Assembly and comparative analysis of the first complete mitochondrial genome of the invasive water hyacinth, Eichhornia crassipes.

Eichhornia crassipes is an aquatic plant in tropical and subtropical regions, renowned for its notorious invasive tendencies. In this study, we assembled the complete mitogenome of E. crassipes into a single circle molecule of 397,361 bp. The mitogenome has 58 unique genes, including 37 protein-coding genes (PCGs), 18 tRNA genes, three rRNA genes, and 47 % GC content. Sixteen (6.93 %) homologous fragments, ranging from 31 bp to 8548 bp, were identified, indicating the transfer of genetic material from chloroplasts to mitochondria. In addition, we detected positive selection in six PCGs (ccmB, ccmC, ccmFC, nad3, nad4 and sdh4), along with the identification of 782 RNA editing sites across 37 mt-PCGs. These findings suggest a potential contribution to the robust adaptation of this invasive plant to the stressful environment. Lastly, we inferred that phylogenetic conflicts of E. crassipes between the plastome and mitogenome may be attributed to the difference in nucleotide substitution rates between the two organelle genomes. In conclusion, our study provided vital genomic resources for further understanding the invasive mechanism of this species and exploring the dynamic evolution of mitogenomes within the monocot clade.

A reference genome of Commelinales provides insights into the commelinids evolution and global spread of water hyacinth (Pontederia crassipes).

Commelinales belongs to the commelinids clade, which also comprises Poales that includes the most important monocot species, such as rice, wheat, and maize. No reference genome of Commelinales is currently available. Water hyacinth (Pontederia crassipes or Eichhornia crassipes), a member of Commelinales, is one of the devastating aquatic weeds, although it is also grown as an ornamental and medical plant. Here, we present a chromosome-scale reference genome of the tetraploid water hyacinth with a total length of 1.22 Gb (over 95% of the estimated size) across 8 pseudochromosome pairs. With the representative genomes, we reconstructed a phylogeny of the commelinids, which supported Zingiberales and Commelinales being sister lineages of Arecales and shed lights on the controversial relationship of the orders. We also reconstructed ancestral karyotypes of the commelinids clade and confirmed the ancient commelinids genome having 8 chromosomes but not 5 as previously reported. Gene family analysis revealed contraction of disease-resistance genes during polyploidization of water hyacinth, likely a result of fitness requirement for its role as a weed. Genetic diversity analysis using 9 water hyacinth lines from 3 continents (South America, Asia, and Europe) revealed very closely related nuclear genomes and almost identical chloroplast genomes of the materials, as well as provided clues about the global dispersal of water hyacinth. The genomic resources of P. crassipes reported here contribute a crucial missing link of the commelinids species and offer novel insights into their phylogeny.

Genetic variation and clonal diversity in floating aquatic plants: Comparative genomic analysis of water hyacinth species in their native range.

Many eukaryotic organisms reproduce by sexual and asexual reproduction. Genetic diversity in populations can be strongly dependent on the relative importance of these two reproductive modes. Here, we compare the amounts and patterns of genetic diversity in related water hyacinths that differ in their propensity for clonal propagation - highly clonal Eichhornia crassipes and moderately clonal E. azurea (Pontederiaceae). Our comparisons involved genotype-by-sequencing (GBS) of 137 E. crassipes ramets from 60 locations (193,495 nucleotide sites) and 118 E. azurea ramets from 53 locations (198,343 nucleotide sites) among six hydrological basins in central South America, the native range of both species. We predicted that because of more prolific clonal propagation, E. crassipes would exhibit lower clonal diversity than E. azurea. This prediction was supported by all measures of clonal diversity that we examined. Eichhornia crassipes also had a larger excess of heterozygotes at variant sites, another signature of clonality. However, genome-wide heterozygosity was not significantly different between the species. Eichhornia crassipes had weaker spatial genetic structure and lower levels of differentiation among hydrological basins than E. azurea, probably because of higher clonality and more extensive dispersal of its free-floating life form. Our findings for E. crassipes contrast with earlier studies from the invasive range which have reported very low levels of clonal diversity and extensive geographic areas of genetic uniformity.

16S rRNA gene profiling of rhizospheric microbial community of Eichhornia crassipes.

