Related Grass Species Research Articles

Grass leaf structural and stomatal trait responses to climate gradients assessed over the 20th century and across the Great Plains, USA.

Abstract. Using herbarium specimens spanning 133 years and field-collected measurements, we assessed intraspecific trait (leaf structural and stomatal) variability from grass species in the Great Plains of North America. We focused on two widespread, closely related grasses from the tribe Paniceae: Dichanthelium oligosanthes subsp. scribnerianum (C3) and Panicum virgatum (C4). Thirty-one specimens per taxon were sampled from local herbaria from the years 1887 to 2013 to assess trait responses across time to changes in atmospheric [CO2] and growing season precipitation and temperature. In 2021 and 2022, the species were measured from eight grasslands sites to explore how traits vary spatially across natural continental precipitation and temperature gradients. Δ13C increased with atmospheric [CO2] for D. oligosanthes but decreased for P. virgatum, likely linked to increases in precipitation in the study region over the past century. Notably, this is the first record of decreasing Δ13C over time for a C4 species illustrating 13C linkages to climate. As atmospheric [CO2] increased, C:N increased and δ15N decreased for both species and %N decreased for D. oligosanthes. Across a large precipitation gradient, D. oligosanthes leaf traits were more responsive to changes in precipitation than those of P. virgatum. In contrast, only two traits of P. virgatum responded to increases in temperature across a gradient: specific leaf area (increase) and leaf dry matter content (decrease). The only shared significant trend between species was increased C:N with precipitation. Our work demonstrates that these closely related grass species with different photosynthetic pathways exhibited various trait responses across temporal and spatial scales, illustrating the key role of scale of inquiry for forecasting leaf trait responses to future environmental change.

Morphology, photosynthetic and molecular mechanisms associated with powdery mildew resistance in Kentucky bluegrass.

Kentucky bluegrass (Poa pratensis L.), one of the most widely used cool-season turfgrasses around the world, is sensitive to powdery mildew (PM; Blumeria graminis). The PM strain identification and regulation mechanisms of Kentucky bluegrass in response to pathogens still remain unclear. Through morphological and molecular analyses, we identified that the pathogen in Kentucky bluegrass was B. graminis f. sp. poae. The infection of B. graminis led to a reduction of the sclerenchyma area, expansion of vesicular cells and movement of chloroplasts. The infected leaves had significantly lower values in net photosynthesis, stomatal conductance and transpiration rate, maximal quantum yield of PSII photochemistry, photochemical quenching and non-regulated energy dissipation compared to mock-inoculated leaves. Expressions of light-harvesting antenna protein genes LHCA and LHCB and photosynthetic electron transport genes petE and petH decreased significantly in infected leaves. Furthermore, upregulations of genes involved in plant-pathogen interaction, such as HSP90, RBOH, and RPM and downregulations of EDS, RPS and WRKY were observed in infected leaves. The findings may help design a feasible approach to effectively control the PM disease in Kentucky bluegrass and other related perennial grass species.

Perspectives for reducing seed shattering in ryegrasses

AbstractIn the last decades, the progress in ryegrass (Lolium spp.) breeding was mainly on agronomic traits such as biomass yield, forage quality or disease resistance. However, for commercial success, a stable and high seed yield is a prerequisite for any cultivar. The realized seed yield is influenced by many different factors such as non‐optimal pollination and fertilization, seed abortion and seed shattering. While seed shattering has been largely eliminated in major cereal crops such as rice, barley or sorghum during domestication, the trait has been largely neglected in ryegrass breeding programs. The close syntenic relationship of cereal and ryegrass genomes offers the opportunity to develop breeding approaches for reducing seed shattering in the latter by transferring knowledge from the former. The objectives of this review are to (1) give an overview on the knowledge of morphology on seed shattering in cereal crops and ryegrasses, (2) compare the genetic background underlying seed shattering in different species, (3) identify putative candidate genes controlling seed shattering in ryegrasses through comparative genomic analysis and (4) give an outlook on new breeding strategies resulting in low seed shattering cultivars of ryegrasses and related forage grass species.

