A new genetic locus for self-compatibility in the outcrossing grass species perennial ryegrass (Lolium perenne).

BACKGROUND: Self-incompatibility (SI) is a physiological mechanism that many flowering plants employ to prevent self-fertilization and maintain heterozygosity. In the grass family this is known to be controlled by a two locus (S-Z) system; however, the SI system is intrinsically leaky. Modifier genes of both the S and Z loci and a further locus, T, are known to override SI leading to self-fertilization and self-seed production. This has implications for the ecological and evolutionary success as well as the commercial breeding of grasses. Here we report a study where the genetic control of self-compatibility (SC) was determined from the results of self-pollinating an F2 population of perennial ryegrass from two independently derived inbred lines produced by single-seed descent. METHODS: In vitro self-pollinations of 73 fertile plants were analysed. A genetic association analysis was made with a panel of 1863 single-nucleotide polymorphism (SNP) markers, generated through genotype-by-sequencing methodology. Markers were placed on a recombination map of seven linkage groups (LGs) created using Joinmap v.5. The seed set on self- and open-pollinated inflorescences was determined on 143 plants, including the 73 plants analysed for self-pollination response. KEY RESULTS: Self-pollinations revealed a bimodal distribution of percentage SC with peaks at 50 and 100 %. A single quantitative trait locus (QTL) was identified with peak association for marker 6S14665z17875_11873 that mapped to LG 6. Peak position was associated with maximum marker segregation distortion. The self-compatible plants were equally fecund after self- and open pollination. CONCLUSIONS: This is the first report in the Poaceae family of an SC locus located on LG 6. This new SC QTL discovery, as well as indicating the complex nature of the pollen-stigma recognition process and its evolutionary significance, provides an additional source of SC for breeding perennial ryegrass.

Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley.

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50-60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24-28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.

Transcriptome characterization and differentially expressed genes under flooding and drought stress in the biomass grasses Phalaris arundinacea and Dactylis glomerata.

BACKGROUND AND AIMS: Perennial grasses are a global resource as forage, and for alternative uses in bioenergy and as raw materials for the processing industry. Marginal lands can be valuable for perennial biomass grass production, if perennial biomass grasses can cope with adverse abiotic environmental stresses such as drought and waterlogging. METHODS: In this study, two perennial grass species, reed canary grass (Phalaris arundinacea) and cocksfoot (Dactylis glomerata) were subjected to drought and waterlogging stress to study their responses for insights to improving environmental stress tolerance. Physiological responses were recorded, reference transcriptomes established and differential gene expression investigated between control and stress conditions. We applied a robust non-parametric method, RoDEO, based on rank ordering of transcripts to investigate differential gene expression. Furthermore, we extended and validated vRoDEO for comparing samples with varying sequencing depths. KEY RESULTS: This allowed us to identify expressed genes under drought and waterlogging whilst using only a limited number of RNA sequencing experiments. Validating the methodology, several differentially expressed candidate genes involved in the stage 3 step-wise scheme in detoxification and degradation of xenobiotics were recovered, while several novel stress-related genes classified as of unknown function were discovered. CONCLUSIONS: Reed canary grass is a species coping particularly well with flooding conditions, but this study adds novel information on how its transcriptome reacts under drought stress. We built extensive transcriptomes for the two investigated C3 species cocksfoot and reed canary grass under both extremes of water stress to provide a clear comparison amongst the two species to broaden our horizon for comparative studies, but further confirmation of the data would be ideal to obtain a more detailed picture.

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.

A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass.

The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic self-incompatibility (SI) mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. Here, we report a fine-mapping approach to identify the male component of the S-locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins. Our results represent a major step forward toward understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S-locus.

Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites.

BACKGROUND: Recent advances in the mapping of biochemical traits have been reported in Lolium perenne. Although the mapped traits, including individual sugars and fatty acids, contribute greatly towards ruminant productivity, organic acids and amino acids have been largely understudied despite their influence on the ruminal microbiome. RESULTS: In this study, we used a targeted gas-chromatography mass spectrometry (GC-MS) approach to profile the levels of 25 polar metabolites from different classes (sugars, amino acids, phenolic acids, organic acids and other nitrogen-containing compounds) present in a L. perenne F2 population consisting of 325 individuals. A quantitative trait (QTL) mapping approach was applied and successfully identified QTLs regulating seven of those polar metabolites (L-serine, L-leucine, glucose, fructose, myo-inositol, citric acid and 2, 3-hydroxypropanoic acid).Two QTL mapping approaches were carried out using SNP markers on about half of the population only and an imputation approach using SNP and DArT markers on the entire population. The imputation approach confirmed the four QTLs found in the SNP-only analysis and identified a further seven QTLs. CONCLUSIONS: These results highlight the potential of utilising molecular assisted breeding in perennial ryegrass to modulate a range of biochemical quality traits with downstream effects in livestock productivity and ruminal digestion.

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.

An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly.

