Assessment of Morphological, Anatomical and Palynological Variation in the Medicinal Plant Disporopsis longifolia Craib (Asparagaceae) for Botanical Quality Control.

Disporopsis longifolia Craib is an Asian medicinal plant belonging to the Asparagaceae family. The plants are well known for their steroidal saponins and phenolic compounds and are traditionally used as tonics for back pain, bellyache, cough, diabetes, asthma, pneumonia and rheumatism. However, they are challenging to identify to species level using morphology. This raises a serious concern for their medicinal applications where botanical quality control is essential. The most appropriate morphological, anatomical and pollen characters for species diagnosis were therefore determined. Synonyms were identified and lectotypification provided. The morphological characters were described from 76 fresh and dried specimens to include a broad range of materials from differing habitats and locations. Paraffin and peeling methods were applied for anatomical studies of leaves and stems and a modified acetolysis method was undertaken for pollen morphology. This paper compares the new character data to published data from other species in the genus, namely D. aspersa, D. fuscopicta, D. jinfushanensis, D. pernyi and D. undulata. This is the first report of such anatomical and pollen morphology characters for D. longifolia. The results provide accurate morphological, anatomical and palynological characters for quality control and are best applied in combination with each other.

Fungal Endophytes Isolated from Elymus repens, a Wild Relative of Barley, Have Potential for Biological Control of Fusarium culmorum and Pyrenophora teres in Barley.

Twenty-four fungal endophytes, isolated from a wild relative of barley, Elymus repens, were screened in barley against an isolate of Fusarium culmorum and an isolate of Pyrenophora teres under controlled conditions. In all experiments, the endophytes were applied individually as seed dressings. Five endophytes could significantly reduce symptoms of Fusarium culmorum (Periconia macrospinosa E1 and E2, Epicoccum nigrum E4, Leptodontidium sp. E7 and Slopeiomyces cylindrosporus E18). In particular, treatment with Periconia macrospinosa E1 significantly reduced Fusarium symptoms on roots by 29-63% in two out of four experiments. Using, a gfp transformed isolate of P. macrospinosa E1, it was possible to show that the fungus was present on roots 14 days after sowing, coinciding with the disease scoring. To test for a potential systemic effect of the seed treatment, eight endophyte isolates were tested against the leaf pathogen Pyrenophora teres. Three isolates could significantly reduce symptoms of P. teres (Lasiosphaeriaceae sp. E10, Lindgomycetaceae sp. E13 and Leptodontidium sp. E16). Seed treatment with Lasiosphaeriaceae sp. E10 reduced net blotch leaf lesion coverage by 89%, in one out of three experiments. In conclusion, specific endophyte isolates exerted varying degrees of protection in the different experiments. Nevertheless, data suggest that endophytic strains from E. repens in a few cases are antagonistic against F. culmorum and P. teres, but otherwise remain neutral when introduced to a barley host in a controlled environment.

Assessing Genotypic and Environmental Effects on Endophyte Communities of Fraxinus (Ash) Using Culture Dependent and Independent DNA Sequencing.

Fraxinus excelsior populations are in decline due to the ash dieback disease Hymenoscyphus fraxineus. It is important to understand genotypic and environmental effects on its fungal microbiome to develop disease management strategies. To do this, we used culture dependent and culture independent approaches to characterize endophyte material from contrasting ash provenances, environments, and tissues (leaves, roots, seeds). Endophytes were isolated and identified using nrITS, LSU, or tef DNA loci in the culture dependent assessments, which were mostly Ascomycota and assigned to 37 families. Few taxa were shared between roots and leaves. The culture independent approach used high throughput sequencing (HTS) of nrITS amplicons directly from plant DNA and detected 35 families. Large differences were found in OTU diversity and community composition estimated by the contrasting approaches and these data need to be combined for estimations of the core endophyte communities. Species richness and Shannon index values were highest for the leaf material and the French population. Few species were shared between seed and leaf tissue. PCoA and NMDS of the HTS data showed that seed and leaf microbiome communities were highly distinct and that there was a strong influence of Fraxinus species identity on their fungal community composition. The results will facilitate a better understanding of ash fungal ecology and are a step toward identifying microbial biocontrol systems to minimize the impact of the disease.

Hidden Fungi: Combining Culture-Dependent and -Independent DNA Barcoding Reveals Inter-Plant Variation in Species Richness of Endophytic Root Fungi in Elymus repens.