Molecular characterization of the cytokinin responsive type-B response regulators in perennial ryegrass highlights LpARR10 conferring salt tolerance via trans-activating SOS1 and SOS3

The type-B response regulator (ARR) family members represent the elements of two-component regulatory systems and play crucial roles in the cytokinin signaling, plant growth, and stress responses. The objective of this study was to characterize type-B ARR genes from perennial ryegrass, and to investigate their roles in regulating to salt tolerance. Twelve type-B ARRs, namely LpARR1-12, were identified in perennial ryegrass. They were clustered into three subgroups and were unevenly distributed on chromosomes. The stress responsive elements (SREs) and hormone responsive elements (HREs) were identified in LpARRs promoters and the transcription of LpARRs genes were responsive to various abiotic stress (heat, salt, and osmotic) and hormonal (cytokinin, ABA, and ethylene) treatments. LpARR10 was strong induced by salt stress, therefore it was selected for function analysis. Overexpressing LpARR10 in Arabidopsis led to a significant improvement in salt tolerance. The LpARR10-overexpressing (LpARR10-OE) lines exhibited lower levels of Na+ content compared to WT plants, and the expression levels of SOS1 and SOS3 were significantly higher in the LpARR10-OE lines. Results of yeast auto-transactivation and plant cell transcriptional activity assays shows LpARR10 is a nucleus localized transcriptional activator and directly bind and transactivated the promoter of SOS1 and SOS3. Together, LpARR10 enhances salt tolerance may via trans-activating SOS1/SOS3 in Arabidopsis. Knowledge gained in this study will facilitate further functional analysis of LpARRs for molecular breeding of ryegrass and other related grass species.

Effect of ecological factors on the grass dynamics at Point Calimere Wildlife Sanctuary, India

Grass dynamics play a major role in the density and diversity of grazing mammals. To understand the drivers of grass quality and quantity, we assessed the height, cover, soft-texture, green leaves, and reproductive phase of grass species in relation to 13 ecological covariates belonging to climate, vegetation, human disturbance, and wild herbivores at Point Calimere Wildlife Sanctuary, southern India during November 2018─September 2020. From the 1,024 quadrates, we recorded 22 grass species and 10 sedges. The grass parameters varied significantly among habitats and between seasons. The grass height and grass cover were more in open scrub, while the soft-textured green grasses were more in grasslands. All the grass parameters except reproductive stage were highest during the wet season. The general linear model (GLM) based analysis on the covariate effect on grass quantity and quality demonstrated that among the 13 covariates compared, Prosopis, an alien invasive species, is the major driver, with negative influence on both grass quantity; the cover, and grass quality; soft-texture and greenness of grass. The feral horse, an alien invasive, negatively influenced grass height. Earlier studies have also shown the devastating effects of these exotics on native flora and fauna at Point Calimere, and measures suggested by these studies are recommended to safeguard natural communities in the area.

The mechanisms underpinning lateral gene transfer between grasses

Societal Impact StatementLateral gene transfer (LGT) refers to the transmission of genetic material without sexual reproduction. LGT is widespread in a number of plant species, including grasses. But how these genes of foreign origin got there is presently unknown. In this review, we show that transformation techniques used to genetically modify organisms could occur in the wild and be responsible for the frequently observed grass‐to‐grass LGTs. The distinction between natural evolutionary processes and genetic engineering might be arbitrary, and its validity will be further debated as agricultural biotechnology becomes more widely used and examples of “natural genetic engineering” through LGT increase.SummaryLateral gene transfer (LGT) is the transmission of genetic material among species without sexual reproduction. LGT was initially thought to be restricted to prokaryotes, but it has since been reported in a wide range of eukaryotes, including plants. Grasses seem to be particularly prone to LGT and frequently exchange genes among species. However, the mechanism(s) facilitating these transfers in this economically and ecologically important group of plants are debated. Here, we review vector‐mediated, direct tissue‐to‐tissue contact, wide‐crossing and reproductive contamination LGT mechanisms and discuss the likelihood of each in light of recent studies. Of particular relevance are transformation approaches that require minimal human intervention to transfer DNA among grasses in the lab that could mimic the mechanisms facilitating grass‐to‐grass LGT in the wild. These approaches include relatively simple techniques, such as pollen tube pathway‐mediated transformation, that take advantage of the permeability of the reproductive process to introduce alien genetic material from a third individual into an embryo. This process could be easily mirrored in the wild where pollen from one species lands on the stigma of another, acting as a source of alien DNA that can ultimately contaminate the reproductive process. This contamination is likely to be prevalent in wind pollinated species such as grasses, where the rates of illegitimate pollination will be high. In conclusion, plant transformation methods requiring minimal intervention are likely paralleled in the wild where they act as the mechanism underpinning LGT between distantly related grass species.

