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 A₈ 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.     

Effects of endophyte infection on drought stress tolerance of Lolium perenne accessions from the Mediterranean region

Perennial ryegrass (Lolium perenne) is a cool-season grass species that is often infected by the leaf-inhabiting endophyte Neotyphodium lolii. This particular endophyte is asexual and has the potential to impact host survival, growth and reproduction. The objective was to assess the potential costs or benefits of endophyte infection on drought stress tolerance of native perennial ryegrass accessions originally collected from Italy, Morocco, Tunisia, and Turkey. Sixty infected (E+) individuals from each accession were planted in a greenhouse. Half of these individuals were treated with a systemic fungicide to eliminate the endophyte (E−). For two drought periods water was withheld for 10-14 days and then allowed a one week recovery period following each. In some accessions under drought, E+ plants had more tillers, greater tiller lengths, total dry mass and green shoot mass than E− plants, suggesting a positive effect of endophyte infection on host growth. Total tiller length and the number of tillers showed significant population × treatment × infection interactions for 4 of 6 populations. This work is one of the few that documents the effects of endophyte infection for a common forage grass species from wild populations native to its distributional range. The results demonstrate that endophyte infection can help ameliorate abiotic stress such as drought and there may be a selective advantage for grasses from certain Mediterranean regions.     

Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.     

Fructan:fructan 1-fructosyltransferase,a key enzyme for biosynthesis of graminan oligomers in hardened wheat

Fructans play important roles not only as a carbon source for survival under persistent snow cover but also as agents that protect against various stresses in overwintering plants. Complex fructans having both ß-(2,1)- and ß-(2,6)-linked fructosyl units accumulate in wheat (Triticum aestivum L.) during cold hardening. We detected fructan: fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) activity for catalyzing the formation and extension of ß-(2,1)-linked fructans in hardened wheat tissues, cloned cDNAs (wft3 and wft4) of 1-FFT, and analyzed the enzymatic properties of a wft3 recombinant protein (Wft3m) produced by yeast. Wft3m transferred ß-(2,1)-linked fructosyl units to phlein, an extension of sucrose through ß-(2,6)-linked fructosyl units, as well as to inulin, an extension of sucrose through ß-(2,1)-linked fructosyl units, but could not efficiently synthesize long inulin oligomers. Incubation of a mixture of Wft3m and another recombinant protein of wheat, sucrose:fructan 6-fructosyltransferase (6-SFT), with sucrose and 1-kestotriose produced fructans similar to those that accumulated in hardened wheat tissues. The results demonstrate that 1-FFT produces branches of ß-(2,1)-linked fructosyl units to phlein and graminan oligomers synthesized by 6-SFT and contributes to accumulation of fructans containing ß-(2,1)- and ß-(2,6)-linked fructosyl units. In combination with sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and 6-SFT, 1-FFT is necessary for fructan synthesis in hardened wheat.     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

氧化石墨烯对多年生黑麦草逆境生理及光合特征的影响

为探讨不同浓度氧化石墨烯（GO）对多年生黑麦草生长、生理及光合特征的影响,该文采用盆栽试验,在土壤中添加0、10、20、30、40、50 mg·g^-1 GO进行多年生黑麦草培养,并测定植物生长指标、光合色素含量、保护酶活性、丙二醛（MDA）含量、叶片质膜透性、可溶性蛋白含量和光合参数。结果表明：（1）10、20 mg·g^-1 GO处理对多年生黑麦草生长无显著影响;30～50 mg·g^-1 GO处理对多年生黑麦草生长具有抑制作用,在50 mg·g^-1 GO浓度下多年生黑麦草株高和生物量均最小,较对照分别降低了16.8%和27.1%。（2）当GO浓度达到30 mg·g^-1时,总叶绿素和类胡萝卜素的含量显著降低,在50 mg·g^-1 GO处理时达到最低。（3）高浓度的GO处理(40、50 mg·g^-1)虽降低了多年生黑麦草的叶片净光合速率（P_n）、气孔导度（G_s）和蒸腾速率（T_r     

Effects of Temperature on Rate of Feeding of the Plant Parasitic Nematodes Rotylenchus robustus,Xiphinema diversicaudatum,and Hemicycliophora conida

Rotylenchus robustus, Xiphinema diversicaudatum, and Hemicycgiophora conida were observed feeding over a range of temperatures on perennial rye-grass (Lolium perenne) seedlings grown on agar plates. R. robustus fed between 0.5 and 42.5 C, X. diversicaudatum between 5.0 and 37.0 C and H. conida between 5.0 and 34.0 C. Between 10 and 25 C there was a direct relationship between temperature and rate of esophageal bulb contractions. Above 25 C the number of esophageal contractions/min did not increase at the same rate and eventually decreased. At the extremes of temperature range, abnormal feeding behaviour was observed. Rates of esophageal bulb contraction did not differ in the different nematode life stages and sexes, or at different feeding sites on the roots.     

