Using phylogeny to investigate the origins of the Cape flora: the importance of taxonomic, gene and genome sampling strategies

Phylogenetic methods hold great promise for the reconstruction of the transition from precursor to modern flora and the identification of underlying factors which drive the process. The phylogenetic methods presently used to address the question of the origin of the Cape flora of South Africa are considered here. The sampling requirements of each of these methods, which include dating of diversifications using calibrated molecular trees, sister pair comparisons, lineage through time plots and biogeographical optimizations are reviewed. Sampling of genes, genomes and species are considered. Although increased higher-level studies and increased sampling are required for robust interpretation, it is clear that much progress is already made. It is argued that despite the remarkable richness of the flora, the Cape flora is a valuable model system to demonstrate the utility of phylogenetic methods in determining the history of a modern flora.

Molecular characterization of Iranian wheat stripe virus shows its taxonomic position as a distinct species in the genus Tenuivirus

The full lengths of three genome segments of Iranian wheat stripe virus (IWSV) were amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR) using a primer complementary to tenuivirus conserved terminal sequences. The segments were sequenced and found to comprise 3469, 2337, and 1831 nt, respectively. The gene organization of these segments is similar to that of other known tenuiviruses, each displaying an ambisense coding strategy. IWSV segments, however, are different from those of other viruses with respect to the number of nucleotides and deduced amino acid sequence for each ORF. Depending on the segment, the first 16-22 nt at the 5' end and the first 16 nt at the 3' end are highly conserved among IWSV and rice hoja blanca virus (RHBV), rice stripe virus (RSV) and maize stripe virus ( MStV). In addition, the first 15-18 nt at the 5' end are complementary to the first 16-18 nt at the 3' end. Phylogenetic analyses showed close similarity and a common ancestor for IWSV, RHBV, and Echinochloa hoja blanca virus (EHBV). These findings confirm the position of IWSV as a distinct species in the genus Tenuivirus.

The slow road to the eukaryotic genome

The eukaryotic genome is a mosaic of eubacterial and archaeal genes in addition to those unique to itself. The mosaic may have arisen as the result of two prokaryotes merging their genomes, or from genes acquired from an endosymbiont of eubacterial origin. A third possibility is that the eukaryotic genome arose from successive events of lateral gene transfer over long periods of time. This theory does not exclude the endosymbiont, but questions whether it is necessary to explain the peculiar set of eukaryotic genes. We use phylogenetic studies and reconstructions of ancestral first appearances of genes on the prokaryotic phylogeny to assess evidence for the lateral gene transfer scenario. We find that phylogenies advanced to support fusion can also arise from a succession of lateral gene transfer events. Our reconstructions of ancestral first appearances of genes reveal that the various genes that make up the eukaryotic mosaic arose at different times and in diverse lineages on the prokaryotic tree, and were not available in a single lineage. Successive events of lateral gene transfer can explain the unusual mosaic structure of the eukaryotic genome, with its content linked to the immediate adaptive value of the genes its acquired. Progress in understanding eukaryotes may come from identifying ancestral features such as the eukaryotic splicesome that could explain why this lineage invaded, or created, the eukaryoticniche.

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background: Rhizobium leguminosarum is an alpha-proteobacterial N-2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841. Results: The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes ( Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were overrepresented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens. Conclusion: Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.

Identifying adaptive genetic divergence among populations from genome scans

The identification of signatures of natural selection in genomic surveys has become an area of intense research, stimulated by the increasing ease with which genetic markers can be typed. Loci identified as subject to selection may be functionally important, and hence (weak) candidates for involvement in disease causation. They can also be useful in determining the adaptive differentiation of populations, and exploring hypotheses about speciation. Adaptive differentiation has traditionally been identified from differences in allele frequencies among different populations, summarised by an estimate of F-ST. Low outliers relative to an appropriate neutral population-genetics model indicate loci subject to balancing selection, whereas high outliers suggest adaptive (directional) selection. However, the problem of identifying statistically significant departures from neutrality is complicated by confounding effects on the distribution of F-ST estimates, and current methods have not yet been tested in large-scale simulation experiments. Here, we simulate data from a structured population at many unlinked, diallelic loci that are predominantly neutral but with some loci subject to adaptive or balancing selection. We develop a hierarchical-Bayesian method, implemented via Markov chain Monte Carlo (MCMC), and assess its performance in distinguishing the loci simulated under selection from the neutral loci. We also compare this performance with that of a frequentist method, based on moment-based estimates of F-ST. We find that both methods can identify loci subject to adaptive selection when the selection coefficient is at least five times the migration rate. Neither method could reliably distinguish loci under balancing selection in our simulations, even when the selection coefficient is twenty times the migration rate.

