Zhibiao Nan

Global transcriptome sequencing using the Illumina platform and the development of EST-SSR markers in autotetraploid alfalfa.

Background Alfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. The large size and complexity of the alfalfa genome has delayed the development of genomic resources for alfalfa research. Second-generation Illumina transcriptome sequencing is an efficient method for generating a global transcriptome sequence dataset for gene discovery and molecular marker development in alfalfa. Methodology/Principal Findings More than 28 million sequencing reads (5.64 Gb of clean nucleotides) were generated by Illumina paired-end sequencing from 15 different alfalfa tissue samples. In total, 40,433 unigenes with an average length of 803 bp were obtained by de novo assembly. Based on a sequence similarity search of known proteins, a total of 36,684 (90.73%) unigenes were annotated. In addition, 1,649 potential EST-SSRs were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to amplify sequences in initial screening tests, and 27 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 10 alfalfa accessions. Conclusions/Significance The present study provided global sequence data for autotetraploid alfalfa and demonstrates the Illumina platform is a fast and effective approach to EST-SSR markers development in alfalfa. The use of these transcriptome datasets will serve as a valuable public information platform to accelerate studies of the alfalfa genome.

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.

Migration, remittances, and agricultural productivity in small farming systems in Northwest China

Purpose - The purpose of this study is to explore the relationship between migration, remittances and agricultural productivity by applying the new economics of labor migration model in the context of north-west China. The specific objectives are to examine the impacts of rural out-migration on agricultural productivity in various farming systems, and whether remittances have been reinvested in agriculture. Design/methodology/approach - Cross-sectional household survey data from three townships were analyzed with the three-stage least squares (3SLS) regression model. Findings - In multi-cropping small farming systems, at least in the short run, the loss resulting from losing family labour on lower-return grain crop production is likely to be offset by the gain from investing in capital-intensive and profitable cash crop production. Originality/value - This study provides empirical evidence for the MELM theory. It expands Taylor

Role of the lens in physical dormancy in seeds of Sophora alopecuroides L. (Fabaceae) from north-west China

Abstract Although many studies have been done on seeds with physical dormancy, i.e. water-impermeable seed or fruit coat, the primary site of water entry into seeds after dormancy-breaking treatments is still controversial. In this work, the role of the lens in physical dormancy breaking was examined. The present study investigated the primary site of water entry and changes in seed-surface features for seeds of Sophora alopecuroides L. following various periods of scarification with sulfuric acid, hot-water treatments, and exposure to field conditions. Most seed coats first cracked in the hilum when pre-treated with sulfuric acid and after field exposure, but they cracked in both the hilum and extrahilar regions when treated with hot water. After pre-treatment, seeds first became slowly permeable in the hilum or extrahilar region and not in the lens. Acid scarification for 35 or 50 min caused the lens to crack, thus causing seeds to enter a fast imbibition stage. Seeds exposed to field conditions had further damage to the hilum, causing them to enter a fast imbibition stage. Imbibition time is a key point that determines the primary site of water entry into seed, and at least in part explains various results and interpretations about the role of the lens in physical dormancy in previous research.

New agricultural practices in the Loess Plateau of China do not reduce colonisation by arbuscular mycorrhizal or root invading fungi and do not carry a yield penalty

Agricultural practices aimed to reduce soil erosion and improve crop yield have been suggested to influence the activity of arbuscular mycorrhizal (AM) and root pathogenic fungi. We conducted a two-year field survey to investigate the effect of recently introduced agricultural practices on crop yield, AM colonisation and percentage isolation of root-invading fungi on the heavily eroded Loess Plateau of China. A rotation of maize (Zea mays L.), winter wheat (Triticum aestivum L.) and soybean (Glycine max L.) replaced monoculture of winter wheat. No-tillage (NT), and return of previous crop residues to the field in tilled (CTR) and non-tilled (NTR) systems replaced conventional tillage (CT). Yield, biomass and phosphorus content of the crops showed similar trends. Residue application increased yields of maize and soybean independent of tillage treatment in 2004, but only under CT in 2005. CT slightly increased maize yield. Neither residue application nor tillage treatment affected yield of wheat. None of the treatments influenced total percent isolation of root-invading fungi from wheat roots. The increase of some individual pathogenic fungi in NT did not translate into reduction of yield by disease. Importantly, the recommended practices did not have a penalty on yield while maintaining high levels of AM colonisation.

Effects of cadmium stress on seed germination and seedling growth of Elymus dahuricus infected with the Neotyphodium endophyte

Various cadmium (Cd) concentrations (0, 50, 100, 200 and 300 μmol L−1) affected Elymus dahuricus seed germination, seedling growth, antioxidative enzymes activities (AEA), and amounts of malondialdehyde (MDA) and proline present. These influences were determined for separate E. dahuricus cohorts known to be either infected (E+) or non-infected (E−) by a Neotyphodium endophyte. Under high Cd concentrations (100, 200 and 300 μmol L−1), E+ specimens showed a significantly (P<0.05) higher germination rate and index, as well as higher values for shoot length, root length and dry biomass. However, the germination rate and index, root length and dry weight did not show a significant (P<0.05) difference under the low Cd concentrations (0 and 50 μmol L−1). AEA and proline content increased, as did MDA content, in the E+ (vs. E−) specimens under high Cd concentrations. There was no significant (P>0.05) difference under low Cd concentrations. Endophyte infection was concluded to be of benefit to E. dahuricus exposed to high Cd concentrations.