The rhizosphere of a plant is an important interface for the plant-microbe interaction that plays a significant role in the uptake and removal of heavy metal from contaminated sites. Eichhornia crassipes is a free-floating macrophyte and a well-known metal hyperaccumulator. It is a promising plant, which harbors a diverse microbial community in its rhizosphere. Therefore it is hypothesized that it can be a good habitat for microorganisms that supports plant growth and increases its phytoremediation potential. The rhizospheric DNA was extracted from the procured plant samples. The library was prepared and sequenced using the Illumina platform. 16S rRNA data from the Next Generation Sequencing (NGS) platform was analyzed using the QIIME software package. Alpha diversity was estimated from statistical indices i.e. Shannon index, Chao1 index, and observed species. The rarefaction plots, rank abundance curve, krona graph, and heat map were generated to study the rhizospheric community in detail. Metagenome consisted of 225,408 flash reads, 185,008 non-chimeric sequences with 17,578 Operational Taxonomic Units (OTU's), and 4622 OTU's without singletons. The data of present study are available at NCBI Bioproject (PRJNA631882). The taxonomic analysis of OTU's showed that the sequences belonged to major Phyla revealing the dominance of Proteobacteria, Bacteroidetes, Cyanobacteria, and Verrucomicrobia. The most abundant Genera in the sampled rhizosphere recorded were Thiothrix and Flavobacterium.

A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence.

BACKGROUND: Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. RESULTS: A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5'-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of ß-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. CONCLUSIONS: EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

The genetic architecture of tristyly and its breakdown to self-fertilization.

The floral polymorphism tristyly involves three style morphs with a reciprocal arrangement of stigma and anther heights governed by two diallelic loci (S and M). Tristyly functions to promote cross-pollination, but modifications to stamen position commonly cause transitions to selfing. Here, we integrate whole-genome sequencing and genetic mapping to investigate the genetic architecture of the M locus and the genetic basis of independent transitions to selfing in tristylous Eichhornia paniculata. We crossed independently derived semi-homostylous selfing variants of the long- and mid-styled morph fixed for alternate alleles at the M locus (ssmm and ssMM, respectively), and backcrossed the F1 to the parental ssmm genotype. We phenotyped and genotyped 462 backcross progeny using 1450 genotyping-by-sequencing (GBS) markers and performed composite interval mapping to identify quantitative trait loci (QTL) governing style-length and anther-height variation. A QTL associated with the primary style-morph differences (style length and anther height) mapped to linkage group 5 and spanned ~13-27.5 Mbp of assembled sequence. Bulk segregant analysis identified 334 genes containing SNPs potentially linked to the M locus. The stamen modifications characterizing each selfing variant were governed by loci on different linkage groups. Our results provide an important step towards identifying the M locus and demonstrate that transitions to selfing have originated by independent sets of mating-system modifier genes unlinked to the M locus, a pattern inconsistent with a recombinational origin of selfing variants at a putative supergene.

Expression of Echmr gene from Eichhornia offers multiple stress tolerance to Cd sensitive Escherichia coli Δgsh mutants.

The detoxification of heavy metals frequently involves conjugation to glutathione prior to compartmentalization and eflux in higher plants. We have expressed a heavy metal stress responsive (Echmr) gene from water hyacinth, which conferred tolerance to Cd sensitive Escherichia coli Δgsh mutants against heavy metals and abiotic stresses. The recombinant E. coli Δgsh mutant cells showed better growth recovery and survival than control cells under Cd (200 µM), Pb(200 µM), heat shock (50 °C), cold stress at 4 °C for 4 h, and UV-B (20 min) exposure. The enhanced expression of Echmr gene revealed by northern analysis during above stresses further advocates its role in multi-stress tolerance. Heterologous expression of EcHMR from Eichhornia rescued Cd(2+) sensitive E. coli mutants from Cd(2+) toxicity and induced better recovery post abiotic stresses. This may suggests a possible role of Echmr in Cd(II) and desiccation tolerance in plants for enhanced stress response.

Recent mating-system evolution in Eichhornia is accompanied by cis-regulatory divergence.

