High-resolution bulked segregant analysis enables candidate gene identification for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole-genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7484) biparental F2 population segregating for resistance to Xtg. Using chromosome-level genome assemblies as references, we were able to define a ~300 kb region highly associated with resistance on pseudo-chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2-like disease resistance proteins, cysteine-rich kinases, and RGA4- and RGA5-like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy of combining BSA studies in very large populations with whole genome deep sequencing and high-quality genome assemblies to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker-assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.

Cytonuclear interplay in auto- and allopolyploids: a multifaceted perspective from the Festuca-Lolium complex.

Restoring cytonuclear stoichiometry is necessary after whole-genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto- and allopolyploids of the Festuca-Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well-established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post-transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

Fine-Mapping and Comparative Genomic Analysis Reveal the Gene Composition at the S and Z Self-incompatibility Loci in Grasses.

Self-incompatibility (SI) is a genetic mechanism of hermaphroditic plants to prevent inbreeding after self-pollination. Allogamous Poaceae species exhibit a unique gametophytic SI system controlled by two multi-allelic and independent loci, S and Z. Despite intense research efforts in the last decades, the genes that determine the initial recognition mechanism are yet to be identified. Here, we report the fine-mapping of the Z-locus in perennial ryegrass (Lolium perenne L.) and provide evidence that the pollen and stigma components are determined by two genes encoding DUF247 domain proteins (ZDUF247-I and ZDUF247-II) and the gene sZ, respectively. The pollen and stigma determinants are located side-by-side and were genetically linked in 10,245 individuals of two independent mapping populations segregating for Z. Moreover, they exhibited high allelic diversity as well as tissue-specific gene expression, matching the expected characteristics of SI determinants known from other systems. Revisiting the S-locus using the latest high-quality whole-genome assemblies revealed a similar gene composition and structure as found for Z, supporting the hypothesis of a duplicated origin of the two-locus SI system of grasses. Ultimately, comparative genomic analyses across a wide range of self-compatible and self-incompatible Poaceae species revealed that the absence of a functional copy of at least one of the six putative SI determinants is accompanied by a self-compatible phenotype. Our study provides new insights into the origin and evolution of the unique gametophytic SI system in one of the largest and economically most important plant families.

Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

Effects of atmospheric CO2 concentration on transpiration and leaf elongation responses to drought in Triticum aestivum, Lolium perenne and Festuca arundinacea.

BACKGROUND AND AIMS: Leaf elongation is vital for Poaceae species' productivity, influenced by atmospheric CO2 concentration ([CO2]) and climate-induced water availability changes. Although [CO2] mitigates the effects of drought on reducing transpiration per unit leaf area, it also increases total leaf area and water use. These complex interactions associated with leaf growth pose challenges in anticipating climate change effects. This study aims to assess [CO2] effects on leaf growth response to drought in perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea) and wheat (Triticum aestivum). METHODS: Plants were cultivated in growth chambers with [CO2] at 200 or 800 ppm. At leaf six to seven unfolding, half of the plants were subjected to severe drought treatment. Leaf elongation rate (LER) was measured daily, whereas plant transpiration was continuously recorded gravimetrically. Additionally, water-soluble carbohydrate (WSC) content along with water and osmotic potentials in the leaf growing zone were measured at drought onset, mid-drought and leaf growth cessation. KEY RESULTS: Elevated [CO2] mitigated drought impacts on LER and delayed growth cessation across species. A positive correlation between LER and soil relative water content (SRWC) was observed. At the same SRWC, perennial grasses exhibited a higher LER with elevated [CO2], likely due to enhanced stomatal regulation. Despite stomatal closure and WSC accumulation, CO2 did not influence nighttime water potential or osmotic potential. The marked increase in leaf area across species resulted in similar (wheat and tall fescue) or higher (ryegrass) total water use by the experiment's end, under both watered and unwatered conditions. CONCLUSIONS: In conclusion, elevated [CO2] mitigates the adverse effects of drought on leaf elongation in three Poaceae species, due to its impact on plant transpiration. Overall, these findings provide valuable insights into CO2 and drought interactions that may help anticipate plant responses to climate change.

