• Characterisation of Miscanthus genetic resources: a combined analysis of plastid and nuclear microsatellites, nrDNA sequences, flow cytometry and morphology.

      De Cesare, Mariateresa; Teagasc Walsh Fellowship Programme; National Development Plan (2012)
      Miscanthus is a highly important forage and horticultural genus of perennial grasses (Poaceae) primarily native to South East Asia. Miscanthus is under intense global investigation as a biomass source for renewable energy production and several breeding initiatives are underway to develop new genotypes optimized for improved biomass and tolerance to a range of environmental stress conditions. A collection of 128 accessions belonging to the genus Miscanthus was established in Oak Park, Teagasc, Carlow, in 2008 and was investigated for morphological and molecular variation. Morphological traits were measured at the end of the second growing season and were compared with herbarium specimens of Miscanthus. Vegetative and inflorescence traits were scored and analysed using basic summary statistics, tests of normality and Principal Components Analysis (PCA). A large degree of morphological variation was recorded in the collections. The PCA of herbarium specimens was able to separate some species from others but there was also considerable overlap among species in the ordination, especially M. sacchariflorus, M. sinensis, M. condensatus and M. floridulus. These are known to be closely related and can interbreed. The PCA of the specimens from the Oak Park collection was less informative because of missing data due to lack of inflorescences (accessions did not flower). It was clear that morphology alone is often insufficient to distinguish taxa especially when inflorescence characters and ploidy information is lacking. The ploidy level of the accessions in the collection was evaluated through flow cytometry. The ploidy included di-, tri- and tetraploids. All individuals labelled as M. ×giganteus showed a triploid status, together with the newly bred M. sacchariflorus×M. sinensis hybrids. Most M. sinensis were diploids. Miscanthus sinensis Tea-62 was triploid and comparable to the value of the M. ×giganteus. A different situation was found for other non-diploid M. sinensis, in particular four M. sinensis ‘Goliath’ and the M. sinensis ‘Zebrinus’ Tea-33. In these the ratio measured by the flowcytometer was in between the values of the triploid M. giganteus and tetraploid M. sacchariflorus standards. The ‘Goliath-like’ hybrid is likely an autotriploid with three M. sinensis haploid sets, whereas M. ×giganteus is an allotriploid that is supposed to have two genomes from M. sinensis and one from M. sacchariflorus, which has a lower amount of DNA per haploid genome. DNA sequences of the internal transcribed spacer of the nrDNA were obtained for 76 genotypes in the collection and compared for polymorphism. The SNPs were particularly VI useful for differentiating M. sinensis, M. sacchariflorus and M. ×giganteus accessions and in combination with ploidy and morphology offer high potential for taxon identification. To gather more markers for population level diversity and differentiation studies, new microsatellite markers for both plastid and nuclear genomes were developed. For the development of plastid markers the chloroplast genome information of Saccharum officinarum was used. The nuclear SSRs (nSSRs) were developed from the sequences of 192 clones obtained from microsatellite enriched library. New primer pairs for the amplification of nineteen nuclear loci and six chloroplast loci were developed. Both chloroplast (cpSSR) and nSSR primers were used to characterise DNA variation, to help establish gene pools and to better understand hybridization and introgression. Huge genotypic variation was found within the genus, mostly in the species M. sinensis. The markers showed wide utility across a large number of Miscanthus species and also some closely related genera. The analysis of the cpSSRs showed a high number of different haplotypes but with a clear bias in allele composition between M. sinensis and the two species M. sacchariflorus and M. ×giganteus, thus confirming M. sacchariflorus as the maternal lineage of the hybrid M. xgiganteus. The nSSRs were found to be highly polymorphic across the collection and transferable to closely related genera such as Saccharum. The new markers were also used in UPGMA clustering and Bayesian structuring analysis to group individuals according to their similarity. Three major clusters of individuals were defined using the Bayesian STRUCTURE analysis with nuclear markers (nSSRs) and two with plastid markers (cpSSRs). In conclusion, the morphological, ploidy, sequence and microsatellite results highlighted the high level of diversity still unexplored in the genus and have clarified taxon identity of many accessions in the collection. A large set of new markers have been developed for the plant breeding and systematics community. The newly developed markers will be useful to further explore this diversity and to select useful traits for breeding of new and improved genotypes for biomass production.
    • Effect of Harvest Timing and Soil Moisture Content on Compaction, Growth and Harvest Yield in a Miscanthus Cropping System