The evolution of predominant self-fertilization from cross-fertilization in plants is accompanied by diverse changes to morphology, ecology and genetics, some of which likely result from regulatory changes in gene expression. We examined changes in gene expression during early stages in the transition to selfing in populations of animal-pollinated Eichhornia paniculata with contrasting mating patterns. We crossed plants from outcrossing and selfing populations and tested for the presence of allele-specific expression (ASE) in floral buds and leaf tissue of F1 offspring, indicative of cis-regulatory changes. We identified 1365 genes exhibiting ASE in floral buds and leaf tissue. These genes preferentially expressed alleles from outcrossing parents. Moreover, we found evidence that genes exhibiting ASE had a greater nonsynonymous diversity compared to synonymous diversity in the selfing parents. Our results suggest that the transition from outcrossing to high rates of self-fertilization may have the potential to shape the cis-regulatory genomic landscape of angiosperm species, but that the changes in ASE may be moderate, particularly during the early stages of this transition.

Revealing crosstalk of plant and fungi in the symbiotic roots of sewage-cleaning Eichhornia crassipes using direct de novo metatranscriptomic analysis.

Cultivation and environmental changes can induce development of novel phenotypes in plants. For example, the root morphology of cultivated purple root Eichhornia crassipes differs remarkably from normal Eichhornia crassipes and also shows an enhanced ability to absorb heavy metal from groundwater. However, the changes in gene expression associated with these processes are unknown because of the lack of information on its large and unsequenced genome and its complex plant-rhizosphere symbiotic system. To investigate these gene expression changes, we applied a new strategy, direct de novo metatranscriptome analysis. Using this approach, we assembled the metatranscriptome of the entire rhizosphere and identified species-specific differentially expressed genes (DEGs) via hyper-accurate algorithms, showing a polarized plant/fungus distribution: the plant genes were responsible for morphological changes to the root system, offering a greater volume and surface area that hosts more fungi; while genes associated with heavy metal response in the fungus Fusarium were upregulated more than 3600-fold. These results suggested a distinct and synergistic functional response by the plant and fungal transcriptomes, indicating significant plant/fungal crosstalk during environmental changes. This study demonstrates that the metatranscriptomic approach adopted here offers a cost-efficient strategy to study symbiosis systems without the need for a priori genomic knowledge.

Molecular characterization of the endophytic fungal community associated with Eichhornia azurea (Kunth) and Eichhornia crassipes (Mart.) (Pontederiaceae) native to the Upper Paraná River floodplain, Brazil.

Endophytic fungi live in the interior of healthy plants without causing them any damage. These fungi are of biotechnological interest; they may be used in the biological control of pests and plant diseases, and in the pharmaceutical industry. The aquatic macrophytes Eichhornia azurea (Kunth) and Eichhornia crassipes (Mart.) belong to the Pontederiaceae family. The first is a fixed-floating species and the second is a free-floating species that is known for its phytoremediation potential. The fungal endophytes associated with the leaves of E. azurea and E. crassipes, native to the Upper Paraná River floodplain, Brazil, were isolated. The sequencing of the ITS1-5.8S-ITS2 region of ribosomal DNA was performed and the nucleotide sequences obtained were compared with those available in the GenBank database for the molecular identification of the isolates. The construction of phylogenetic trees was performed using the MEGA5 software. The results showed that high colonization frequencies were obtained from the 610 foliar fragments sampled from each plant: 87.86% for E. azurea and 88.85% for E. crassipes. At the genus level, it was possible to identify 19 fungal endophytes belonging to the genera Alternaria, Bipolaris, Cercospora, Diaporthe, Gibberella, Pestalotiopsis, Plectosphaerella, Phoma, and Saccharicola. Two other endophytes were identified at the species level (Microsphaeropsis arundinis). Genera Bipolaris, Cercospora, Microsphaeropsis, and Phoma were found as endophytes in the two macrophytes and the other genera were host-specific, being isolated from only one macrophyte, proving that there is a small difference in the endophytic diversity of the two Eichhornia species analyzed.

Physiological and proteomics analyses reveal the mechanism of Eichhornia crassipes tolerance to high-concentration cadmium stress compared with Pistia stratiotes.

