All roads lead to weediness: Patterns of genomic divergence reveal extensive recurrent weedy rice origins from South Asian Oryza.

Weedy rice (Oryza spp.), a weedy relative of cultivated rice (O. sativa), infests and persists in cultivated rice fields worldwide. Many weedy rice populations have evolved similar adaptive traits, considered part of the 'agricultural weed syndrome', making this an ideal model to study the genetic basis of parallel evolution. Understanding parallel evolution hinges on accurate knowledge of the genetic background and origins of existing weedy rice groups. Using population structure analyses of South Asian and US weedy rice, we show that weeds in South Asia have highly heterogeneous genetic backgrounds, with ancestry contributions both from cultivated varieties (aus and indica) and wild rice. Moreover, the two main groups of weedy rice in the USA, which are also related to aus and indica cultivars, constitute a separate origin from that of Asian weeds. Weedy rice populations in South Asia largely converge on presence of red pericarps and awns and on ease of shattering. Genomewide divergence scans between weed groups from the USA and South Asia, and their crop relatives are enriched for loci involved in metabolic processes. Some candidate genes related to iconic weedy traits and competitiveness are highly divergent between some weed-crop pairs, but are not shared among all weed-crop comparisons. Our results show that weedy rice is an extreme example of recurrent evolution, and suggest that most populations are evolving their weedy traits through different genetic mechanisms.

Population Structure in the Model Grass Is Highly Correlated with Flowering Differences across Broad Geographic Areas.

The small, annual grass (L.) Beauv., a close relative of wheat ( L.) and barley ( L.), is a powerful model system for cereals and bioenergy grasses. Genome-wide association studies (GWAS) of natural variation can elucidate the genetic basis of complex traits but have been so far limited in by the lack of large numbers of well-characterized and sufficiently diverse accessions. Here, we report on genotyping-by-sequencing (GBS) of 84 , seven , and three accessions with diverse geographic origins including Albania, Armenia, Georgia, Italy, Spain, and Turkey. Over 90,000 high-quality single-nucleotide polymorphisms (SNPs) distributed across the Bd21 reference genome were identified. Our results confirm the hybrid nature of the genome, which appears as a mosaic of -like and -like sequences. Analysis of more than 50,000 SNPs for the accessions revealed three distinct, genetically defined populations. Surprisingly, these genomic profiles are associated with differences in flowering time rather than with broad geographic origin. High levels of differentiation in loci associated with floral development support the differences in flowering phenology between populations. Genome-wide association studies combining genotypic and phenotypic data also suggest the presence of one or more photoperiodism, circadian clock, and vernalization genes in loci associated with flowering time variation within populations. Our characterization elucidates genes underlying population differences, expands the germplasm resources available for , and illustrates the feasibility and limitations of GWAS in this model grass.

More than one way to evolve a weed: parallel evolution of US weedy rice through independent genetic mechanisms.

Many different crop species were selected for a common suite of 'domestication traits', which facilitates their use for studies of parallel evolution. Within domesticated rice (Oryza sativa), there has also been independent evolution of weedy strains from different cultivated varieties. This makes it possible to examine the genetic basis of parallel weed evolution and the extent to which this process occurs through shared genetic mechanisms. We performed comparative QTL mapping of weediness traits using two recombinant inbred line populations derived from crosses between an indica crop variety and representatives of each of the two independently evolved weed strains found in US rice fields, strawhull (S) and blackhull awned (B). Genotyping-by-sequencing provided dense marker coverage for linkage map construction (average marker interval <0.25 cM), with 6016 and 13 730 SNPs mapped in F5 lines of the S and B populations, respectively. For some weediness traits (awn length, hull pigmentation and pericarp pigmentation), QTL mapping and sequencing of underlying candidate genes confirmed that trait variation was largely attributable to individual loci. However, for more complex quantitative traits (including heading date, panicle length and seed shattering), we found multiple QTL, with little evidence of shared genetic bases between the S and B populations or across previous studies of weedy rice. Candidate gene sequencing revealed causal genetic bases for 8 of 27 total mapped QTL. Together these findings suggest that despite the genetic bottleneck that occurred during rice domestication, there is ample genetic variation in this crop to allow agricultural weed evolution through multiple genetic mechanisms.