BACKGROUND AND AIMS: High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing. METHODS: The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40 kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach. KEY RESULTS: The assembly comprises 424 750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212 bp and a contig N50 of 3790 bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200 Mb of the reference assembly. CONCLUSIONS: The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.

Network measures for characterising team adaptation processes.

The aim of this study was to advance the conceptualisation of team adaptation by applying social network analysis (SNA) measures in a field study of a paediatric cardiac surgical team adapting to changes in task complexity and ongoing dynamic complexity. Forty surgical procedures were observed by trained human factors researchers, and communication processes amongst team members were recorded. Focusing on who talked to whom, team communication structures, in response to changing task demands, were characterised by various network measures. Results showed that in complex procedures, the communication patterns were more decentralised and flatter. Also, in critical transition phases of the procedure, communication was characterised by higher information sharing and participation. We discuss implications for team adaptation theory and teamwork observation methods. PRACTITIONER SUMMARY: The reasons for this study were to advance our conceptualisation of team adaptation processes and to further quantify team observation methods. We found that the surgical team studied adapted to complexity of surgical procedures by adopting flatter communication patterns. We quantified team observation methods by applying SNA techniques.

Comparative exomics of Phalaris cultivars under salt stress.

BACKGROUND: Reed canary grass (Phalaris arundinacea) is an economically important forage and bioenergy grass of the temperate regions of the world. Despite its economic importance, it is lacking in public genomic data. We explore comparative exomics of the grass cultivars in the context of response to salt exposure. The limited data set poses challenges to the computational pipeline. METHODS: As a prerequisite for the comparative study, we generate the Phalaris reference transcriptome sequence, one of the first steps in addressing the issue of paucity of processed genomic data in this species. In addition, the differential expression (DE) and active-but-stable genes for salt stress conditions were analyzed by a novel method that was experimentally verified on human RNA-seq data. For the comparative exomics, we focus on the DE and stable genic regions, with respect to salt stress, of the genome. RESULTS AND CONCLUSIONS: In our comparative study, we find that phylogeny of the DE and stable genic regions of the Phalaris cultivars are distinct. At the same time we find the phylogeny of the entire expressed reference transcriptome matches the phylogeny of only the stable genes. Thus the behavior of the different cultivars is distinguished by the salt stress response. This is also reflected in the genomic distinctions in the DE genic regions. These observations have important implications in the choice of cultivars, and their breeding, for bio-energy fuels. Further, we identified genes that are representative of DE under salt stress and could provide vital clues in our understanding of the stress handling mechanisms in general.

A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover.

BACKGROUND: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. RESULTS: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. CONCLUSIONS: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a "core set" of ancestrally derived homeolog specific variants in white clover.

Localized hypermutation and associated gene losses in legume chloroplast genomes.

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume "inverted repeat loss" clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Demand-driven biogas production from Upflow Anaerobic Sludge Blanket (UASB) reactors to balance the power grid.

Future energy systems necessitate dispatchable renewable energy to balance electrical grids with high shares of intermittent renewables. Biogas from anaerobic digestion (AD) can generate electricity on-demand. High-rate methanogenic reactors, such as the Upflow Anaerobic Sludge Blanket (UASB), can react quicker to variations in feeding as compared to traditional AD systems. In this study, experimental trials validated the feasibility of operating the UASB in a demand-driven manner. The UASB was operated with leachate produced from a hydrolysis reactor treating grass silage. The UASB demonstrated a high degree of flexibility in responding to variable feeding regimes. The intra-day biogas production rate could be increased by up to 123% under 4 hours in demand-driven operation, without significant deterioration in performance. A model based on kinetic analysis was developed to help align demand-driven operation with the grid. The findings suggest significant opportunities for UASBs to provide positive and negative balance to the power grid.

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species.

BACKGROUND AND AIMS: Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne 'Cashel'. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species. METHODS: Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species. KEY RESULTS: All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A(8) mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively. CONCLUSIONS: The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to their ability to monitor genetic diversity within breeding pools, to trace maternal inheritance and to distinguish closely related species.

An innovative food system approach to diversifying protein intake: Protein-I: Shared Island sustainable healthy nutrition.

There is a need to transform our current food system if we are to feed the rapidly expanding global population while maintaining planetary health. Within the island of Ireland, there is an urgent need to diversify the foods that currently contribute to our populations' protein intake. A Shared Island Innovative Food System approach is required to achieve this in a manner that is sustainable and provides benefits to producers, consumers and other supply chain participants. The Protein-I project employs such an approach, with the paradigm focusing on production of plant food through to human health, while paying particular attention to the development of the rural bioeconomy. Using an interdisciplinary approach, the team will develop strategies to maximise sustainable plant protein production in a traceable/transparent fashion and assess the impact of changes to existing value chains and the development of new value chains for the rural economy. A smart supply chain technology solution tailored to the needs of the agri-food industry will be developed and tested. Additionally, we will co-design consumer-led approaches to diversify plant protein intake, model the impact of changes at the population level and perform human interventions to demonstrate efficacy in terms of achieving adequate nutrition and improved health. Comprehensive engagement with stakeholders is embedded throughout the whole project to embrace the multi-actor approach. Overall, the project will be a key step towards future-proofing our food system on the island of Ireland and moving towards protecting planetary and population health, within the context of a just transition.

