction. By contrast, iteroparous perennial plants flower multiple times over several years, and partition their resources between reproduction and persistence. To which extent evolutionary transitions between life-cycle strategies are internally constrained at the developmental, genetic and phylogenetic level is unknown. Here we study the evolution of life-cycle strategies in the grass subfamily Pooideae and test if transitions between them are facilitated by evolutionary precursors. We integrate ecological, life-cycle strategy and growth data in a phylogenetic framework. We investigate if growth traits are candidates for a precursor. Species in certain Pooideae clades are predisposed to evolve annuality from perenniality, potentially due to the shared inheritance of specific evolutionary precursors. Seasonal dry climates, which have been linked to annuality, were only able to select for transitions to annuality when the precursor was present. Allocation of more resources to above-ground rather than below-ground growth is a candidate for the precursor. Our findings support the hypothesis that only certain lineages can respond quickly to changing external conditions by switching their life-cycle strategy, likely due to the presence of evolutionary precursors.

Successive evolutionary steps drove Pooideae grasses from tropical to temperate regions.

Angiosperm adaptations to seasonally cold climates have occurred multiple times independently. However, the observation that less than half of all angiosperm families are represented in temperate latitudes suggests internal constraints on the evolution of cold tolerance/avoidance strategies. Similar to angiosperms as a whole, grasses are primarily tropical, but one major clade, subfamily Pooideae, radiated extensively within temperate regions. It is posited that this Pooideae niche transition was facilitated by an early origin of long-term cold responsiveness around the base of the subfamily, and that a set of more ancient pathways enabled evolution of seasonal cold tolerance. To test this, we compared transcriptome-level responses of disparate Pooideae to short-/long-term cold and with those previously known in the subtropical grass rice (Ehrhartoideae). Analyses identified several highly conserved cold-responsive 'orthogroups' within our focal Pooideae species that originated successively during the diversification of land plants, predominantly via gene duplication. The majority of conserved Pooideae cold-responsive genes appear to have ancient roles in stress responses, with most of the orthogroups also being sensitive to cold in rice. However, a subgroup of genes was likely co-opted de novo early in the Pooideae. These results highlight a plausible stepwise evolutionary trajectory for cold adaptations across Pooideae.

Variability of Pyrrolizidine Alkaloid Occurrence in Species of the Grass Subfamily Pooideae (Poaceae).

Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites found in various unrelated angiosperm lineages including cool-season grasses (Poaceae, subfamily Pooideae). Thesinine conjugates, saturated forms of PA that are regarded as non-toxic, have been described to occur in the two grass species Lolium perenne and Festuca arundinacea (Poaceae, subfamily Pooideae). In a wider screen, we tested various species of the Pooideae lineage, grown under controlled conditions, for their ability to produce thesinine conjugates or related structures. Using an LC-MS based targeted metabolomics approach we were able to show that PA biosynthesis in grasses is limited to a group of very closely related Pooideae species that produce a limited diversity of PA structures. High variability in PA levels was observed even between individuals of the same species. These individual accumulation patterns are discussed with respect to a possible function and evolution of this type of alkaloid.

Are diversification rates and chromosome evolution in the temperate grasses (Pooideae) associated with major environmental changes in the Oligocene-Miocene?

The Pooideae are a highly diverse C3 grass subfamily that includes some of the most economically important crops, nested within the highly speciose core-pooid clade. Here, we build and explore the phylogeny of the Pooideae within a temporal framework, assessing its patterns of diversification and its chromosomal evolutionary changes in the light of past environmental transformations. We sequenced five plastid DNA loci, two coding (ndhF, matk) and three non-coding (trnH-psbA, trnT-L and trnL-F), in 163 Poaceae taxa, including representatives for all subfamilies of the grasses and all but four ingroup Pooideae tribes. Parsimony and Bayesian phylogenetic analyses were conducted and divergence times were inferred in BEAST using a relaxed molecular clock. Diversification rates were assessed using the MEDUSA approach, and chromosome evolution was analyzed using the chromEvol software. Diversification of the Pooideae started in the Late-Eocene and was especially intense during the Oligocene-Miocene. The background diversification rate increased significantly at the time of the origin of the Poodae + Triticodae clade. This shift in diversification occurred in a context of falling temperatures that potentially increased ecological opportunities for grasses adapted to open areas around the world. The base haploid chromosome number n = 7 has remained stable throughout the phylogenetic history of the core pooids and we found no link between chromosome transitions and major diversification events in the Pooideae.

Evolution of the miR5200-FLOWERING LOCUS T flowering time regulon in the temperate grass subfamily Pooideae.

Flowering time is a carefully regulated trait controlled primarily through the action of the central genetic regulator, FLOWERING LOCUS T (FT). Recently it was demonstrated that a microRNA, miR5200, targets the end of the second exon of FT under short-day photoperiods in the grass subfamily Pooideae, thus preventing FT transcripts from reaching threshold levels under non-inductive conditions. Pooideae are an interesting group in that they rapidly diversified from the tropics into the northern temperate region during a major global cooling event spanning the Eocene-Oligocene transition. We hypothesize that miR5200 photoperiod-sensitive regulation of Pooideae flowering time networks assisted their transition into northern seasonal environments. Here, we test predictions derived from this hypothesis that miR5200, originally found in bread wheat and later identified in Brachypodium distachyon, (1) was present in the genome of the Pooideae common ancestor, (2) is transcriptionally regulated by photoperiod, and (3) is negatively correlated with FT transcript abundance, indicative of miR5200 regulating FT. Our results demonstrate that miR5200 did evolve at or around the base of Pooideae, but only acquired photoperiod-regulated transcription within the Brachypodium lineage. Based on expression profiles and previous data, we posit that the progenitor of miR5200 was co-regulated with FT by an unknown mechanism.

Similarities and differences in the nuclear genome organization within Pooideae species revealed by comparative genomic in situ hybridization (GISH).

In this paper, we highlight the affinity between the genomes of key representatives of the Pooideae subfamily, revealed at the chromosomal level by genomic in situ hybridization (GISH). The analyses were conducted using labeled probes from each species to hybridize with chromosomes of every species used in this study based on a "round robin" rule. As a result, the whole chromosomes or chromosome regions were distinguished or variable types of signals were visualized to prove the different levels of the relationships between genomes used in this study. We observed the unexpected lack of signals in secondary constrictions of rye (RR) chromosomes probed by triticale (AABBRR) genomic DNA. We have also identified unlabeled chromosome regions, which point to species-specific sequences connected with disparate pathways of chromosome differentiation. Our results revealed a conservative character of coding sequence of 35S rDNA among selected species of the genera Aegilops, Brachypodium, Festuca, Hordeum, Lolium, Secale, and Triticum. In summary, we showed strong relationships in genomic DNA sequences between species which have been previously reported to be phylogenetically distant.