Cadmium (Cd) pollution is an environmental problem worldwide. Phytoremediation is a convenient method of removing Cd from both soil and water, but its efficiency is still low, especially in aquatic environments. Scientists have been trying to improve the ability of plants to absorb and accumulate Cd based on interactions between plants and Cd, especially the mechanism by which plants resist Cd. Eichhornia crassipes and Pistia stratiotes are aquatic plants commonly used in the phytoremediation of heavy metals. In the present study, we conducted physiological and biochemical analyses to compare the resistance of these two species to Cd stress at 100 mg/L. E. crassipes showed stronger resistance and was therefore used for subsequent comparative proteomics to explore the potential mechanism of E. crassipes tolerance to Cd stress at the protein level. The expression patterns of proteins in different functional categories revealed that the physiological activities and metabolic processes of E. crassipes were affected by exposure to Cd stress. However, when some proteins related to these processes were negatively inhibited, some analogous proteins were induced to compensate for the corresponding functions. As a result, E. crassipes could maintain more stable physiological parameters than P. stratiotes. Many stress-resistance substances and proteins, such as proline and heat shock proteins (HSPs) and post translational modifications, were found to be involved in the protection and repair of functional proteins. In addition, antioxidant enzymes played important roles in ROS detoxification. These findings will facilitate further understanding of the potential mechanism of plant response to Cd stress at the protein level.

The evolution of selfing is accompanied by reduced efficacy of selection and purging of deleterious mutations.

The transition from outcrossing to selfing is predicted to reduce the genome-wide efficacy of selection because of the lower effective population size (Ne) that accompanies this change in mating system. However, strongly recessive deleterious mutations exposed in the homozygous backgrounds of selfers should be under strong purifying selection. Here, we examine estimates of the distribution of fitness effects (DFE) and changes in the magnitude of effective selection coefficients (Nes) acting on mutations during the transition from outcrossing to selfing. Using forward simulations, we investigated the ability of a DFE inference approach to detect the joint influence of mating system and the dominance of deleterious mutations on selection efficacy. We investigated predictions from our simulations in the annual plant Eichhornia paniculata, in which selfing has evolved from outcrossing on multiple occasions. We used range-wide sampling to generate population genomic datasets and identified nonsynonymous and synonymous polymorphisms segregating in outcrossing and selfing populations. We found that the transition to selfing was accompanied by a change in the DFE, with a larger fraction of effectively neutral sites (Nes < 1), a result consistent with the effects of reduced Ne in selfers. Moreover, an increased proportion of sites in selfers were under strong purifying selection (Nes > 100), and simulations suggest that this is due to the exposure of recessive deleterious mutations. We conclude that the transition to selfing has been accompanied by the genome-wide influences of reduced Ne and strong purifying selection against deleterious recessive mutations, an example of purging at the molecular level.

Mercury heavy-metal-induced physiochemical changes and genotoxic alterations in water hyacinths [Eichhornia crassipes (Mart.)].

Mercury heavy metal pollution has become an important environmental problem worldwide. Accumulation of mercury ions by plants may disrupt many cellular functions and block normal growth and development. To assess mercury heavy metal toxicity, we performed an experiment focusing on the responses of Eichhornia crassipes to mercury-induced oxidative stress. E. crassipes seedlings were exposed to varying concentrations of mercury to investigate the level of mercury ions accumulation, changes in growth patterns, antioxidant defense mechanisms, and DNA damage under hydroponics system. Results showed that plant growth rate was significantly inhibited (52 %) at 50 mg/L treatment. Accumulation of mercury ion level were 1.99 mg/g dry weight, 1.74 mg/g dry weight, and 1.39 mg/g dry weight in root, leaf, and petiole tissues, respectively. There was a decreasing trend for chlorophyll a, b, and carotenoids with increasing the concentration of mercury ions. Both the ascorbate peroxidase and malondialdehyde contents showed increased trend in leaves and roots up to 30 mg/L mercury treatment and slightly decreased at the higher concentrations. There was a positive correlation between heavy metal dose and superoxide dismutase, catalase, and peroxidase antioxidative enzyme activities which could be used as biomarkers to monitor pollution in E. crassipes. Due to heavy metal stress, some of the normal DNA bands were disappeared and additional bands were amplified compared to the control in the random amplified polymorphic DNA (RAPD) profile. Random amplified polymorphic DNA results indicated that genomic template stability was significantly affected by mercury heavy metal treatment. We concluded that DNA changes determined by random amplified polymorphic DNA assay evolved a useful molecular marker for detection of genotoxic effects of mercury heavy metal contamination in plant species.