The root endophyte community of the grass species Elymus repens was investigated using both a culture-dependent approach and a direct amplicon sequencing method across five sites and from individual plants. There was much heterogeneity across the five sites and among individual plants. Focusing on one site, 349 OTUs were identified by direct amplicon sequencing but only 66 OTUs were cultured. The two approaches shared ten OTUs and the majority of cultured endophytes do not overlap with the amplicon dataset. Media influenced the cultured species richness and without the inclusion of 2% MEA and full-strength MEA, approximately half of the unique OTUs would not have been isolated using only PDA. Combining both culture-dependent and -independent methods for the most accurate determination of root fungal species richness is therefore recommended. High inter-plant variation in fungal species richness was demonstrated, which highlights the need to rethink the scale at which we describe endophyte communities.

First Report of the Ash Sawfly, Tomostethus nigritus, Established on Fraxinus excelsior in the Republic of Ireland.

This is the first report of the ash sawfly, Tomostethus nigritus, in the Republic of Ireland. We observed defoliated leaves of Fraxinus excelsior L. and T. nigritus larvae at a forestry plantation in Co. Kildare. Morphological observation of the larvae and DNA analysis using mitochondrial COI barcoding confirmed the identification of this pest of ash.

Fungal Endophytes for Grass Based Bioremediation: An Endophytic Consortium Isolated from Agrostis stolonifera Stimulates the Growth of Festuca arundinacea in Lead Contaminated Soil.

Bioremediation is an ecologically-friendly approach for the restoration of heavy metal-contaminated sites and can exploit environmental microorganisms such as bacteria and fungi. These microorganisms are capable of removing and/or deactivating pollutants from contaminated substrates through biological and chemical reactions. Moreover, they interact with the natural flora, protecting and stimulating plant growth in these harsh conditions. In this study, we isolated a group of endophytic fungi from Agrostis stolonifera grasses growing on toxic waste from an abandoned lead mine (up to 47,990 Pb mg/kg) and identified them using DNA sequencing (nrITS barcoding). The endophytes were then tested as a consortium of eight strains in a growth chamber experiment in association with the grass Festuca arundinacea at increasing concentrations of lead in the soil to investigate how they influenced several growth parameters. As a general trend, plants treated with endophytes performed better compared to the controls at each concentration of heavy metal, with significant improvements in growth recorded at the highest concentration of lead (800 galena mg/kg). Indeed, this set of plants germinated and tillered significantly earlier compared to the control, with greater production of foliar fresh and dry biomass. Compared with the control, endophyte treated plants germinated more than 1-day earlier and produced 35.91% more plant tillers at 35 days-after-sowing. Our results demonstrate the potential of these fungal endophytes used in a consortium for establishing grassy plant species on lead contaminated soils, which may result in practical applications for heavy metal bioremediation.

Genome biology of the paleotetraploid perennial biomass crop Miscanthus.

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. × giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses.

Global gene flow releases invasive plants from environmental constraints on genetic diversity.

When plants establish outside their native range, their ability to adapt to the new environment is influenced by both demography and dispersal. However, the relative importance of these two factors is poorly understood. To quantify the influence of demography and dispersal on patterns of genetic diversity underlying adaptation, we used data from a globally distributed demographic research network comprising 35 native and 18 nonnative populations of Plantago lanceolata Species-specific simulation experiments showed that dispersal would dilute demographic influences on genetic diversity at local scales. Populations in the native European range had strong spatial genetic structure associated with geographic distance and precipitation seasonality. In contrast, nonnative populations had weaker spatial genetic structure that was not associated with environmental gradients but with higher within-population genetic diversity. Our findings show that dispersal caused by repeated, long-distance, human-mediated introductions has allowed invasive plant populations to overcome environmental constraints on genetic diversity, even without strong demographic changes. The impact of invasive plants may, therefore, increase with repeated introductions, highlighting the need to constrain future introductions of species even if they already exist in an area.

Two new species of Phyllanthus (Phyllanthaceae) from Thailand.

Two Phyllanthus species are newly described from a limestone mountain in the north of Thailand. The first species, P. huamotensis Pornp., Chantar. & J.Parn., sp. nov., is one of the most distinct Phyllanthus species easily distinguished by its reddish branchlets and stem, conspicuous reddish venation, especially on the lower leaf surface, red sepals with long fimbriate margin and red capsule with papillose-puberulous surface. The second species, P. chantaranothaii Pornp., J.Parn. & Hodk., sp. nov., is similar to P. pulcher Wall. ex Müll.Arg., but it is distinguished by its puberulous upper leaf surface and pistillate flowers which have red, narrowly lanceolate sepals with a white, long fimbriate margin, puberulous outer side as well as puberulous pedicel.

Genomic prediction of crown rust resistance in Lolium perenne.

