Pei Tian

Generation and analysis of expressed sequence tags in perennial ryegrass (Lolium perenne L.)

Abstract We report here the generation of 29 cDNA libraries of perennial ryegrass ( Lolium perenne ) representing a range of plant organs and developmental stages as well as the single-pass DNA sequencing of randomly selected clones to establish a genomic resource of expressed sequence tags (ESTs) for this key temperate forage grass. Over 44 000 ESTs were produced, analysed by blast searches, categorised functionally, subjected to a cluster analysis leading to the identification of a unigene set corresponding to 14 767 genes. This unigene set, representing approximately half or one third of all expressed sequences in ryegrass, was compared to the Arabidopsis and rice proteome and used to develop a unigene cDNA microarray for genome-wide gene expression analysis in grasses.

Effect of the endophyte Neotyphodium lolii on susceptibility and host physiological response of perennial ryegrass to fungal pathogens

The effect of the endophyte Neotyphodium lolii on susceptibility of perennial ryegrass (Lolium perenne) to ten fungal pathogens in detached leaves was studied. The pathogens were Alternaria alternata, Ascochyta leptospora, Bipolaris sorokiniana, Curvularia lunata, Fusarium acuminatum, F. avenaceum, F. chlamydosporum, F. solani, F. oxysporum, and Gliocladium roseum. In addition, the effect of the endophyte on four pathogens (A. alternata, B. sorokiniana, Curvularia lunata and F. avenaceum) in living plants was studied, and changes in host superoxide dismutase (SOD) or peroxidases (POD) activity were examined. The total lengths of lesions on detached leaves were greater (P < 0.05) on E- plants than on E+ plants except for A. leptospora although differences between E+ and E- were not consistently significant at all sample times (days after inoculation).The numbers of lesions were greater (P < 0.05) and the lesions were larger (P < 0.05) on intact E- plants than on intact E+ plants for all of the four pathogens. SOD enzyme activity was significantly greater (P < 0.05) in E+ plants than in E- plants only for A. alternata, C. lunata, and F. avenaceum. POD enzyme activity was significantly greater (P < 0.05) in E+ plants than in E- plants only for C. lunata, B. sorokiniana and the uninoculated control.

Genome-Wide Analysis of Codon Usage Bias in Epichloe festucae

Analysis of codon usage data has both practical and theoretical applications in understanding the basics of molecular biology. Differences in codon usage patterns among genes reflect variations in local base compositional biases and the intensity of natural selection. Recently, there have been several reports related to codon usage in fungi, but little is known about codon usage bias in Epichloë endophytes. The present study aimed to assess codon usage patterns and biases in 4870 sequences from Epichloë festucae, which may be helpful in revealing the constraint factors such as mutation or selection pressure and improving the bioreactor on the cloning, expression, and characterization of some special genes. The GC content with 56.41% is higher than the AT content (43.59%) in E. festucae. The results of neutrality and effective number of codons plot analyses showed that both mutational bias and natural selection play roles in shaping codon usage in this species. We found that gene length is strongly correlated with codon usage and may contribute to the codon usage patterns observed in genes. Nucleotide composition and gene expression levels also shape codon usage bias in E. festucae. E. festucae exhibits codon usage bias based on the relative synonymous codon usage (RSCU) values of 61 sense codons, with 25 codons showing an RSCU larger than 1. In addition, we identified 27 optimal codons that end in a G or C.

Characterisation of novel perennial ryegrass host–Neotyphodium endophyte associations

Abstract. The temperate pasture grass Lolium perenne L. is commonly found in symbiotic association with the asexual fungal endophyte Neotyphodium lolii. Levels of endophyte colonisation and alkaloid content were evaluated in associations formed by plant genotypes from cv. Bronsyn with the standard endophyte (SE) and five distinct commercial endophyte strains. Bronsyn–SE produced all of the measured alkaloids (lolitrem B, peramine, and ergovaline). Bronsyn–AR1 produced only peramine, while Bronsyn–AR37 produced none of the tested alkaloids. Bronsyn–NEA2, Bronsyn–NEA3, and Bronsyn–NEA6 produced both ergovaline and peramine. Both endophyte strain and host genotype exerted significant effects on alkaloid production. Analysis of endophyte colonisation using qPCR revealed differences between each association. With the exception of Bronsyn–AR1 and Bronsyn–NEA3, host genotype also significantly affected colonisation levels. Phenotypic performance of each association was also assessed, based on measurement of morphological traits under glasshouse conditions in hydroponic culture. Significant variation due to different endophyte and host genotypes was observed. Collectively, these studies confirm that differences in both endophyte and host genotypes contribute to host–endophyte performance in a complex interactive manner.