Studies on the effects of oxadiargyl on direct-seeded rice and Echinochloa crus-galli under drained and flooded conditions

Dose–response experiments were conducted in glasshouse pot experiments to investigate the selectivity of oxadiargyl, a recently introduced herbicide, in direct-seeded rice under both aerobic and anaerobic conditions. Crop sensitivity to oxadiargyl was comparatively greater for wet-seeded (anaerobic) than for dry-seeded rice (aerobic). Likewise, greater efficacy against Echinochloa crus-galli (L.) was also observed under anaerobic conditions. These results indicate greater activity of oxadiargyl under anaerobic conditions, but that application pre-sowing with subsequent flooding would reduce selectivity in wet-seeded rice. The results are discussed in relation to rice production in Mediterranean agriculture.

Attenuating mutations in the influenza virus genome which may increase the safety of vaccine production

Influenza virus epidemics occur on an annual basis and cause severe disease in the very young and old. The vaccine administered to high-risk groups is generated by amplifying reassortant viruses, with chronologically relevant viral surface antigens, in eggs. Every 20 years or so, influenza pandemics occur causing widespread fatality in all age groups. These viruses display novel viral surface antigens acquired from a zoonotic source, and vaccination against them poses new issues since production of large amounts of a respiratory virus containing novel surface antigens could be dangerous for those involved in manufacture. To minimise risks, it is advisable to use a virus whose genetic backbone is highly attenuated in man. Traditionally, the A/PR/8/34 strain of virus is used, however, the genetic basis of its attenuation is unclear. Cold-adapted (CA) strains of the influenza virus are all based on the H2N2 subtype, itself a virus with pandemic potential, and again the genetic basis of temperature sensitivity is not yet established. Reverse genetics technology allows us to engineer designer influenza viruses to order. Using this technology, we have been investigating mutations in several different gene segments to effectively attenuate potential vaccine strains allowing the safe production of vaccine to protect against the next pandemic. (C) 2003 Elsevier B.V. All rights reserved.

Enhancing the oxidative stability of rice crackers by addition of the ethanolic extract of phytochemicals from Cratoxylum formosum Dyer

Craloxylum formosum Dyer is consumed throughout the year as food and medicine in Thailand. It contains large amounts of chlorogenic acid and quinic acid derivatives. The antioxidative activity of the extract was studied in refined soybean oil coating on rice crackers without any seasoning. They were stored in accelerated oxidation conditions at 40 degrees C, 80% relative humidity (RH) in the dark for 18 days. The oxidative state of each sample was monitored by analyzing of the peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) as well as by odor analysis by quantitative descriptive analysis (QDA). The C formosum extract was more effective than alpha-tocopherol due to metal ions present in the crackers, which resulted in alpha-tocopherol being less effective as an antioxidant. Sensory odor attributes of rice crackers were related more closely to TBARS than to PV values by linear regression analysis. The present study indicated that C. formosum extract was a promising source of a natural food antioxidant and was effective in inhibiting lipid oxidation in rice crackers.

Kinetics of high pressure facilitated starch gelatinisation in Thai glutinous rice

The combined effect of pressure and temperature on the rate of gelatinisation of starch present in Thai glutinous rice was investigated. Pressure was found to initiate gelatinisation when its value exceeded 200 MPa at ambient temperature. On the other hand, complete gelatinisation was observed at 500 and 600 MPa at 70 degrees C, when the rice was soaked in water under these conditions for 120 min. A first-order kinetic model describing the rate of gelatinisation was developed to estimate the values of the rate constants as a function of pressure and temperature in the range: 0.1-600 MPa and 20-70 degrees C. The model, based on the well-known Arrhenius and Eyring equations, assumed the form [GRAPHICS] The constants k(0), E-a, and Delta V were found to take values: 31.19 s(-1), 37.89 kJ mol(-1) and -9.98 cm(3) mol(-1), respectively. It was further noted that the extent of gelatinisation occurring at any time, temperature and pressure, could be exclusively correlated with the grain moisture content. (c) 2006 Elsevier Ltd. All rights reserved.