Genome of Paspalum vaginatum and the role of trehalose mediated autophagy in increasing maize biomass

A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

100 Years of Chromosome Research in Rye, Secale L.

Although microscopy and genetics were still in their infancy, there are cytological results produced a hundred years ago that are still relevant today. Since the 1920s, rye has been a subject of chromosome research. It started by plotting its mitotic and meiotic chromosomes to determine genome size. After controversial evidence, it became clear that the base number is n = 7. However, structural differences exist between species within the genus Secale. Some rye populations even carry accessory chromosomes evolutionary derived from the A genome. The development of tetraploid strains significantly promoted chromosome analysis. Various techniques have tried to stabilize the disturbed chromosome pairing of the induced tetraploids. Although slight improvements could be achieved, they did not lead to a breakthrough. However, the various aneuploid derivatives of the polyploids found major advances in the genetic analysis of rye. Trisomics, telo-trisomics, and reciprocal translocation have served as important tools for gene mapping. Since the 1970s, various chromosome banding techniques have stimulated scientific progress. The seven haploid chromosomes could be diagnosed unequivocally, not only in S. cereale but also in related species. These findings led to a clear homoeologous assignment to the genomes of related grass species such as wheat, barley, rice, etc. Current applications of in situ fluorescence staining methods, such as GISH and FISH, allow even more precise results, depending on the specificity of the DNA samples. Advanced preparation techniques are supplemented by the variety of innovations in the field of molecular genome analysis. They replace complex cytological examinations. In this way, introgressions can be safely detected by DNA markers and be much more detailed. In addition, CRISPR/CAS-mediated chromosome engineering will become an important method of the future.

Genome-Wide Identification and Characterization of the LpSAPK Family Genes in Perennial Ryegrass Highlight LpSAPK9 as an Active Regulator of Drought Stress.

SAPK/SnRK2 family genes play crucial roles in plant growth, development, and abiotic stress responses. The objective of this study was to identify and characterize the LpSAPK genes in perennial ryegrass (Lolium perenne L.). The results showed that there are 10 LpSAPKs in perennial ryegrass that could be classified into three groups with similar genic (exon–intron) structures to their orthologous genes in Arabidopsis and other grass species. Ka/Ks analysis suggested that the LpSAPKs and their orthologs were under purifying selection to maintain their conserved function during evolution. Nine out of ten LpSAPKs were localized in the cytoplasm and nucleus with the exception of LpSAPK5 which was only observed in the cytoplasm. Most LpSAPKs were responsive to various abiotic stress and hormonal (ABA, cytokinin, and ethylene) treatments but were downregulated in leaves and upregulated in roots, suggesting that there were unknown cis elements in promoters of these genes or unidentified post-transcriptional mechanism responsible for the tissue-dependent stress-regulated expression of these LpSAPKs. Furthermore, LpSAPK9 was identified as a candidate positive regulator in drought tolerance using a yeast ectopic expression system, and LpSAPK9 showed contrasting expression changes in drought-sensitive and -tolerant ryegrass varieties, suggesting that expression levels of LpSAPK9 were related to ryegrass drought tolerance. These results will facilitate further functional analysis of LpSAPKs for molecular breeding of ryegrass and other related grass species.