Advances in Research on Epichloe endophytes in Chinese Native Grasses

Epichloë fungal endophytes are broadly found in cool-season grasses. The symbiosis between these grasses and Epichloë may improve the abiotic and biotic resistance of the grass plant, but some Epichloë species produce alkaloids that are toxic for livestock. Therefore, it is important to understand the characteristics of the grass-Epichloë s symbiosis so that the beneficial aspects can be preserved and the toxic effects to livestock can be avoided. Since the 1990s, Chinese researchers have conducted a series of studies on grass-Epichloë symbiosis. In this review, we describe the current state of Epichloë endophyte research in Chinese native grasses. We found that more than 77 species of native grasses in China are associated with Epichloë endophytes. In addition, we review the effects of various Epichloë species on native grass responses to abiotic and biotic stress, phylogeny, and alkaloid production. We provide an overview of the study of Epichloë species on native grasses in China and directions for future research.

Discovery and characterisation of novel asexual Epichloë endophytes from perennial ryegrass (Lolium perenne L.)

Abstract. Asexual fungal endophytes of the genus Epichloë form mutually beneficial associations with cool-season pasture grasses such as ryegrasses (Lolium spp.). Alkaloid production by the fungus confers both beneficial (deterrence of invertebrate herbivory) and detrimental (toxicity to mammalian livestock) attributes. A few novel strains with desirable metabolite profiles have been advanced into commercial production by inoculation of perennial ryegrass cultivars. In the present study, an integrated process for discovery of novel endophytes based on exploitation of genotypic information has been designed and implemented. A survey of genetic diversity was performed on a large-scale, customised germplasm collection (containing 244 accessions) in order to identify previously uncharacterised endophyte genotypes. Preliminary qualitative metabolic profiling in the endogenous genetic background permitted elimination of undesirable combinations, and definition of a subset of priority candidates. A novel method was developed for inoculation of endophytes into meristem-culture-derived callus tissue of single genotypes from multiple perennial ryegrass cultivars, in order to allow isogenic comparisons with respect to both host and endophyte genotype. Beneficial toxin profiles were confirmed for associations formed with the grass genotypic panel, and semi-quantitative metabolite analysis provided evidence for genotype-specific effects of both host and genotype on levels of alkaloid production. Vegetative stability was also assessed over both shorter and longer terms. A final set of three prioritised candidates was obtained, two of which (belonging to the known taxa E. festucae var. lolii and LpTG-2) produce the alkaloids ergovaline and peramine. The third candidate endophyte, belonging to a putative novel taxon, solely produces representatives of an additional alkaloid class, the epoxy-janthitrems.

Stability and viability of novel perennial ryegrass host–Neotyphodium endophyte associations

Abstract. The temperate pasture grass Lolium perenne L. is commonly found in association with the fungal endophyte Neotyphodium lolii. Viability of both seed and endophyte was evaluated by inoculation of individual genotypes from the host cultivar Bronsyn with six distinct endophyte strains. The resulting populations were subjected to either long-term storage under various temperature conditions, or accelerated ageing (AA) treatments. High temperature storage was detrimental to endophyte viability. The AA variables (relative humidity [RH] and time) and subsequent temperature and duration of storage significantly (P < 0.05) affected both seed germination and endophyte viability. Significant interaction effects between the AA treatment and storage conditions were also observed. Endophyte viability following AA treatment was inversely correlated with both increasing RH and duration of treatment. Differential responses between different endophytes within the Bronsyn host were also apparent. The standard endophyte (SE) strain and the novel endophytes AR1 and AR37 exhibited higher viability than NEA2, NEA3, and NEA6 during seed storage, suggesting the importance of identification and selection for compatible symbiotic associations in agronomic varieties. The observed similarity between assessments of endophyte viability after AA treatments and following long-term storage confirmed the capacity of moderate conditions (e.g. 80% RH for 7 days or 100% RH for 4 days) to predict variation in viability between different endophyte strains.

