Differential gene expression and gene ontologies associated with increasing water-stress in leaf and root transcriptomes of perennial ryegrass (Lolium perenne)

Perennial ryegrass (Lolium perenne) is a forage and amenity grass species widely cultivated in temperate regions worldwide. As such, perennial ryegrass populations are exposed to a range of environmental conditions and stresses on a seasonal basis and from year to year. One source of potential stress is limitation on water availability. The ability of these perennial grasses to be able to withstand and recover after periods of water limitation or drought can be a key component of grassland performance. Thus, we were interested in looking at changes in patterns of gene expression associated with increasing water stress. Clones of a single genotype of perennial ryegrass were grown under non-flowering growth room conditions in vermiculite supplemented with nutrient solution. Leaf and root tissue was sampled at 4 times in quadruplicate relating to estimated water contents of 35%, 15%, 5% and 1%. RNA was extracted and RNAseq used to generate transcriptome profiles at each sampling point. Transcriptomes were assembled using the published reference genome sequence and differential gene expression analysed using 3 different programmes, DESeq2, edgeR and limma (with the voom transformation), individually and in combination, deriving Early, Middle and Late stage comparisons. Identified differentially expressed genes were then associated with enriched GO terms using BLAST2GO. For the leaf, up-regulated differentially expressed genes were strongly associated with GO terms only during the Early stage and the majority of GO terms were associated with only down-regulated genes at the Middle or Late stages. For the roots, few differentially expressed genes were identified at either Early or Middle stages. Only one replicate at 1% estimated water content produced high quality data for the root, however, this indicated a high level of differential expression. Again the majority of enriched GO terms were associated with down-regulated genes. The performance of the different analysis programmes and the annotations associated with identified differentially expressed genes is discussed

An investigation of genotype-phenotype association in a festulolium forage grass population containing genome-spanning <i>Festuca pratensis</i> chromosome segments in a <i>Lolium perenne</i> background

Alien chromosome introgression is used for the transfer of beneficial traits in plant breeding. For temperate forage grasses, much of the work in this context has focused on species within the ryegrasses (Lolium spp.) and the closely related fescues (Festuca spp.) particularly with a view to combining high forage quality with reliability and enhanced environmental services. We have analysed a L. perenne (perennial ryegrass) population containing the majority of a F. pratensis (meadow fescue) genome as introgressed chromosome segments to identify a) marker-trait associations for nutrient use and abiotic stress response across the family, and b) to assess the effects of introgression of F. pratensis genomic regions on phenotype. Using container-based assays and a system of flowing solution culture, we looked at phenotype responses, including root growth, to nitrogen and phosphorus status in the growing medium and abiotic stresses within this festulolium family. A number of significant marker/trait associations were identified across the family for root biomass on chromosomes 2, 3 and 5 and for heading date on chromosome 2. Of particular interest was a region on chromosome 2 associated with increased root biomass in phosphorus-limited conditions derived from one of the L. perenne parents. A genotype containing F. pratensis chromosome 4 as a monosomic introgression showed increased tiller number, shoot and root growth and genotypes with F. pratensis chromosome segment introgressions at different ends of chromosome 4 exhibited differential phenotypes across a variety of test conditions. There was also a general negative correlation between the extent of the F. pratensis genome that had been introgressed and root-related trait performances. We conclude that 1) the identification of alleles affecting root growth has potential application in forage grass breeding and, 2) F. pratensis introgressions can enhance quantitative traits, however, introgression can also have more general negative effects

B chromosomes are associated with redistribution of genetic recombination towards lower-recombination chromosomal regions in perennial ryegrass

Supernumerary ‘B’ chromosomes are non-essential components of the genome present in a range of plant and animal species – including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT marker genetic maps for 6 perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progenies and their parents we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared to zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, i.e., pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders

A comparison of shared patterns of differential gene expression and gene ontologies in response to water-stress in roots and leaves of four diverse genotypes of Lolium and Festuca spp. temperate pasture grasses