Comparative Transcriptional Profiling Provides Insights into the Evolution and Development of the Zygomorphic Flower of Vicia sativa (Papilionoideae)

Background Vicia sativa (the common vetch) possesses a predominant zygomorphic flower and belongs to the subfamily Papilionoideae, which is related to Arabidopsis thaliana in the eurosid II clade of the core eudicots. Each vetch flower consists of 21 concentrically arranged organs: the outermost five sepals, then five petals and ten stamens, and a single carpel in the center. Methodology/Principal Findings We explored the floral transcriptome to examine a genome-scale genetic model of the zygomorphic flower of vetch. mRNA was obtained from an equal mixture of six floral organs, leaves and roots. De novo assembly of the vetch transcriptome using Illumina paired-end technology produced 71,553 unigenes with an average length of 511 bp. We then compared the expression changes in the 71,553 unigenes in the eight independent organs through RNA-Seq Quantification analysis. We predominantly analyzed gene expression patterns specific to each floral organ and combinations of floral organs that corresponded to the traditional ABC model domains. Comparative analyses were performed in the floral transcriptomes of vetch and Arabidopsis, and genomes of vetch and Medicago truncatula. Conclusions/Significance Our comparative analysis of vetch and Arabidopsis showed that the vetch flowers conform to a strict ABC model. We analyzed the evolution and expression of the TCP gene family in vetch at a whole-genome level, and several unigenes specific to three different vetch petals, which might offer some clues toward elucidating the molecular mechanisms underlying floral zygomorphy. Our results provide the first insights into the genome-scale molecular regulatory network that controls the evolution and development of the zygomorphic flower in Papilionoideae.

Effects of the endophyte Epichloë festucae var. lolii of perennial ryegrass (Lolium perenne) on indicators of oxidative stress from pathogenic fungi during seed germination and seedling growth

To determine whether infection by a fungal endophyte of the genus Epichloë has a modifying influence on pathogenic fungal stress, endophyte-infected (E+) and endophyte-free (E-) seeds of perennial ryegrass (Lolium perenne), inoculated with the ryegrass pathogens Alternaria alternata, Ascochyta leptospora, Bipolaris sorokiniana, Curvularia lunata and Fusarium avenaceum were used for germination experiments and quantification of oxidative stress. Four measures of oxidative stress were used: malonaldehyde (MDA) content, peroxidases (POD) and superoxide dismutase (SOD) activity, and proline content. MDA content, an indicator of membrane lipid peroxidation damage, was significantly lower in E+ seedlings than in E- seedlings under stress from the test fungi. POD activity of E+ seedlings exposed to A. alternata, B. sorokiniana and C. lunata was significantly higher than similarly challenged E- seedlings. SOD activity of E+ seedlings was significantly higher than E- seedlings after exposure to A. alternata, B. sorokiniana, C. lunata and As. leptospora. Proline content of E+ seedlings exposed to the five pathogens was significantly higher than E- seedlings. Interestingly, these effects were present in the absence of disease symptoms apart from some root browning of E+ and E- seedlings exposed to B. sorokiniana. The results of this study showed that the seed-borne endophyte E. festucae var. lolii has potential to enhance establishment of perennial ryegrass by providing protection against pathogenic fungi during the critical early stages of seed germination and subsequent growth.

Effects of cutting frequency and height on alkaloid production in endophyte-infected drunken horse grass (Achnatherum inebrians)

An evaluation was performed on the influence of mowing height (2.5, 5.0 or 7.5 cm) and mowing frequency (weekly or fortnightly) on levels of ergot alkaloids (ergine and ergonovine) formed in drunken horse grass, Achnatherum inebrians, grown under greenhouse conditions. Samples were taken monthly and alkaloids were extracted and analyzed by reverse-phase HPLC. Alkaloid levels increased more or less linearly with plant age over the first four months following establishment. Levels were higher in samples cut fortnightly compared to those cut weekly, and were higher when plants were cut at a mowing height of 7.5 cm vs. 2.5 cm. In most cases, the highest alkaloid levels observed were almost three times those of the lowest. If plant protection applications are developed for the endophytic fungus, Neotyphodium gansuense, it will be necessary to be aware of the potential role of plant husbandry practices (e.g., defoliation frequency and intensity) for reducing or enhancing levels of plant alkaloids. Similar husbandry factors may affect alkaloid levels in other Neotyphodium-grass associations, which would be interesting for further study.

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.