Microdochium paspali, a new species causing seashore paspalum disease in southern China

A new species of Microdochium was identified as the causal agent of leaf blight of seashore paspalum (Paspalum vaginatum), a turf grass widely used in tropical and subtropical golf courses. In 2010 foliar necrosis and canopy thinning were observed on 11 surveyed golf courses in Hainan province, China, especially on fairways and putting greens. The infected leaves initially appeared water-soaked and dark green, rapidly faded to yellow or became chlorotic and quickly died, resulting in a sparse appearance in infected areas, leading to the disease name “sparse leaf patch.” Isolates with rich and light pink to yellow mycelia and salmon-colored pionnotes were cultured from diseased turf foliage. Pathogenicity was demonstrated by inoculating these isolates onto “seaspray” seashore paspalum. Phylogenetic analysis based on the nuc rDNA internal transcribed spacer 1–5.8S–internal transcribed spacer 2 region (ITS), translation elongation factor 1-α (TEF1-α) and β-tubulin (BenA) indicated these isolates formed as a distinct clade within Microdochium (Xylariales). Further microscopic examination demonstrated that the species was morphologically distinct from three similar species of Microdochium. The name Microdochium paspali sp. nov. is proposed for this novel fungal pathogen.

Comprehensive analysis of correlations among codon usage bias, gene expression, and substitution rate in Arachis duranensis and Arachis ipaënsis orthologs

The relationship between evolutionary rates and gene expression in model plant orthologs is well documented. However, little is known about the relationships between gene expression and evolutionary trends in Arachis orthologs. We identified 7,435 one-to-one orthologs, including 925 single-copy and 6,510 multiple-copy sequences in Arachis duranensis and Arachis ipaënsis. Codon usage was stronger for shorter polypeptides, which were encoded by codons with higher GC contents. Highly expressed coding sequences had higher codon usage bias, GC content, and expression breadth. Additionally, expression breadth was positively correlated with polypeptide length, but there was no correlation between gene expression and polypeptide length. Inferred selective pressure was also negatively correlated with both gene expression and expression breadth in all one-to-one orthologs, while positively but non-significantly correlated with gene expression in sequences with signatures of positive selection. Gene expression levels and expression breadth were significantly higher for single-copy genes than for multiple-copy genes. Similarly, the gene expression and expression breadth in sequences with signatures of purifying selection were higher than those of sequences with positive selective signatures. These results indicated that gene expression differed between single-copy and multiple-copy genes as well as sequences with signatures of positive and purifying selection.

Differential evolutionary patterns and expression levels between sex-specific and somatic tissue-specific genes in peanut

The patterns of evolution and expression of tissue-specific genes are poorly understood beyond sex-specific genes. Accordingly, we identified 3,191 tissue-specific genes and 38,745 common genes using 22 RNA-seq datasets from cultivated peanut. The expression levels of tissue-specific genes were significantly lower than those of common genes. Further, the expression levels of sex-specific genes were significantly higher than those of somatic tissue-specific genes. Among sex-specific genes, the expression levels of gynoecium-specific genes were significantly higher than those of androecium-specific genes. Function-specific genes were lacking among tissue-specific genes, and tissue-specific gene annotations overlapped among different tissues. Duplicate gene pairs were classified as homogeneous pairs expressed within the same tissue or heterogeneous pairs expressed in different tissues. Heterogeneous gene pairs evolved more rapidly than homogeneous gene pairs. In addition, somatic tissue-specific genes evolved faster than sex-specific genes. Molecular signatures of selection indicated that somatic tissue-specific genes have mainly experienced relaxed selection, while sex-specific genes have been under stronger selective constraint. Somatic tissue-specific genes had higher codon usage bias than sex-specific genes. These contrasting patterns between somatic tissue-specific and sex-specific genes provide new insights into the basic biology and evolution of peanut, an important crop.