Sucrose-Phosphate Synthase and Sucrose Synthase contribute to refoliation in ryegrass, a grassland fructan-accumulating species.

The perennity of grassland species such as Lolium perenne greatly depends on their ability to regrow after cutting or grazing. Refoliation largely relies on the mobilization of fructans in the remaining tissues and on the associated sucrose synthesis and transport towards the basal leaf meristems. However, nothing is known yet about the sucrose synthesis pathway. Sucrose Phosphate Synthase (SPS) and Sucrose Synthase (SuS) activities, together with their transcripts, were monitored during the first hours after defoliation along the leaf axis of mature leaf sheaths and elongating leaf bases (ELB) where the leaf meristems are located. In leaf sheaths, which undergo a sink-source transition, fructan and sucrose contents declined while SPS and SuS activities increased, along with the expression of LpSPSA, LpSPSD.2, LpSuS1, LpSuS2, and LpSuS4. In ELB, which continue to act as a strong carbon sink, SPS and SuS activities increased to varying degrees while the expression of all the LpSPS and LpSuS genes decreased after defoliation. SPS and SuS both contribute to refoliation but are regulated differently depending on the source or sink status of the tissues. Together with fructan metabolism, they represent key determinants of ryegrass perennity and, more generally, of grassland sustainability.

Effects and mechanisms of microbial ecology and diversity on phytoremediation of cadmium-contaminated soil under the influence of biodegradable organic acids.

In recent years, heavy metal pollution has become a global environmental problem and poses a great threat to the health of people and ecosystems. Therefore, strategies for the effective remediation of Cd from contaminated soil are urgently needed. In this study, ryegrass was utilized as a remediation plant, and its remediation potential was enhanced through the application of Citric Acid (CA) in conjunction with Bacillus megaterium (B. megaterium). The P3 treatment (CA + Bacillus megaterium) exhibited a significantly higher efficiency in promoting cadmium extraction by ryegrass, resulting in a 1.79-fold increase in shoot cadmium accumulation compared to the control group (CK) with no Bacillus megaterium or CA. Moreover, the P3 treatment led to an increased abundance of Actinobacteriota, Acidobacteriota, and Patescibacteria in the rhizosphere. The concentration of amino derivatives (such as betaine, sulfolithocholylglycine, N-alpha-acetyl-lysine, glycocholic acid, arginyl-threonine) showed significant upregulation following the P3 treatment. In summary, this study proposes a viable approach for phytoremediation of soil contaminated with cadmium by harnessing the mobilizing abilities of soil bacteria.

Our aim was to gain a comprehensive understanding of the mechanisms involved in phytoremediation. These findings contribute to the existing knowledge by providing insights into the mechanism of phytoremediation in Cd-contaminated soil. They are expected to serve as a theoretical foundation for further elucidation of the phytoremediation mechanisms employed in Cd-contaminated soil.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

18 O enrichment of sucrose and photosynthetic and nonphotosynthetic leaf water in a C3 grass-atmospheric drivers and physiological relations.

The 18 O enrichment (Δ18 O) of leaf water affects the Δ18 O of photosynthetic products such as sucrose, generating an isotopic archive of plant function and past climate. However, uncertainty remains as to whether leaf water compartmentation between photosynthetic and nonphotosynthetic tissue affects the relationship between Δ18 O of bulk leaf water (Δ18 OLW ) and leaf sucrose (Δ18 OSucrose ). We grew Lolium perenne (a C3 grass) in mesocosm-scale, replicated experiments with daytime relative humidity (50% or 75%) and CO2 level (200, 400 or 800 µmol mol-1 ) as factors, and determined Δ18 OLW , Δ18 OSucrose and morphophysiological leaf parameters, including transpiration (Eleaf ), stomatal conductance (gs ) and mesophyll conductance to CO2 (gm ). The Δ18 O of photosynthetic medium water (Δ18 OSSW ) was estimated from Δ18 OSucrose and the equilibrium fractionation between water and carbonyl groups (εbio ). Δ18 OSSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ18 Oe ) with adjustments that correlated with gas exchange parameters (gs or total conductance to CO2 ). Isotopic mass balance and published work indicated that nonphotosynthetic tissue water was a large fraction (~0.53) of bulk leaf water. Δ18 OLW was a poor proxy for Δ18 OSucrose , mainly due to opposite Δ18 O responses of nonphotosynthetic tissue water (Δ18 Onon-SSW ) relative to Δ18 OSSW , driven by atmospheric conditions.