BACKGROUND: Genomic selection (GS) can accelerate genetic gains in breeding programmes by reducing the time it takes to complete a cycle of selection. Puccinia coronata f. sp lolli (crown rust) is one of the most widespread diseases of perennial ryegrass and can lead to reductions in yield, persistency and nutritional value. Here, we used a large perennial ryegrass population to assess the accuracy of using genome wide markers to predict crown rust resistance and to investigate the factors affecting predictive ability. RESULTS: Using these data, predictive ability for crown rust resistance in the complete population reached a maximum of 0.52. Much of the predictive ability resulted from the ability of markers to capture genetic relationships among families within the training set, and reducing the marker density had little impact on predictive ability. Using permutation based variable importance measure and genome wide association studies (GWAS) to identify and rank markers enabled the identification of a small subset of SNPs that could achieve predictive abilities close to those achieved using the complete marker set. CONCLUSION: Using a GWAS to identify and rank markers enabled a small panel of markers to be identified that could achieve higher predictive ability than the same number of randomly selected markers, and predictive abilities close to those achieved with the entire marker set. This was particularly evident in a sub-population characterised by having on-average higher genome-wide linkage disequilibirum (LD). Higher predictive abilities with selected markers over random markers suggests they are in LD with QTL. Accuracy due to genetic relationships will decay rapidly over generations whereas accuracy due to LD will persist, which is advantageous for practical breeding applications.

From Concept to Commerce: Developing a Successful Fungal Endophyte Inoculant for Agricultural Crops.

The development of endophyte inoculants for agricultural crops has been bedevilled by the twin problems of a lack of reliability and consistency, with a consequent lack of belief among end users in the efficacy of such treatments. We have developed a successful research pipeline for the production of a reliable, consistent and environmentally targeted fungal endophyte seed-delivered inoculant for barley cultivars. Our approach was developed de novo from an initial concept to source candidate endophyte inoculants from a wild relative of barley, Hordeum murinum (wall barley). A careful screening and selection procedure and extensive controlled environment testing of fungal endophyte strains, followed by multi-year field trials has resulted in the validation of an endophyte consortium suitable for barley crops grown on relatively dry sites. Our approach can be adapted for any crop or environment, provided that the set of first principles we have developed is followed. Here, we report how we developed the successful pipeline for the production of an economically viable fungal endophyte inoculant for barley cultivars.

Origin of year-long bean (Phaseolus dumosus Macfady, Fabaceae) from reticulated hybridization events between multiple Phaseolus species.

Background and Aims Improved understanding of the secondary gene pools of crops is essential for advancing genetic gain in breeding programmes. Common bean, Phaseolus vulgaris, is a staple crop with several wild relatives in its secondary gene pool. The year-long bean, P. dumosus, an important crop in Guatemala, is considered particularly closely related to P. vulgaris and a potential source of novel variation. However, the genetic diversity and relationship to other Phaseolus species of P. dumosus remain unclear. Methods We conducted the first comprehensive investigation of P. dumosus genetic diversity using both nuclear and chloroplast genome markers. Our nuclear marker set included over 700 markers present within the Phaseolus DArT (Diversity Arrays Technology) array, which we applied to P. dumosus and other relatives of P. vulgaris (including every secondary gene pool species: P. acutifolius, P. albescens, P. coccineus and P. costaricensis). Key Results Phaseolus dumosus arose from hybridization of P. vulgaris and P. coccineus, followed by at least two later hybridizations with sympatric congener populations. Existing P. dumosus collections have low genetic diversity. Conclusions The under-utilized crop P. dumosus has a complex hybrid origin. Further sampling in the region in which it arose may uncover additional germplasm for introgressing favourable traits into crops within the P. vulgaris gene pool.

Markers associated with heading and aftermath heading in perennial ryegrass full-sib families.

BACKGROUND: Heading and aftermath heading are important traits in perennial ryegrass because they impact forage quality. So far, genome-wide association analyses in this major forage species have only identified a small number of genetic variants associated with heading date that overall explained little of the variation. Some possible reasons include rare alleles with large phenotypic affects, allelic heterogeneity, or insufficient marker density. We established a genome-wide association panel with multiple genotypes from multiple full-sib families. This ensured alleles were present at the frequency needed to have sufficient statistical power to identify associations. We genotyped the panel via partial genome sequencing and performed genome-wide association analyses with multi-year phenotype data collected for heading date, and aftermath heading. RESULTS: Genome wide association using a mixed linear model failed to identify any variants significantly associated with heading date or aftermath heading. Our failure to identify associations for these traits is likely due to the extremely low linkage disequilibrium we observed in this population. However, using single marker analysis within each full-sib family we could identify markers and genomic regions associated with heading and aftermath heading. Using the ryegrass genome we identified putative orthologs of key heading genes, some of which were located in regions of marker-trait associations. CONCLUSION: Given the very low levels of LD, genome wide association studies in perennial ryegrass populations are going to require very high SNP densities. Single marker analysis within full-sibs enabled us to identify significant marker-trait associations. One of these markers anchored proximal to a putative ortholog of TFL1, homologues of which have been shown to play a key role in continuous heading of some members of the rose family, Rosaceae.