Identification of gene fragments related to nitrogen deficiency in Eichhornia crassipes (Pontederiaceae) / Identificación de fragmentos de genes relacionados con la deficiencia de nitrógeno en Eichhornia crassipes (Pontederiaceae)

Eichhornia crassipes is an aquatic plant native to the Amazon River Basin. It has become a serious weed in freshwater habitats in rivers, lakes and reservoirs both in tropical and warm temperate areas worldwide. Some research has stated that it can be used for water phytoremediation, due to its strong assimilation of nitro- gen and phosphorus, and the accumulation of heavy metals, and its growth and spread may play an important role in environmental ecology. In order to explore the molecular mechanism of E. crassipes to responses to nitrogen deficiency, we constructed forward and reversed subtracted cDNA libraries for E. crassipes roots under nitrogen deficient condition using a suppressive subtractive hybridization (SSH) method. The forward subtraction included 2 100 clones, and the reversed included 2 650 clones. One thousand clones were randomly selected from each library for sequencing. About 737 (527 unigenes) clones from the forward library and 757 (483 unigenes) clones from the reversed library were informative. Sequence BlastX analysis showed that there were more transporters and adenosylhomocysteinase-like proteins in E. crassipes cultured in nitrogen deficient medium; while, those cultured in nitrogen replete medium had more proteins such as UBR4-like e3 ubiquitin- protein ligase and fasciclin-like arabinogalactan protein 8-like, as well as more cytoskeletal proteins, including actin and tubulin. Cluster of Orthologous Group (COG) analysis also demonstrated that in the forward library, the most ESTs were involved in coenzyme transportation and metabolism. In the reversed library, cytoskeletal ESTs were the most abundant. Gene Ontology (GO) analysis categories demonstrated that unigenes involved in binding, cellular process and electron carrier were the most differentially expressed unigenes between the forward and reversed libraries. All these results suggest that E. crassipes can respond to different nitrogen status by efficiently regulating and controlling some transporter gene expressions, certain metabolism processes, specific signal transduction pathways and cytoskeletal construction.

Se ha convertido en una maleza importante en hábitats de agua dulce en ríos, lagos y embalses, tanto en zonas tropicales como templadas de todo el mundo. Algunas investigaciones han indicado que se puede utilizar para la fitorremediación de agua, debido a su fuerte asimilación de nitrógeno y fósforo, y la acumulación de metales pesados, su crecimiento y propagación puede desempeñar un papel importante en la ecología ambiental. Con el fin de explorar el mecanismo molecular de respuesta a la deficiencia de nitrógeno en E. crassipes, se construyeron bibliotecas de cDNA mediante síntesis adelantada y retrasada para raíces de E. crassipes en condiciones de deficiencia de nitrógeno mediante el método de hibridación supresiva sustractiva (SSH). Para este estudio se utilizaron 2 100 clones de síntesis adelantada y 2 650 de síntesis retrasada. De la biblioteca se escogieron al azar mil clones, 737 (527 unigenes) de síntesis adelanta- da y 757 (483 unigenes) de síntesis retrasada que fueron informativos. El análisis BLASTX mostró que había más transportadores y proteínas adenosilhomocisteinasa en E. crassipes cultivadas en un medio deficiente de nitrógeno; mientras que las cultivadas en un medio repleto de nitróge- no tenían más proteínas como UBR4 e3 ubiquitina-proteína ligasa y la proteína arabinogalactano 8 tipo fasciclina, así como otras proteínas del citoesqueleto, incluyendo la actina y la tubulina. Clúster del Grupo Ortológico (COG) también demostró que en la biblioteca de síntesis adelan- tada, la mayoría de los marcadores de secuencia expresada (ESTs) estaban involucrados en el transporte de coenzimas y el metabolismo.

Identification of gene fragments related to nitrogen deficiency in Eichhornia crassipes (Pontederiaceae).