Ryegrasses (Lolium spp.) and fescues (Festuca spp.) are closely related and widely cultivated perennial forage grasses. As such, resilience in the face of abiotic stresses is an important component of their traits. We have compared patterns of differentially expressed genes (DEGs) in roots and leaves of two perennial ryegrass genotypes and a single genotype of each of a festulolium (predominantly Italian ryegrass) and meadow fescue with the onset of water stress, focussing on overall patterns of DEGs and gene ontology terms (GOs) shared by all four genotypes. Plants were established in a growing medium of vermiculite watered with nutrient solution. Leaf and root material were sampled at 35% (saturation) and, as the medium dried, at 15%, 5% and 1% estimated water contents (EWCs) and RNA extracted. Differential gene expression was evaluated comparing the EWC sampling points from RNAseq data using a combination of analysis methods. For all genotypes, the greatest numbers of DEGs were identified in the 35/1 and 5/1 comparisons in both leaves and roots. In total, 566 leaf and 643 root DEGs were common to all 4 genotypes, though a third of these leaf DEGs were not regulated in the same up/down direction in all 4 genotypes. For roots, the equivalent figure was 1% of the DEGs. GO terms shared by all four genotypes were often enriched by both up- and down-regulated DEGs in the leaf, whereas generally, only by either up- or down-regulated DEGs in the root. Overall, up-regulated leaf DEGs tended to be more genotype-specific than down-regulated leaf DEGs or root DEGs and were also associated with fewer GOs. On average, only 5-15% of the DEGs enriching common GO terms were shared by all 4 genotypes, suggesting considerable variation in DEGs between related genotypes in enacting similar biological processes

An Increasing Need for Productive and Stress Resilient Festulolium Amphiploids:What Can Be Learnt from the Stable Genomic Composition of Festuca pratensis subsp. apennina (De Not.) Hegi?

Genome composition of Festuca pratensis subsp. apennina (De Not.) Hegi, a tetraploid fescue species native to the tall forbs communities of south-eastern Europe at altitudes between 1100 and 2200m a.s.l. has been the subject of some debate by grass taxonomists. Our cytogenetic analyses including fluorescence in situ hybridisation with probes for genomic DNA and selected DNA repeats revealed the species to be allotetraploid and derived from interspecific hybridization between F. pratensis Huds., a species confined to grassland at lower altitudes, and a so far unknown Festuca species. Besides tetraploids, triploids and pentaploids were found growing in Alpine meadows in close association with F. pratensis subsp. apennina. Triploid cytotypes predominated at many sites in Switzerland and Romania, and in some localities, they were the only cytotypes observed. Cytogenetic analyses revealed the triploids to be hybrids between diploid F. pratensis and tetraploid Festuca pratensis subsp. apennina, while the pentaploid cytotypes originated from hybridization between F. pratensis subsp. apennina and hexaploid F. arundinacea Schreb., a closely-related species growing in a close vicinity to F. pratensis subsp. apennina. Parental genomes of F. pratensis subsp. apennina and of the triploid and pentaploid hybrids showed no evidence of homoeologous chromosome pairing and interspecific recombination, supporting previous observation of a disomic inheritance at meiosis, where chromosome pairing was restricted to bivalent associations. A hypothesis is presented that a chromosome pairing regulator(s), reported previously in other polyploid broad-leaved fescue species of the Festuca subg. Schedonorus, is present and functional in F. pratensis subsp. apennina. It is likely that a common ancestors’ genome that carries the chromosome pairing regulator(s) is present in all polyploid broad-leaved fescue species, and its acquisition was a key event that enabled speciation, and development of a polyploid series within Festuca. Identification of a functional chromosome pairing regulator capable of stabilizing advantageous genome combinations in hybrids within the Lolium-Festuca complex would greatly assist in development of stable Festulolium cultivars. Its expression within Festulolium amphiploid cultivars would assist strategies aimed at climate-proofing productive European grasslands to combat exposures to stress conditions

A Comparison of Differential Gene Expression in Response to the Onset of Water Stress Between Three Hybrid Brachiaria Genotypes