High pressure induced water uptake characteristics of Thai glutinous rice

Glutinous rice (or sticky rice) has to be soaked in water over an extended period of time before cooking. Soaking provides some of the water needed for starch gelatinisation to occur during cooking. The extent of water uptake during soaking is known to be influenced by temperature. This paper explores the use of very high pressures up to 600 MPa to accelerate water uptake kinetics during soaking. Changes occurring in length, diameter and moisture content were determined as a function of soaking time, pressure and temperature. The results show that length and diameter are positively correlated with all three parameters. However, the expansion ratios are not very high: the maximum length expansion ratio observed was 1.2, while the maximum diameter expansion ratio was 1. 1. Given these low values, it was possible to model water uptake kinetics by using the well-known Fickian model applied to a finite cylinder, assuming uniform average dimensions and effective diffusion coefficient. The results showed that the overall rates of water uptake and the equilibrium moisture content increased with pressure and temperature. The effective diffusion coefficient, on the other hand, did not follow the same trend. Temperature influenced the effective diffusion coefficient below 300 MPa, but had a marginal effect at higher pressures. Moreover, the effective diffusion coefficient increased with temperature between 20 and 50 degrees C, but dropped at higher temperatures. This drop can be attributed to the gelatinisation of starch, which restricts the transport of water. Regardless, it is possible to increase the quantity of water absorbed by rice and the rate at which it is absorbed, by using high pressures and temperatures. (c) 2004 Elsevier Ltd. All rights reserved.

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

The impact of genome defense on mobile elements in Microbotryum

Repeat induced point mutation (RIP), a mechanism causing hypermutation of repetitive DNA sequences in fungi, has been described as a ‘genome defense’ which functions to inactivate mobile elements and inhibit their deleterious effects on genome stability. Here we address the interactions between RIP and transposable elements in the Microbotryum violaceum species complex. Ten strains of M. violaceum, most of which belong to different species of the fungus, were all found to contain intragenomic populations of copia-like retrotransposons. Intragenomic DNA sequence variation among the copia-like elements was analyzed for evidence of RIP. Among species with RIP, there was no significant correlation between the frequency of RIP-induced mutations and inferred transposition rate based on diversity. Two strains of M. violaceum, from two different plant species but belonging to the same fungal lineage, contained copia-like elements with very low diversity, as would result from a high transposition rate, and these were also unique in showing no evidence of the hypermutation patterns indicative of the RIP genome defense. In this species, evidence of RIP was also absent from a Class II helitron-like transposable element. However, unexpectedly the absolute repetitive element load was lower than in other strains.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting thatmost effectors represent species-specific adaptations.

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.

Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.)

Episodes of high temperature at anthesis, which in rice is the most sensitive stage to temperature, are expected to occur more frequently in future climates. The morphology of the reproductive organs and pollen number, and changes in anther protein expression, were studied in response to high temperature at anthesis in three rice (Oryza sativa L.) genotypes. Plants were exposed to 6 h of high (38 °C) and control (29 °C) temperature at anthesis and spikelets collected for morphological and proteomic analysis. Moroberekan was the most heat-sensitive genotype (18% spikelet fertility at 38 °C), while IR64 (48%) and N22 (71%) were moderately and highly heat tolerant, respectively. There were significant differences among the genotypes in anther length and width, apical and basal pore lengths, apical pore area, and stigma and pistil length. Temperature also affected some of these traits, increasing anther pore size and reducing stigma length. Nonetheless, variation in the number of pollen on the stigma could not be related to measured morphological traits. Variation in spikelet fertility was highly correlated (r=0.97, n=6) with the proportion of spikelets with ≥20 germinated pollen grains on the stigma. A 2D-gel electrophoresis showed 46 protein spots changing in abundance, of which 13 differentially expressed protein spots were analysed by MS/MALDI-TOF. A cold and a heat shock protein were found significantly up-regulated in N22, and this may have contributed to the greater heat tolerance of N22. The role of differentially expressed proteins and morphology during anther dehiscence and pollination in shaping heat tolerance and susceptibility is discussed.