Eichhornia crassipes is an aquatic plant native to the Amazon River Basin. It has become a serious weed in freshwater habitats in rivers, lakes and reservoirs both in tropical and warm temperate areas worldwide. Some research has stated that it can be used for water phytoremediation, due to its strong assimilation of nitrogen and phosphorus, and the accumulation of heavy metals, and its growth and spread may play an important role in environmental ecology. In order to explore the molecular mechanism of E. crassipes to responses to nitrogen deficiency, we constructed forward and reversed subtracted cDNA libraries for E. crassipes roots under nitrogen deficient condition using a suppressive subtractive hybridization (SSH) method. The forward subtraction included 2,100 clones, and the reversed included 2,650 clones. One thousand clones were randomly selected from each library for sequencing. About 737 (527 unigenes) clones from the forward library and 757 (483 unigenes) clones from the reversed library were informative. Sequence BlastX analysis showed that there were more transporters and adenosylhomocysteinase-like proteins in E. crassipes cultured in nitrogen deficient medium; while, those cultured in nitrogen replete medium had more proteins such as UBR4-like e3 ubiquitin-protein ligase and fasciclin-like arabinogalactan protein 8-like, as well as more cytoskeletal proteins, including actin and tubulin. Cluster of Orthologous Group (COG) analysis also demonstrated that in the forward library, the most ESTs were involved in coenzyme transportation and metabolism. In the reversed library, cytoskeletal ESTs were the most abundant. Gene Ontology (GO) analysis categories demonstrated that unigenes involved in binding, cellular process and electron carrier were the most differentially expressed unigenes between the forward and reversed libraries. All these results suggest that E. crassipes can respond to different nitrogen status by efficiently regulating and controlling some transporter gene expressions, certain metabolism processes, specific signal transduction pathways and cytoskeletal construction.

Genomic consequences of transitions from cross- to self-fertilization on the efficacy of selection in three independently derived selfing plants.

BACKGROUND: Transitions from cross- to self-fertilization are associated with increased genetic drift rendering weakly selected mutations effectively neutral. The effect of drift is predicted to reduce selective constraints on amino acid sequences of proteins and relax biased codon usage. We investigated patterns of nucleotide variation to assess the effect of inbreeding on the accumulation of deleterious mutations in three independently evolved selfing plants. Using high-throughput sequencing, we assembled the floral transcriptomes of four individuals of Eichhornia (Pontederiaceae); these included one outcrosser and two independently derived selfers of E. paniculata, and E. paradoxa, a selfing outgroup. The dataset included ~8000 loci totalling ~3.5 Mb of coding DNA. RESULTS: Tests of selection were consistent with purifying selection constraining evolution of the transcriptome. However, we found an elevation in the proportion of non-synonymous sites that were potentially deleterious in the E. paniculata selfers relative to the outcrosser. Measurements of codon usage in high versus low expression genes demonstrated reduced bias in both E. paniculata selfers. CONCLUSIONS: Our findings are consistent with a small reduction in the efficacy of selection on protein sequences associated with transitions to selfing, and reduced selection in selfers on synonymous changes that influence codon usage.

De novo sequence assembly and characterization of the floral transcriptome in cross- and self-fertilizing plants.

BACKGROUND: The shift from cross-fertilization to predominant self-fertilization is among the most common evolutionary transitions in the reproductive biology of flowering plants. Increased inbreeding has important consequences for floral morphology, population genetic structure and genome evolution. The transition to selfing is usually characterized by a marked reduction in flower size and the loss of traits involved in pollinator attraction and the avoidance of self-fertilization. Here, we use short-read sequencing to assemble, de novo, the floral transcriptomes of three genotypes of Eichhornia paniculata, including an outcrosser and two genotypes from independently derived selfers, and a single genotype of the sister species E. paradoxa. By sequencing mRNA from tissues sampled at various stages of flower development, our goal was to sequence and assemble the floral transcriptome and identify differential patterns of gene expression. RESULTS: Our 24 Mbp assembly resulted in ~27,000 contigs that averaged ~900 bp in length. All four genotypes had highly correlated gene expression, but the three E. paniculata genotypes were more correlated with one another than each was to E. paradoxa. Our analysis identified 269 genes associated with floral development, 22 of which were differentially expressed in selfing lineages relative to the outcrosser. Many of the differentially expressed genes affect floral traits commonly altered in selfing plants and these represent a set of potential candidate genes for investigating the evolution of the selfing syndrome. CONCLUSIONS: Our study is among the first to demonstrate the use of Illumina short read sequencing for de novo transcriptome assembly in non-model species, and the first to implement this technology for comparing floral transcriptomes in outcrossing and selfing plants.