Brachiaria (Trin.) Griseb. (syn. Urochloa P. Beauv.) is a C4 grass genus belonging to the Panicoideae. Native to Africa, these grasses are now widely grown as forages in tropical areas worldwide and are the subject of intensive breeding, particularly in South America. Tolerance to abiotic stresses such as aluminum and drought are major breeding objectives. In this study, we present the transcriptomic profiling of leaves and roots of three Brachiaria interspecific hybrid genotypes with the onset of water stress, Br12/3659-17 (gt-17), Br12/2360-9 (gt-9), and Br12/3868-18 (gt-18), previously characterized as having good, intermediate and poor tolerance to drought, respectively, in germplasm evaluation programs. RNA was extracted from leaf and root tissue of plants at estimated growing medium water contents (EWC) of 35, 15, and 5%. Differentially expressed genes (DEGs) were compared between different EWCs, 35/15, 15/5, and 35/5 using DESeq2. Overall, the proportions of DEGs enriched in all three genotypes varied in a genotype-dependent manner in relation to EWC comparison, with intermediate and sensitive gt-9 and gt-18 being more similar to each other than to drought tolerant gt-17. More specifically, GO terms relating to carbohydrate and cell wall metabolism in the leaves were enriched by up-regulated DEGs in gt-9 and gt-18, but by down-regulated DEGs in gt-17. Across all genotypes, analysis of DEG enzyme activities indicated an excess of down-regulated putative apoplastic peroxidases in the roots as water stress increased. This suggests that changes in root cell-wall architecture may be an important component of the response to water stress in Brachiaria

Impacts of abiotic stresses on the physiology and metabolism of cool-season grasses:A review

Grasslands cover more than 70% of the world's agricultural land playing a pivotal role in global food security, economy, and ecology due to their flexibility and functionality. Climate change, characterized by changes in temperature and precipitation patterns, and by increased levels of greenhouse gases in the atmosphere, is anticipated to increase both the frequency and severity of extreme weather events, such as drought, heat waves, and flooding. Potentially, climate change could severely compromise future forage crop production and should be considered a direct threat to food security. This review aimed to summarize our current understanding of the physiological and metabolic responses of temperate grasses to those abiotic stresses associated with climate change. Primarily, substantial decreases in photosynthetic rates of cool‐season grasses occur as a result of high temperatures, water‐deficit or water‐excess, and elevated ozone, but not CO2 concentrations. Those decreases are usually attributed to stomatal and non‐stomatal limitations. Additionally, while membrane instability and reactive oxygen species production was a common feature of the abiotic stress response, total antioxidant capacity showed a stress‐specific response. Furthermore, climate change‐related stresses altered carbohydrate partitioning, with implications for biomass production. While water‐deficit stress, increased CO2, and ozone concentrations resulted in higher carbohydrate content, the opposite occurred under conditions of heat stress and flooding. The extent of damage is greatly dependent on location, as well as the type and intensity of stress. Fortunately, temperate forage grass species are highly heterogeneous. Consequently, through intra‐ and in particular inter‐specific plant hybridization (e.g., Festuca x Lolium hybrids) new opportunities are available to harness, within single genotypes, gene combinations capable of combating climate change

Integrating a newly developed BAC-based physical mapping resource for Lolium perenne with a genome-wide association study across a L. Perenne European ecotype collection identifies genomic contexts associated with agriculturally important traits

Background and Aims Lolium perenne (perennial ryegrass) is the most widely cultivated forage and amenity grass species in temperate areas worldwide and there is a need to understand the genetic architectures of key agricultural traits and crop characteristics that deliver wider environmental services. Our aim was to identify genomic regions associated with agriculturally important traits by integrating a bacterial artificial chromosome (BAC)-based physical map with a genome-wide association study (GWAS). Methods BAC-based physical maps for L. perenne were constructed from similar to 212 000 high-information-content fingerprints using Fingerprint Contig and Linear Topology Contig software. BAC clones were associated with both BAC-end sequences and a partial minimum tiling path sequence. A panel of 716 L. perenne diploid genotypes from 90 European accessions was assessed in the field over 2 years, and genotyped using a Lolium Infinium SNP array. The GWAS was carried out using a linear mixed model implemented in TASSEL, and extended genomic regions associated with significant markers were identified through integration with the physical map. Key Results Between similar to 3600 and 7500 physical map contigs were derived, depending on the software and probability thresholds used, and integrated with similar to 35 k sequenced BAC clones to develop a resource predicted to span the majority of the L. perenne genome. From the GWAS, eight different loci were significantly associated with heading date, plant width, plant biomass and water-soluble carbohydrate accumulation, seven of which could be associated with physical map contigs. This allowed the identification of a number of candidate genes. Conclusions Combining the physical mapping resource with the GWAS has allowed us to extend the search for candidate genes across larger regions of the L. perenne genome and identified a number of interesting gene model annotations. These physical maps will aid in validating future sequence-based assemblies of the L. perenne genome.UK Biotechnology and Biological Sciences Research Council [BB/J004405/1, BB/CSP1730/1, BB/G012342/1]; Germinal Holdings (UK); Syngenta (UK); Vialactia Biosciences (NZ)Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]