Early response mechanisms of perennial ryegrass (Lolium perenne) to phosphorus deficiency.

BACKGROUND AND AIMS: Improving phosphorus (P) nutrient efficiency in Lolium perenne (perennial ryegrass) is likely to result in considerable economic and ecological benefits. To date, research into the molecular and biochemical response of perennial ryegrass to P deficiency has been limited, particularly in relation to the early response mechanisms. This study aimed to identify molecular mechanisms activated in response to the initial stages of P deficiency. METHODS: A barley microarray was successfully used to study gene expression in perennial ryegrass and this was complemented with gas chromatography-mass spectrometry metabolic profiling to obtain an overview of the plant response to early stages of P deficiency. KEY RESULTS: After 24 h of P deficiency, internal phosphate concentrations were reduced and significant alterations were detected in the metabolome and transcriptome of two perennial ryegrass genotypes. Results indicated a replacement of phospholipids with sulfolipids and the utilization of glycolytic bypasses in response to P deficiency in perennial ryegrass. CONCLUSIONS: The transcriptome and metabolome of perennial ryegrass undergo changes in response to reductions in P supply after 24 h.

Progress towards elucidating the mechanisms of self-incompatibility in the grasses: further insights from studies in Lolium.

BACKGROUND AND SCOPE: Self-incompatibility (SI) in flowering plants ensures the maintenance of genetic diversity by ensuring outbreeding. Different genetic and mechanistic systems of SI among flowering plants suggest either multiple origins of SI or considerable evolutionary diversification. In the grasses, SI is based on two loci, S and Z, which are both polyallelic: an incompatible reaction occurs only if both S and Z alleles are matched in individual pollen with alleles of the pistil on which they alight. Such incompatibility is referred to as gametophytic SI (GSI). The mechanics of grass GSI is poorly understood relative to the well-characterized S-RNase-based single-locus GSI systems (Solanaceae, Rosaceae, Plantaginaceae), or the Papaver recognition system that triggers a calcium-dependent signalling network culminating in programmed cell death. There is every reason to suggest that the grass SI system represents yet another mechanism of SI. S and Z loci have been mapped using isozymes to linkage groups C1 and C2 of the Triticeae consensus maps in Secale, Phalaris and Lolium. Recently, in Lolium perenne, in order to finely map and identify S and Z, more closely spaced markers have been developed based on cDNA and repeat DNA sequences, in part from genomic regions syntenic between the grasses. Several genes tightly linked to the S and Z loci were identified, but so far no convincing candidate has emerged. RESEARCH AND PROGRESS: From subtracted Lolium immature stigma cDNA libraries derived from S and Z genotyped individuals enriched for SI potential component genes, kinase enzyme domains, a calmodulin-dependent kinase and a peptide with several calcium (Ca(2+)) binding domains were identified. Preliminary findings suggest that Ca(2+) signalling and phosphorylation may be involved in Lolium GSI. This is supported by the inhibition of Lolium SI by Ca(2+) channel blockers lanthanum (La(3+)) and verapamil, and by findings of increased phosphorylation activity during an SI response.

Experimental comparison of two methods to study barley responses to partial submergence.

BACKGROUND: Crop yield is dependent on climate conditions, which are becoming both more variable and extreme in some areas of the world as a consequence of global climate change. Increased precipitation and flooding events are the cause of important yield losses due to waterlogging or (partial) submergence of crops in the field. Our ability to screen efficiently and quickly for varieties that have increased tolerance to waterlogging or (partial) submergence is important. Barley, a staple crop worldwide, is particularly sensitive to waterlogging. Screening for waterlogging tolerant barley varieties has been ongoing for many years, but methods used to screen vary greatly, from the type of soil used to the time at which the treatment is applied. This variation makes it difficult to cross-compare results. RESULTS: Here, we have devised a scoring system to assess barley tolerance to waterlogging and compare two different methods when partial submergence is applied with either water or a starch solution at an early developmental stage, which is particularly sensitive to waterlogging or partial submergence. The use of a starch solution has been previously shown to result in more reducing soil conditions and has been used to screen for waterlogging tolerance. CONCLUSIONS: Our results show that the two methods provide similar results to qualitatively rank varieties as tolerant or sensitive, while also affecting plants differently, in that application of a starch solution results in stronger and earlier symptoms than applying partial submergence with water.

Physiological and transcriptional response to drought stress among bioenergy grass Miscanthus species.

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.

EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass (Lolium spp.).

BACKGROUND: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm. RESULTS: A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2. CONCLUSIONS: The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.