Variation in sequences containing microsatellite motifs in the perennial biomass and forage grass, Phalaris arundinacea (Poaceae).

Forty three microsatellite markers were developed for further genetic characterisation of a forage and biomass grass crop, for which genomic resources are currently scarce. The microsatellite markers were developed from a normalized EST-SSR library. All of the 43 markers gave a clear banding pattern on 3% Metaphor agarose gels. Eight selected SSR markers were tested in detail for polymorphism across eleven DNA samples of large geographic distribution across Europe. The new set of 43 SSR markers will help future research to characterise the genetic structure and diversity of Phalaris arundinacea, with a potential to further understand its invasive character in North American wetlands, as well as aid in breeding work for desired biomass and forage traits. P. arundinacea is particularly valued in the northern latitude as a crop with high biomass potential, even more so on marginal lands.

Profundae diversitas: the uncharted genetic diversity in a newly studied group of fungal root endophytes.

Endophytes associated with crops have potential as beneficial inoculants in agriculture, but little is known about their genetic diversity and phylogenetic relationships. We carried out the first ever ecological and phylogenetic survey of the culturable fungal root endophytes of a wild barley species. Fungal root endophytes were isolated from 10 populations of wall barley (Hordeum murinum), and 112 taxa of fungi were identified based on internal transcribed spacer sequence similarity. We found representatives from 8 orders, 12 families and 18 genera. Within this group, only 34 isolates (30% of the total) could be confidently assigned to a species, and 23 of the isolates (21% of the total) had no significant match to anything deposited in GenBank (based on <85% sequence similarity). These results suggest a high proportion of novel fungi, with 28% not assigned to a known fungal order. This includes three endophytes that have been shown to significantly improve agronomic traits in cultivated barley. This study has, therefore, revealed a profound diversity of fungal root endophytes in a single wild relative of barley. Extrapolating from this, the study highlights the largely unknown, hugely diverse and potentially useful resource of crop wild relative endophytes.

Genetic diversity and floral width variation in introduced and native populations of a long-lived woody perennial.

Populations of introduced species in their new environments are expected to differ from native populations, due to processes such as genetic drift, founder effects and local adaptation, which can often result in rapid phenotypic change. Such processes can also lead to changes in the genetic structure of these populations. This study investigated the populations of Rhododendron ponticum in its introduced range in Ireland, where it is severely invasive, to determine both genetic and flower width diversity and differentiation. We compared six introduced Irish populations with two populations from R. ponticum's native range in Spain, using amplified fragment length polymorphism and simple sequence repeat genetic markers. We measured flower width, a trait that may affect pollinator visitation, from four Irish and four Spanish populations by measuring both the width at the corolla tip and tube base (nectar holder width). With both genetic markers, populations were differentiated between Ireland and Spain and from each other in both countries. However, populations displayed low genetic diversity (mean Nei's genetic diversity = 0.22), with the largest proportion (76-93 %) of genetic variation contained within, rather than between, populations. Although corolla width was highly variable between individuals within populations, tube width was significantly wider (>0.5 mm) in introduced, compared with native, populations. Our results show that the same species can have genetically distinct populations in both invasive and native regions, and that differences in floral width may occur, possibly in response to ecological sorting processes or local adaptation to pollinator communities.

Allopolyploidy, diversification, and the Miocene grassland expansion.

The role of polyploidy, particularly allopolyploidy, in plant diversification is a subject of debate. Whole-genome duplications precede the origins of many major clades (e.g., angiosperms, Brassicaceae, Poaceae), suggesting that polyploidy drives diversification. However, theoretical arguments and empirical studies suggest that polyploid lineages may actually have lower speciation rates and higher extinction rates than diploid lineages. We focus here on the grass tribe Andropogoneae, an economically and ecologically important group of C4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the species of the clade, based on sequences of four concatenated low-copy nuclear loci. Genetic allopolyploidy was documented using the characteristic pattern of double-labeled gene trees. At least 32% of the species sampled are the result of genetic allopolyploidy and result from 28 distinct tetraploidy events plus an additional six hexaploidy events. This number is a minimum, and the actual frequency could be considerably higher. The parental genomes of most Andropogoneae polyploids diverged in the Late Miocene coincident with the expansion of the major C4 grasslands that dominate the earth today. The well-documented whole-genome duplication in Zea mays ssp. mays occurred after the divergence of Zea and Sorghum. We find no evidence that polyploidization is followed by an increase in net diversification rate; nonetheless, allopolyploidy itself is a major mode of speciation.