Metabolism of crown tissue is crucial for drought tolerance and recovery after stress cessation in Lolium/Festuca forage grasses.

A process of plant recovery after drought cessation is a complex trait which has not been fully recognized. The most important organ associated with this phenomenon in monocots, including forage grasses, is the crown tissue located between shoots and roots. The crown tissue is a meristematic crossroads for metabolites and other compounds between these two plant organs. Here, for the first time, we present a metabolomic and lipidomic study focused on the crown tissue under drought and recovery in forage grasses, important for agriculture in European temperate regions. The plant materials involve high (HDT) and low drought-tolerant (LDT) genotypes of Festuca arundinacea, and Lolium multiflorum/F. arundinacea introgression forms. The obtained results clearly demonstrated that remodeling patterns of the primary metabolome and lipidome in the crown under drought and recovery were different between HDT and LDT plants. Furthermore, HDT plants accumulated higher contents of primary metabolites under drought in the crown tissue, especially carbohydrates which could function as osmoprotectants and storage materials. On the other hand, LDT plants characterized by higher membranes damage under drought, simultaneously accumulated membrane phospholipids in the crown and possessed the capacity to recover their metabolic functions after stress cessation to the levels observed in HDT plants.

First Report of Pseudopithomyces palmicola Causing Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely cultivated for the production of forage, hay and silage worldwide, due to its high nutritional value and palatability (Feng et al. 2021). It has been infected by a number of foliar fungal diseases caused by different fungal pathogens (Xue et al. 2017, 2020; Victoria Arellano et al. 2021; Liu et al. 2023). In August 2021, three Pseudopithomyces isolates with similar colony characteristics were isolated from fresh samples of leaf spots on Italian ryegrass that was collected from the Forage Germplasm Nursery, Maming town of Qujing city, Yunnan province, China (25.53833°N, 103.60278°E). For specific isolation, tissue pieces (about 0.5 cm × 1 cm) from symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed three times in sterilized distilled water and air dried, plated on potato dextrose agar (PDA), and incubated at 25°C for 3 to 7 days in the dark. Following initial isolation, a representative isolate, KM42, was selected for further study. Colonies growing on PDA were cottony, white to grey, 53.8 to 56.9 mm diameter after 6 days in the dark at 25°C, with white and regular edge; in reverse, colonies were brown to dark brown in the center, and pale yellow toward the periphery with concentric rings. Under near-UV light at room temperature (20 ± 3°C), colonies were incubated on PDA for ten days to produce conidia. Conidia were globose or ellipsoid to amygdaloid, 1- to 3-transversely septate, 0- to 2-vertical septate, light brown to brown, measuring 11.6 to 24.4 × 7.7 to 16.8 µm (avg. 17.3 × 10.9 µm). The internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA(ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (OQ875842 for ITS, OQ875844 for LSU, and OQ883943 for RPB2). BLAST analysis of all these three segments showed 100% (ITS: MF804527), 100% (LSU: KU554630), and 99.4% (RPB2: MH249030) identity with sequences of reported isolate CBS 143931 (= UC22) of Pseudopithomyces palmicola (Lorenzini et al. 2016; Liu et al. 2018). To fulfill Koch's postulates, four 12-week-old healthy Italian ryegrass plants were spray-inoculated separately with a mycelial suspension of about 5.4 × 102 CFU/ml of an isolate of P. palmicola. In addition, four control plants were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 22°C. Small brown to dark brown spots developed on leaves 10 days after inoculation; symptoms were not observed on control plants. Pathogenicity tests were conducted three times by the same method. The same fungus was re-isolated from the lesions, and confirmed by morphological and molecular methods as described above. To the best of our knowledge, this is the first report of P. palmicola causing leaf spot on Italian ryegrass in China or worldwide. This information will be useful to forage grass managers and plant pathologists in recognizing the disease and developing effective control measures.