Genetic uniformity characterizes the invasive spread of water hyacinth (Eichhornia crassipes), a clonal aquatic plant.

Aquatic plant invasions are often associated with long-distance dispersal of vegetative propagules and prolific clonal reproduction. These reproductive features combined with genetic bottlenecks have the potential to severely limit genetic diversity in invasive populations. To investigate this question we conducted a global scale population genetic survey using amplified fragment length polymorphism markers of the world's most successful aquatic plant invader -Eichhornia crassipes (water hyacinth). We sampled 1140 ramets from 54 populations from the native (South America) and introduced range (Asia, Africa, Europe, North America, Central America and the Caribbean). Although we detected 49 clones, introduced populations exhibited very low genetic diversity and little differentiation compared with those from the native range, and approximately 80% of introduced populations were composed of a single clone. A widespread clone ('W') detected in two Peruvian populations accounted for 70.9% of the individuals sampled and dominated in 74.5% of the introduced populations. However, samples from Bangladesh and Indonesia were composed of different genotypes, implicating multiple introductions to the introduced range. Nine of 47 introduced populations contained clonal diversity suggesting that sexual recruitment occurs in some invasive sites where environmental conditions favour seedling establishment. The global patterns of genetic diversity in E. crassipes likely result from severe genetic bottlenecks during colonization and prolific clonal propagation. The prevalence of the 'W' genotype throughout the invasive range may be explained by stochastic sampling, or possibly because of pre-adaptation of the 'W' genotype to tolerate low temperatures.

Mating-system variation, demographic history and patterns of nucleotide diversity in the Tristylous plant Eichhornia paniculata.

Inbreeding in highly selfing populations reduces effective size and, combined with demographic conditions associated with selfing, this can erode genetic diversity and increase population differentiation. Here we investigate the role that variation in mating patterns and demographic history play in shaping the distribution of nucleotide variation within and among populations of the annual neotropical colonizing plant Eichhornia paniculata, a species with wide variation in selfing rates. We sequenced 10 EST-derived nuclear loci in 225 individuals from 25 populations sampled from much of the geographic range and used coalescent simulations to investigate demographic history. Highly selfing populations exhibited moderate reductions in diversity but there was no significant difference in variation between outcrossing and mixed mating populations. Population size interacted strongly with mating system and explained more of the variation in diversity within populations. Bayesian structure analysis revealed strong regional clustering and selfing populations were highly differentiated on the basis of an analysis of F(st). There was no evidence for a significant loss of within-locus linkage disequilibrium within populations, but regional samples revealed greater breakdown in Brazil than in selfing populations from the Caribbean. Coalescent simulations indicate a moderate bottleneck associated with colonization of the Caribbean from Brazil approximately 125,000 years before the present. Our results suggest that the recent multiple origins of selfing in E. paniculata from diverse outcrossing populations result in higher diversity than expected under long-term equilibrium.

Isolation of a low-sulfur tolerance gene from Eichhornia crassipes using a functional gene-mining approach.

Genes enhancing nutrient utilization efficiency are needed for crop improvement. Here, we report the isolation of a gene conferring low-sulfur tolerance from water hyacinth (Eichhornia crassipes) using a functional gene-mining method. In doing this, an entry cDNA library was constructed from the roots of nutrient-starved water hyacinth using recombination cloning and subsequently shuttled into the plant transformation- and expression-ready vector. The plant transformation- and expression-ready library was transferred into Arabidopsis and a seed library of 50,000 independent transgenic lines was generated. Three transgenic lines with enhanced low-sulfur tolerance were isolated from the seed library. One of the transgenic lines, shl143-1, with improved tolerance to sulfate deficiency and an improved root system was further analyzed. It was found that a water hyacinth jacalin-related lectin gene (EcJRL-1) was overexpressed in shl143-1. Recapitulation analysis confirmed that the overexpression of the EcJRL-1 cDNA caused the phenotype. Therefore, this study demonstrates that a jacalin-related lectin is involved in root elongation under sulfur-deficient conditions.