Salinity Tolerance of Halophytic Grass Puccinellia nuttalliana Is Associated with Enhancement of Aquaporin-Mediated Water Transport by Sodium.

In salt-sensitive plants, root hydraulic conductivity is severely inhibited by NaCl, rapidly leading to the loss of water balance. However, halophytic plants appear to effectively control plant water flow under salinity conditions. In this study, we tested the hypothesis that Na+ is the principal salt factor responsible for the enhancement of aquaporin-mediated water transport in the roots of halophytic grasses, and this enhancement plays a significant role in the maintenance of water balance, gas exchange, and the growth of halophytic plants exposed to salinity. We examined the effects of treatments with 150 mM of NaCl, KCl, and Na2SO4 to separate the factors that affect water relations and, consequently, physiological and growth responses in three related grass species varying in salt tolerance. The grasses included relatively salt-sensitive Poa pratensis, moderately salt-tolerant Poa juncifolia, and the salt-loving halophytic grass Puccinellia nuttalliana. Our study demonstrated that sustained growth, chlorophyll concentrations, gas exchange, and water transport in Puccinellia nuttalliana were associated with the presence of Na in the applied salt treatments. Contrary to the other examined grasses, the root cell hydraulic conductivity in Puccinellia nuttalliana was enhanced by the 150 mM NaCl and 150 mM Na2SO4 treatments. This enhancement was abolished by the 50 µM HgCl2 treatment, demonstrating that Na was the factor responsible for the increase in mercury-sensitive, aquaporin-mediated water transport. The observed increases in root Ca and K concentrations likely played a role in the transcriptional and (or) posttranslational regulation of aquaporins that enhanced root water transport capacity in Puccinellia nuttalliana. The study demonstrates that Na plays a key role in the aquaporin-mediated root water transport of the halophytic grass Puccinellia nuttalliana, contributing to its salinity tolerance.

Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

Macroevolutionary history predicts flowering time but not phenological sensitivity to temperature in grasses.

Long-term observations show that flowering phenology has shifted in many lineages in response to climate change. However, it remains unclear whether these results can be generalized to predict the presence, direction, or magnitude of responses in lineages for which we lack long time-series data. If phenological responses are phylogenetically conserved, we can extrapolate from species for which we have data to predict the responses of close relatives. While several studies have found that closely related species flower at similar times, fewer have evaluated whether phylogenetically proximal species respond to environmental change similarly. We paired flowering time data from 3161 manually scored herbarium specimens of 72 species of grasses (Poaceae) with historical climate data and analyzed the phylogenetic signal and phylogenetic half-life of phenological sensitivity. We also ran these analyses on a subset of species showing statistically significant sensitivities, in order to assess the role of sampling bias on phylogenetic signal. Closely related grass species tend to flower at similar times, but flowering times respond to temperature changes in species-specific ways. We also show that only including species for which there is strong evidence of phenological shifts results in overestimating phylogenetic signal. In agreement with other recent studies, our results suggest caution in extrapolating from evidence of phylogenetic similarity to predicting shared responses in this ecologically relevant trait. Future work is needed to better understand the discrepancy between the phylogenetic signal in observed phenological shifts and absence of such signal in sensitivity.

Identification and Characterization of Perennial Ryegrass (Lolium perenne) Vernalization Genes.

Perennial ryegrass (Lolium perenne) is a temperate grass species commonly used as pasture for livestock. Flowering (heading) of ryegrass impacts metabolizable energy content and seed yield, therefore this trait is important for both farmers and seed producers. In related grass species, the VRN genes (VRN1-3) have been largely implicated in the determination of vernalization response and are responsible for much of the intra-species variation in this trait. Many other important flowering-time regulators have been cataloged in the model grass Brachypodium distachyon; however, in several cases, such as VRN2, their ryegrass homologs have not been well-characterized. Here, ryegrass homologs of important flowering time genes from B. distachyon were identified through available synteny data and sequence similarity. Phylogenetic analysis of VRN3/FT-like and VRN2-like genes was performed to elucidate these families further. The expression patterns of these genes were assessed during vernalization. This confirmed the key roles played by LpVRN1 and LpFT3 in the promotion of flowering. Furthermore, two orthologs of VRN2 identified here, as well as an ortholog of CO9, were expressed prior to vernalization, and were repressed in flowering plants, suggesting a role in floral repression. Significant variability in expression of these flowering pathway genes in diverse genotypes was detected and may underlie variation in flowering time and vernalization response.