Structural elucidation and antitumor activity of a fructan from Cyathula officinalis Kuan

A fructan named CoPS3 was isolated from Cyathula officinalis Kuan. The structure of CoPS3 was determined by methylation, by the reductive-cleavage method combined with GC-MS analysis, and both 1D and 2D 1H and 13C NMR spectroscopy. These results show that CoPS3 is a graminans-type fructan that is comprised of a beta-D-fructofuranosyl backbone having residues linked (2-->1)- and (2-->6) with branches and an alpha-D-glucopyranose residue on the nonreducing end of the fructan chain. Each branch is terminated by a beta-D-Fruf residue. Bioassay showed that it could inhibit growth of Lewis pulmonary carcinoma implanted in mice.     

Metabolomics of forage plants: a review

Background

Forage plant breeding is under increasing pressure to deliver new cultivars with improved yield, quality and persistence to the pastoral industry. New innovations in DNA sequencing technologies mean that quantitative trait loci analysis and marker-assisted selection approaches are becoming faster and cheaper, and are increasingly used in the breeding process with the aim to speed it up and improve its precision. High-throughput phenotyping is currently a major bottle neck and emerging technologies such as metabolomics are being developed to bridge the gap between genotype and phenotype; metabolomics studies on forages are reviewed in this article.

Scope

Major challenges for pasture production arise from the reduced availability of resources, mainly water, nitrogen and phosphorus, and metabolomics studies on metabolic responses to these abiotic stresses in Lolium perenne and Lotus species will be discussed here. Many forage plants can be associated with symbiotic microorganisms such as legumes with nitrogen fixing rhizobia, grasses and legumes with phosphorus-solubilizing arbuscular mycorrhizal fungi, and cool temperate grasses with fungal anti-herbivorous alkaloid-producing Neotyphodium endophytes and metabolomics studies have shown that these associations can significantly affect the metabolic composition of forage plants. The combination of genetics and metabolomics, also known as genetical metabolomics can be a powerful tool to identify genetic regions related to specific metabolites or metabolic profiles, but this approach has not been widely adopted for forages yet, and we argue here that more studies are needed to improve our chances of success in forage breeding.

Conclusions

Metabolomics combined with other ‘-omics’ technologies and genome sequencing can be invaluable tools for large-scale geno- and phenotyping of breeding populations, although the implementation of these approaches in forage breeding programmes still lags behind. The majority of studies using metabolomics approaches have been performed with model species or cereals and findings from these studies are not easily translated to forage species. To be most effective these approaches should be accompanied by whole-plant physiology and proof of concept (modelling) studies. Wider considerations of possible consequences of novel traits on the fitness of new cultivars and symbiotic associations need also to be taken into account.     

Leaf and root respiration of Lolium perenne populations selected for contrasting leaf respiration rates are affected by intra- and interpopulation interactions

Autotoxicity and allelopathy affect the yield of GL66 and GL72, two populations of perennial ryegrass (Lolium perenne L. cv. S23) that were originally selected for contrasting rates of mature leaf dark respiration, but under conditions where allelopathic effects could not occur and autotoxic effects were minimal. To investigate if the respiration properties are also affected, the populations were subjected to two conditions (monoculture and mixed culture) and two treatments (grown in `renewed', i.e. solution completely replaced every week, and `replenished', i.e. nutrients added to the solution every week, to compensate for the nutrients that had been absorbed from the solution). When plants were grown in mixed culture with a `renewed' nutrient solution, the rate of mature leaf respiration in the absence of inhibitors was higher in GL66 than in GL72. In GL66, this rate was unaffected by condition or treatment, whereas in GL72 it increased in the `replenished' cultures, due to autotoxic and allelopathic effects. In contrast with GL66, for GL72 the effect of an inhibitor of the alternative path (salicylhydroxamic acid, SHAM) differed between conditions and treatments. Effects of an inhibitor of the cytochrome path (KCN) were affected by treatment in both populations. The increase in the rate of root respiration of GL66 due to autotoxic and allelopathic effects was accompanied by a higher rate of the SHAM-resistant component, i.e. maximum activity of the cytochrome path. Roots of GL72 showed a higher control respiration rate and a higher rate of the SHAM-resistant component when long-term accumulation of root exudates was allowed to occur in mixed cultures (allelopathy). We conclude that allelochemicals can modify both leaf and root respiration and that there is no consistent correlation between yield and respiration for GL66 and GL72 in the present study. This shows that selection for low rates of mature leaf respiration is not an appropriate method to select for high-yielding cultivars in perennial ryegrass.     

Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.     

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.     

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.¹ The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.