Differences in the Characteristics and Pathogenicity of Pyrenophora Species Associated with Seeds of Italian Ryegrass.

Pyrenophora is a genus of pathogens that cause leaf damage and a common seedborne fungus of Italian ryegrass (Lolium multiflorum Lam.). To elucidate the main seedborne Pyrenophora species, 36 seed samples of Italian ryegrass were collected; in total, 113 strains, representing 12.3% of all isolated strains (921), were identified as Pyrenophora species using the identity of ITS sequences in NCBI and the similarity of morphological characteristics. P. dictyoides (97) was the most frequent species. By pure culture technique, 24 representative pure isolates were obtained for further study. Based on DNA analysis of multiple loci (ITS, LSU, GPDH, CHS-1, and RPB1) and morphological characters, eight Pyrenophora species were identified, P. avenicola, P. chaetomioides, P. dictyoides, P. lolii, P. nobleae, P. teres, P. triseptata, and P. tritici-repentis; among them, P. avenicola, P. tritici-repentis, and P. triseptata were newly reported on Italian ryegrass worldwide. Seed inoculation showed that P. dictyoides, P. lolii, and P. teres remarkably decreased the final germination percentages and germination indexes compared with control treatments (P ≤ 0.05); and plant inoculation showed that P. dictyoides, P. lolii, and P. nobleae could cause typical brown spot in vivo with a higher infection rate (P ≤ 0.05). In conclusion, pathogenicity tests showed that all Pyrenophora species could both inhibit seed germination and infect Italian ryegrass to different degrees; among them, P. dictyoides was the most important seedborne pathogen based on the combination of its isolation and infection rate, followed by P. lolii and P. nobleae. The data generated in this study are helpful for the accurate identification of Pyrenophora species and the development of seedborne disease management strategies.

High Diversity of Epicoccum Associated with Leaf Spot on Italian Ryegrass in Southwestern China: Three New Species and Six New Records.

Italian ryegrass is widely cultivated for the productions of forage, hay and silage, due to its high nutritional value and good palatability. Leaf spots caused by fungi pose a serious threat to forage crops. In order to expand knowledge of fungi causing leaf spot in ryegrass (Lolium multiforum) in Sichuan, Yunnan, Chongqing and Guizhou of southwestern China, a comprehensive survey was undertaken from 2015 to 2022. Survey discovered that Epicoccum leaf spot (ELS) was a common and widespread disease, more serious at the late stage of growth (after late May); symptomatic leaf samples collected from the four different provinces were analyzed, and a total of 202 Epicoccum isolates were obtained; based on both multilocus phylogeny (ITS, LSU, TUB2, and RPB2) and morphology, 10 Epicoccum species were finally identified, including three novel species (E. endololii sp. nov., E. lolii sp. nov. and E. loliicola sp. nov.), six new host records (E. draconis, E. endophyticum, E. oryzae, E. plurivorum, E. thailandicum and E. tobaicum), and an unknown species (Epicoccum sp.1). Pathogenicity tests showed that E. endophyticum, E. endololii and Epicoccum sp.1 were non-pathogenic to Italian ryegrass, which were confirmed as endophytes in this study; other six species could infect Italian ryegrass and cause leaf lesions to different degrees, of which E. draconis was more aggressive (P ≤ 0.05). Coupling with the isolation rates and geographical distributions of these species, E. plurivorum was the predominant pathogen in Yunnan while E. oryzae and E. tobaicum in other three provinces. This work provides an initial understanding of the taxonomies, virulence and distributions of Epicoccum species associated with ELS of southwestern China, and lays a solid foundation for the diagnosis in the field, and scientific control of ELS on Italian ryegrass.

Overexpression of Lolium multiflorum LmMYB1 Enhances Drought Tolerance in Transgenic Arabidopsis.