High levels of gene flow and genetic diversity in Irish populations of Salix caprea L. inferred from chloroplast and nuclear SSR markers.

BACKGROUND: Salix caprea is a cold-tolerant pioneer species that is ecologically important in Europe and western and central Asia. However, little data is available on its population genetic structure and molecular ecology. We describe the levels of geographic population genetic structure in natural Irish populations of S. caprea and determine the extent of gene flow and sexual reproduction using both chloroplast and nuclear simple sequence repeats (SSRs). RESULTS: A total of 183 individuals from 21 semi-natural woodlands were collected and genotyped. Gene diversity across populations was high for the chloroplast SSRs (H T = 0.21-0.58) and 79 different haplotypes were discovered, among them 48% were unique to a single individual. Genetic differentiation of populations was found to be between moderate and high (mean G ST = 0.38). For the nuclear SSRs, G ST was low at 0.07 and observed heterozygosity across populations was high (H O = 0.32-0.51); only 9.8% of the genotypes discovered were present in two or more individuals. For both types of markers, AMOVA showed that most of the variation was within populations. Minor geographic pattern was confirmed by a Bayesian clustering analysis. Gene flow via pollen was found to be approximately 7 times more important than via seeds. CONCLUSIONS: The data are consistent with outbreeding and indicate that there are no significant barriers for gene flow within Ireland over large geographic distances. Both pollen-mediated and seed-mediated gene flow were found to be high, with some of the populations being more than 200 km apart from each other. These findings could simply be due to human intervention through seed trade or accidental transportation of both seeds and pollen. These results are of value to breeders wishing to exploit natural genetic variation and foresters having to choose planting material.

High levels of variation in Salix lignocellulose genes revealed using poplar genomic resources.

BACKGROUND: Little is known about the levels of variation in lignin or other wood related genes in Salix, a genus that is being increasingly used for biomass and biofuel production. The lignin biosynthesis pathway is well characterized in a number of species, including the model tree Populus. We aimed to transfer the genomic resources already available in Populus to its sister genus Salix to assess levels of variation within genes involved in wood formation. RESULTS: Amplification trials for 27 gene regions were undertaken in 40 Salix taxa. Twelve of these regions were sequenced. Alignment searches of the resulting sequences against reference databases, combined with phylogenetic analyses, showed the close similarity of these Salix sequences to Populus, confirming homology of the primer regions and indicating a high level of conservation within the wood formation genes. However, all sequences were found to vary considerably among Salix species, mainly as SNPs with a smaller number of insertions-deletions. Between 25 and 176 SNPs per kbp per gene region (in predicted exons) were discovered within Salix. CONCLUSIONS: The variation found is sizeable but not unexpected as it is based on interspecific and not intraspecific comparison; it is comparable to interspecific variation in Populus. The characterisation of genetic variation is a key process in pre-breeding and for the conservation and exploitation of genetic resources in Salix. This study characterises the variation in several lignocellulose gene markers for such purposes.

Nuclear SSR markers for Miscanthus, Saccharum, and related grasses (Saccharinae, Poaceae).

PREMISE OF THE STUDY: We developed nuclear simple sequence repeat (SSR) markers for the characterization of the biomass crop Miscanthus, especially M. sacchariflorus, M. sinensis, and M. ×giganteus, and tested for cross-species amplification. • METHODS AND RESULTS: Twenty-nine SSR markers (di- and tetranucleotide repeats) were developed from DNA sequences obtained from 192 clones from an enriched genomic library of M. sinensis. All markers were successfully amplified in M. sacchariflorus, M. sinensis, and M. ×giganteus, and 19 amplified across a broad range of Miscanthus species. Polymorphism information content and expected heterozygosity values (19 locus sample) were 0.88 and 0.89, respectively, for M. sinensis, 0.48 and 0.54 for M. sacchariflorus, and were the lowest in M. ×giganteus (0.33, 0.41). Thirteen out of 19 primer pairs showed cross-species amplification in non-Miscanthus sensu stricto taxa. • CONCLUSIONS: The new set of 29 SSR markers will be of high value for characterizing Miscanthus germplasm collections, for prebreeding, and for assessing variation in natural populations.