The population dynamics of four grass species in relation to burning in the KwaZulu-Natal Drakensberg

Marked tillers of Heteropogon contortus, Trachypogon spicatus, Tristachya leucothrix and Harpochloa falx were sampled regularly over a two-year period in Highland Sourveld burnt annually in winter and biennially in spring. Parameters recorded were height of shoot apex, tiller mass, number of new lateral tillers and time of flowering. Monitoring of marked tillers before and after each burn enabled the life history of populations of tillers to be followed. In all species investigated, flowering was found to be of minimal importance with most tillers remaining vegetative until death. Shoot apices remained close to the soil surface (<20 mm) enabling them to survive frequent burns. All species exhibited smooth survivorship curves, suggesting that dramatic fluctuations in climate and severe defoliation, as by fire, had little impact on mortality. The differential response of the four grass species was explained by their different reproductive capacities and reproductive rates. The predicted responses to fire regime were verified by changes that took place at the study area from 1981 to 2019, showing the importance of applying appropriate burning regimes to maintain the bud bank. A biennial spring burn was best for maintaining these important grass species at current levels of abundance in the KwaZulu-Natal Drakensberg.

Genetic dissection of Ni toxicity in a spring wheat diversity panel by using 90 K SNP array

Excess Ni intake has harmful implications on human health, which include chronic bronchitis, reduced lung function, and cancer of lung and nasal sinuses. Like other toxic metals, higher Ni accumulation in grains leads to excess intake by humans when the contaminated grains are consumed as food. There is little information about the genetic factors that regulate Ni uptake in plants. To investigate genetic architecture of Ni uptake in leaf and translocation to grain, we performed a genome-wide association study with genotyping from 90 K array in a historical bread wheat diversity panel from Pakistan. We observed that Ni toxicity caused more than 50 % reductions in biological yield and grain yield, other agronomic traits were also partly or severely affected. Genetic association study helped identify 23 SNP-trait associations involved in Ni uptake in leaf and translocation to grains. These 23 SNPs covered 15 genomic loci at chromosomes 1A, 2D, 3B, 4A and 4B of wheat. The favorable alleles of these SNPs were randomly distributed in subpopulations indicating no selection pressure for this trait during breeding improvement. These regions had 283 low-confidence and 248 high-confidence protein coding genes. Among these, 156 were annotated using databases of wheat and closely related grass species. Since there is no previous report on genetic information of Ni uptake and translocation, these results provide sufficient grounds for further research of candidate genes and varietal development.

Inter- and intraspecific variation in grass phytolith shape and size: a geometric morphometrics perspective.

The relative contributions of inter- and intraspecific variation to phytolith shape and size have only been investigated in a limited number of studies. However, a detailed understanding of phytolith variation patterns among populations or even within a single plant specimen is of key importance for the correct taxonomic identification of grass taxa in fossil samples and for the reconstruction of vegetation and environmental conditions in the past. In this study, we used geometric morphometric analysis for the quantification of different sources of phytolith shape and size variation. We used landmark-based geometric morphometric methods for the analysis of phytolith shapes in two extant grass species (Brachypodium pinnatum and B. sylvaticum). For each species, 1200 phytoliths were analysed from 12 leaves originating from six plants growing in three populations. Phytolith shape and size data were subjected to multivariate Procrustes analysis of variance (ANOVA), multivariate regression, principal component analysis and linear discriminant analysis. Interspecific variation largely outweighed intraspecific variation with respect to phytolith shape. Individual phytolith shapes were classified with 83 % accuracy into their respective species. Conversely, variation in phytolith shapes within species but among populations, possibly related to environmental heterogeneity, was comparatively low. Our results imply that phytolith shape relatively closely corresponds to the taxonomic identity of closely related grass species. Moreover, our methodological approach, applied here in phytolith analysis for the first time, enabled the quantification and separation of variation that is not related to species discrimination. Our findings strengthen the role of grass phytoliths in the reconstruction of past vegetation dynamics.