      O'Flynn, Michael G.; Finnan, John; Curley, Edna M.; McDonnell, Kevin; Science Foundation Ireland; CP/E001 (MDPI, 2018-09-22)
      Harvesting Miscanthus × giganteus (J.M. Greef & Deuter ex Hodkinson & Renvoize) after shoot emergence is known to reduce yields in subsequent seasons. This research was conducted in Miscanthus to assess the effects on crop response and soil compaction of annually repeated traffic, applied both before new growth in the rhizomes (early harvest) and after shoot emergence (late harvest), at two different soil moisture contents. While an annual early harvest, yields more than a late harvest, because damage to new shoots is avoided, soil compaction may be increased following repeated harvests. Five treatments were tested: (a) An untrafficked control, (b) early-traffic on soil with typical soil moisture content (SMC) (early-normal), (c) early-traffic on soil with elevated SMC (early-elevated), (d) late-traffic on soil with typical SMC (late-normal) and (e) late-traffic on soil with elevated SMC (late-wet). The experiment was conducted on a Gleysol in Co. Dublin, Ireland during 2010 and 2011. Crop response effects were assessed by measuring stem numbers, stem height, trafficked zone biomass yield (November) and overall stem yield (January). Compaction effects were assessed by measuring penetration resistance, bulk density and water infiltration rate. Trafficked zone biomass yield in the early-dry and early-wet treatments was, respectively, 18% and 23% lower than in the control, but was, respectively, 39% and 31% higher than in the late-dry treatment. Overall, stem yield was significantly lower in the late-normal and late-wet treatments (10.4 and 10.1 tdm ha−1 respectively) when compared with the control (12.4 tdm ha−1), but no significant difference was recorded in overall stem yield between both early-traffic treatments and the control. Penetration resistance values were significantly higher in all trafficked treatments when compared with the control at depths of 0.15 m (≥54–61%) and 0.30 m (≥27–57%) and were significantly higher in 2011 when compared with 2010 at depths of 0.15 and 0.30 m. Baler system traffic in Miscanthus significantly reduced yields and significantly increased compaction annually. Miscanthus harvested early, on a dry soil, yielded 1.1 tdm ha−1 more than when harvested late on a dry soil. The yield advantage increased to 1.3 tdm ha−1 when early harvesting on a soil with 40–43% moisture content was compared with late harvesting on a wetter soil (51–52% moisture content). In this study, the magnitude of yield losses from compaction or other causes in early harvests was substantially lower than the yield losses, which resulted from shoot damage in late harvests. It is likely in similar climates that the results of this study would also apply to other perennial crops growing in similar soil types
    • Physiological and Transcriptional Response to Drought Stress Among Bioenergy Grass Miscanthus Species

      Vega, Jose J. De; Teshome, Abel; Klaas, Manfred; Grant, Jim; Finnan, John; Barth, Susanne; European Union; FP7-KBBE-2011-5-289461; CLNE/2017/364 (Biomed Central, 2020-07-28)
      Background: Miscanthus is a commercial lignocellulosic biomass crop owing to its high biomass productivity, particularly in the temperate regions. This study was conducted to elucidate physiological and molecular responses of four Miscanthus species subjected to well-watered and droughted greenhouse conditions. Results: A signicant biomass loss was observed under drought conditions for all genotypes. A sterile M. x giganteus showed a lower reduction in biomass yield under drought conditions compared to the control than the other species. Under well-watered conditions, biomass yield was as good as or better than control conditions in all species tested. M. sinensis was more tolerant than M. sacchariorus to both water stress conditions. 4,389 of the 67,789 genes (6.4%) in the reference genome were differentially expressed among four Miscanthus species. Most of the genes were differentially expressed in a single species, but the enrichment analysis of gene ontology (GO) terms revealed that the same biological processes were regulated in all the species during stress conditions. Namely, upregulated differentially expressed genes were signicantly involved in sucrose and starch metabolism, redox, and water and glycerol homeostasis and channel activity. Multiple copies of starch metabolic enzymes BAM and waxy GBSS-I were strongly up-regulated in drought stress in all Miscanthus genotypes. Twelve aquaporins (PIP1, PIP2 and NIP2) were also up-regulated in drought stress across genotypes. On the other hand, downregulated differentially expressed genes were signicantly involved in protein kinase activity, cell receptor signalling and phosphorylation. Conclusions: Findings in the present study can assist in implementing molecular breeding approaches of drought resistant Miscanthus and its domestication.
    • Physiological and transcriptional response to drought stress among bioenergy grass Miscanthus species

      De Vega, Jose J.; Teshome, Abel; Klaas, Manfred; Grant, Jim; Finnan, John; Barth, Susanne; European Union; Marie Sklodowska-Curie Actions COFUND CAROLINE; UK Research Council; FP7-KBBE-2011-5-289461; et al. (Biomed Central, 2021-03-06)
      Background Miscanthus is a commercial lignocellulosic biomass crop owing to its high biomass productivity, resilience and photosynthetic capacity at low temperature. These qualities make Miscanthus a particularly good candidate for temperate marginal land, where yields can be limited by insufficient or excessive water supply. Differences in response to water stress have been observed among Miscanthus species, which correlated to origin. In this study, we compared the physiological and molecular responses among Miscanthus species under excessive (flooded) and insufficient (drought) water supply in glasshouse conditions. Results A significant biomass loss was observed under drought conditions in all genotypes. M. x giganteus showed a lower reduction in biomass yield under drought conditions compared to the control than the other species. Under flooded conditions, biomass yield was as good as or better than control conditions in all species. 4389 of the 67,789 genes (6.4%) in the reference genome were differentially expressed during drought among four Miscanthus genotypes from different species. We observed the same biological processes were regulated across Miscanthus species during drought stress despite the DEGs being not similar. Upregulated differentially expressed genes were significantly involved in sucrose and starch metabolism, redox, and water and glycerol homeostasis and channel activity. Multiple copies of the starch metabolic enzymes BAM and waxy GBSS-I were strongly up-regulated in drought stress in all Miscanthus genotypes, and 12 aquaporins (PIP1, PIP2 and NIP2) were also up-regulated in drought stress across genotypes. Conclusions Different phenotypic responses were observed during drought stress among Miscanthus genotypes from different species, supporting differences in genetic adaption. The low number of DEGs and higher biomass yield in flooded conditions supported Miscanthus use in flooded land. The molecular processes regulated during drought were shared among Miscanthus species and consistent with functional categories known to be critical during drought stress in model organisms. However, differences in the regulated genes, likely associated with ploidy and heterosis, highlighted the value of exploring its diversity for breeding.