Lolium multiflorum is one of the world-famous forage grasses with rich biomass, fast growth rate and good nutritional quality. However, its growth and forage yield are often affected by drought, which is a major natural disaster all over the world. MYB transcription factors have some specific roles in response to drought stress, such as regulation of stomatal development and density, control of cell wall and root development. However, the biological function of MYB in L. multiflorum remains unclear. Previously, we elucidated the role of LmMYB1 in enhancing osmotic stress resistance in Saccharomyces cerevisiae. Here, this study elucidates the biological function of LmMYB1 in enhancing plant drought tolerance through an ABA-dependent pathway involving the regulation of cell wall development and stomatal density. After drought stress and ABA stress, the expression of LmMYB1 in L. multiflorum was significantly increased. Overexpression of LmMYB1 increased the survival rate of Arabidopsis thaliana under drought stress. Under drought conditions, expression levels of drought-responsive genes such as AtRD22, AtRAB and AtAREB were up-regulated in OE compared with those in WT. Further observation showed that the stomatal density of OE was reduced, which was associated with the up-regulated expression of cell wall-related pathway genes in the RNA-Seq results. In conclusion, this study confirmed the biological function of LmMYB1 in improving drought tolerance by mediating cell wall development through the ABA-dependent pathway and thereby affecting stomatal density.

Exogenous Methyl Jasmonate Mediated MiRNA-mRNA Network Improves Heat Tolerance of Perennial Ryegrass.

Heat stress can hinder the growth of perennial ryegrass (Lolium perenne L.). Methyl jasmonate (MeJA) applied exogenously can increase heat stress tolerance in plants; however, the regulatory mechanisms involved in heat tolerance mediated by MeJA are poorly understood in perennial ryegrass. Here, the microRNA (miRNA) expression profiles of perennial ryegrass were assessed to elucidate the regulatory pathways associated with heat tolerance induced by MeJA. Plants were subjected to four treatments, namely, control (CK), MeJA pre-treatment (T), heat stress treatment (H), and MeJA pre-treatment + heat stress (TH). According to the results, 102 miRNAs were up-regulated in all treatments, with 20, 27, and 33 miRNAs being up-regulated in the T, H, and TH treatment groups, respectively. The co-expression network analysis between the deferentially expressed miRNAs and their corresponding target genes showed that 20 miRNAs modulated 51 potential target genes. Notably, the miRNAs that targeted genes related to with regards to heat tolerance were driven by MeJA, and they were involved in four pathways: novel-m0258-5p mediated signal transduction, novel-m0350-5p mediated protein homeostasis, miR397-z, miR5658-z, and novel-m0008-5p involved in cell wall component, and miR1144-z and miR5185-z dominated chlorophyll degradation. Overall, the findings of this research paved the way for more research into the heat tolerance mechanism in perennial ryegrass and provided a theoretical foundation for developing cultivars with enhanced heat tolerance.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.

Chromosome-scale assembly and annotation of the perennial ryegrass genome.

BACKGROUND: The availability of chromosome-scale genome assemblies is fundamentally important to advance genetics and breeding in crops, as well as for evolutionary and comparative genomics. The improvement of long-read sequencing technologies and the advent of optical mapping and chromosome conformation capture technologies in the last few years, significantly promoted the development of chromosome-scale genome assemblies of model plants and crop species. In grasses, chromosome-scale genome assemblies recently became available for cultivated and wild species of the Triticeae subfamily. Development of state-of-the-art genomic resources in species of the Poeae subfamily, which includes important crops like fescues and ryegrasses, is lagging behind the progress in the cereal species. RESULTS: Here, we report a new chromosome-scale genome sequence assembly for perennial ryegrass, obtained by combining PacBio long-read sequencing, Illumina short-read polishing, BioNano optical mapping and Hi-C scaffolding. More than 90% of the total genome size of perennial ryegrass (approximately 2.55 Gb) is covered by seven pseudo-chromosomes that show high levels of collinearity to the orthologous chromosomes of Triticeae species. The transposon fraction of perennial ryegrass was found to be relatively low, approximately 35% of the total genome content, which is less than half of the genome repeat content of cultivated cereal species. We predicted 54,629 high-confidence gene models, 10,287 long non-coding RNAs and a total of 8,393 short non-coding RNAs in the perennial ryegrass genome. CONCLUSIONS: The new reference genome sequence and annotation presented here are valuable resources for comparative genomic studies in grasses, as well as for breeding applications and will expedite the development of productive varieties in perennial ryegrass and related species.

The Role of Triacylglycerol in the Protection of Cells against Lipotoxicity under Drought in Lolium multiflorum/Festucaarundinacea Introgression Forms.