Uncovering the role of a positive selection site of wax ester synthase/diacylglycerol acyltransferase in two closely related Stipa species in wax ester synthesis under drought stress.

Natural selection drives local adaptations of species to biotic or abiotic environmental stresses. As a result, adaptive phenotypic divergence can evolve among related species living in different habitats. However, the genetic foundation of this divergence process remains largely unknown. Two closely related alpine grass species, Stipa capillacea and Stipa purpurea, are distributed in different rainfall regions of northern Tibet. Here, we analyzed the drought tolerance of these two closely related Stipa species, and found that S. purpurea was more resistance to drought stress than S. capillacea. To further understand the genetic diversity behind their adaptation to drought environments, a comprehensive gene repertoire was generated using PacBio isoform and Illumina RNA sequencing technologies. Bioinformatics analyses revealed that differential transcripts were mainly enriched in the wax synthetic pathway, and a threonine residue at position 239 of WSD1 was identified as having undergone positive selection in S. purpurea. Using heterologous expression in the Saccharomyces cerevisiae mutant H1246, site-directed mutagenesis studies demonstrated that a positive selection site results in changes to the wax esters profile. This difference may play an important role in S. purpurea in response to drought conditions, indicating that S. purpurea has evolved specific strategies involving its wax biosynthetic pathway as part of its long-term adaptation to the Qinghai-Tibet Plateau.

The anatomy of abscission zones is diverse among grass species.

PremiseAbscission zones (AZ) are specialized cell layers that separate plant parts at the organ junction upon developmental or environmental signals. Fruit or seed abscission has been well studied in model species because of its crucial role for seed dispersal. Previous work showed that AZ localization differs among species of Poaceae and that AZ formation is histologically and genetically distinct in three distantly related grass species, refuting the idea of a broadly conserved module. However, whether AZ structure is consistent within subfamilies is unknown.MethodsEleven species were selected from six subfamilies of Poaceae, and their AZ was investigated using paraffin‐embedded, stained material. Observations were added from the literature for an additional six species. Data were recorded on AZ location and whether cells in the AZ were distinguishable by size or lignification. Characteristics of the AZ were mapped on the phylogeny using maximum likelihood.ResultsAbscission zone anatomy and histology vary among species, and characteristics of the AZ do not correlate with phylogeny. Twelve of the seventeen studied species have an AZ in which the cells are significantly smaller than surrounding cells. Of these, eight have differential lignification. Differential lignification is often associated with differential cell size, but not vice versa.ConclusionsNeither smaller cells in the AZ nor differential lignification between the AZ and surrounding cells is required for abscission, although differential cell size and lignification are often correlated. Abscission zone anatomy does not correlate with phylogeny, suggesting its rapid change over evolutionary time.

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species.

Isolation of good quality chloroplast DNA (cpDNA) is a challenge in different plant species, although several methods for isolation are known. Attempts were undertaken to isolate cpDNA from Festuca grass species by using available standard protocols; however, they failed due to difficulties separating intact chloroplasts from the polysaccharides, oleoresin, and contaminated nuclear DNA that are present in the crude homogenate. In this study, we present a quick and inexpensive protocol for isolating intact chloroplasts from seven grass varieties/accessions of five Festuca species using a single layer of 30% Percoll solution. This protocol was successful in isolating high quality cpDNA with the least amount of contamination of other DNA. We performed Illumina MiSeq paired-end sequencing (2 × 300 bp) using 200 ng of cpDNA of each variety/accession. Chloroplast genome mapping showed that 0.28%–11.37% were chloroplast reads, which covered 94%–96% of the reference plastid genomes of the closely related grass species. This improved method delivered high quality cpDNA from seven grass varieties/accessions of five Festuca species and could be useful for other grass species with similar genome complexity.