Triacylglycerol is a key lipid compound involved in maintaining homeostasis of both membrane lipids and free fatty acids (FFA) in plant cells under adverse environmental conditions. However, its role in the process of lipid remodeling has not been fully recognized, especially in monocots, including grass species. For our study, two closely related introgression forms of Lolium multiflorum (Italian ryegrass) and Festuca arundinacea (tall fescue), distinct in their level of drought tolerance, were selected as plant models to study rearrangements in plant lipidome under water deficit and further re-watering. The low drought tolerant (LDT) form revealed an elevated level of cellular membrane damage accompanied by an increased content of polyunsaturated FFA and triacylglycerol under water deficit, compared with the high drought tolerant (HDT) form. However, the LDT introgression form demonstrated also the ability to regenerate its membranes after stress cessation. The obtained results clearly indicated that accumulation of triacylglycerol under advanced drought in the LDT form could serve as a cellular protective mechanism against overaccumulation of toxic polyunsaturated FFA and other lipid intermediates. Furthermore, accumulation of triacylglycerol under drought conditions could serve also as storage of substrates required for further regeneration of membranes after stress cessation. The rearrangements in triacylglycerol metabolism were supported by the upregulation of several genes, involved in a biosynthesis of triacylglycerol. With respect to this process, diacylglycerol O-acyltransferase DGAT2 seems to play the most important role in the analyzed grasses.

RAD-seq as an effective strategy for heterogenous variety identification in plants-a case study in Italian Ryegrass (Lolium multiflorum).

The primary approach for variety distinction in Italian ryegrass is currently the DUS (distinctness, uniformity and stability) test based on phenotypic traits. Considering the diverse genetic background within the population and the complexity of the environment, however, it is challenging to accurately distinguish varieties based on DUS criteria alone. In this study, we proposed the application of high-throughput RAD-seq to distinguish 11 Italian ryegrass varieties with three bulks of 50 individuals per variety. Our findings revealed significant differences among the 11 tested varieties. The PCA, DAPC and STRUCTURE analysis indicated a heterogeneous genetic background for all of them, and the AMOVA analysis also showed large genetic variance among these varieties (ΦST = 0.373), which were clearly distinguished based on phylogenetic analysis. Further nucleotide diversity (Pi) analysis showed that the variety 'Changjiang No.2' had the best intra-variety consistency among 11 tested varieties. Our findings suggest that the RAD-seq could be an effectively alternative method for the variety distinction of Italian ryegrass, as well as a potential tool for open-pollinated varieties (OPVs) of other allogamous species.

Transcriptome characterization of candidate genes for heat tolerance in perennial ryegrass after exogenous methyl Jasmonate application.

Methyl jasmonate (MeJA) plays a role in improving plant stress tolerance. The molecular mechanisms associated with heat tolerance mediated by MeJA are not fully understood in perennial grass species. The study was designed to explore transcriptomic mechanisms underlying heat tolerance by exogenous MeJA in perennial ryegrass (Lolium perenne L.) using RNA-seq. Transcriptomic profiling was performed on plants under normal temperature (CK), high temperature for 12 h (H), MeJA pretreatment (T), MeJA pretreatment + H (T-H), respectively. The analysis of differentially expressed genes (DEGs) showed that H resulted in the most DEGs and T had the least, compared with CK. Among them, the DEGs related to the response to oxygen-containing compound was higher in CKvsH, while many genes related to photosynthetic system were down-regulated. The DEGs related to plastid components was higher in CKvsT. GO and KEGG analysis showed that exogenous application of MeJA enriched photosynthesis related pathways under heat stress. Exogenous MeJA significantly increased the expression of genes involved in chlorophyll (Chl) biosynthesis and antioxidant metabolism, and decreased the expression of Chl degradation genes, as well as the expression of heat shock transcription factor - heat shock protein (HSF-HSP) network under heat stress. The results indicated that exogenous application of MeJA improved the heat tolerance of perennial ryegrass by mediating expression of genes in different pathways, such as Chl biosynthesis and degradation, antioxidant enzyme system, HSF-HSP network and JAs biosynthesis.