The impact of herbicide-resistant rice technology on phenotypic diversity and population structure of United States weedy rice.

The use of herbicide-resistant (HR) Clearfield rice (Oryza sativa) to control weedy rice has increased in the past 12 years to constitute about 60% of rice acreage in Arkansas, where most U.S. rice is grown. To assess the impact of HR cultivated rice on the herbicide resistance and population structure of weedy rice, weedy samples were collected from commercial fields with a history of Clearfield rice. Panicles from each weedy type were harvested and tested for resistance to imazethapyr. The majority of plants sampled had at least 20% resistant offspring. These resistant weeds were 97 to 199 cm tall and initiated flowering from 78 to 128 d, generally later than recorded for accessions collected prior to the widespread use of Clearfield rice (i.e. historical accessions). Whereas the majority (70%) of historical accessions had straw-colored hulls, only 30% of contemporary HR weedy rice had straw-colored hulls. Analysis of genotyping-by-sequencing data showed that HR weeds were not genetically structured according to hull color, whereas historical weedy rice was separated into straw-hull and black-hull populations. A significant portion of the local rice crop genome was introgressed into HR weedy rice, which was rare in historical weedy accessions. Admixture analyses showed that HR weeds tend to possess crop haplotypes in the portion of chromosome 2 containing the ACETOLACTATE SYNTHASE gene, which confers herbicide resistance to Clearfield rice. Thus, U.S. HR weedy rice is a distinct population relative to historical weedy rice and shows modifications in morphology and phenology that are relevant to weed management.

Comparative genomic analysis of Geobacter sulfurreducens KN400, a strain with enhanced capacity for extracellular electron transfer and electricity production.

BACKGROUND: A new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved. RESULTS: Both genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail. CONCLUSIONS: The improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear to be unchanged, but cytochrome folding, localization, and redox potentials may all be affected, which would alter the electrical connection between the cell and the substrate.

Genome sequence of the deltaproteobacterial strain NaphS2 and analysis of differential gene expression during anaerobic growth on naphthalene.

BACKGROUND: Anaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined. RESULTS: Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds. CONCLUSION: This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring.

The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments.

BACKGROUND: Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species. RESULTS: Annotation of the genome sequence of G. bemidjiensis indicates several differences in metabolism compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire, detoxify and avoid oxygen. CONCLUSION: Overall, the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G. bemidjiensis than from the non-subsurface model species studied so far.

De Novo assembly of the complete genome of an enhanced electricity-producing variant of Geobacter sulfurreducens using only short reads.

State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated.We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm.The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another.

Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes.

BACKGROUND: Geobacter species grow by transferring electrons out of the cell--either to Fe(III)-oxides or to man-made substances like energy-harvesting electrodes. Study of Geobacter sulfurreducens has shown that TCA cycle enzymes, inner-membrane respiratory enzymes, and periplasmic and outer-membrane cytochromes are required. Here we present comparative analysis of six Geobacter genomes, including species from the clade that predominates in the subsurface. Conservation of proteins across the genomes was determined to better understand the evolution of Geobacter species and to create a metabolic model applicable to subsurface environments. RESULTS: The results showed that enzymes for acetate transport and oxidation, and for proton transport across the inner membrane were well conserved. An NADH dehydrogenase, the ATP synthase, and several TCA cycle enzymes were among the best conserved in the genomes. However, most of the cytochromes required for Fe(III)-reduction were not, including many of the outer-membrane cytochromes. While conservation of cytochromes was poor, an abundance and diversity of cytochromes were found in every genome, with duplications apparent in several species. CONCLUSIONS: These results indicate there is a common pathway for acetate oxidation and energy generation across the family and in the last common ancestor. They also suggest that while cytochromes are important for extracellular electron transport, the path of electrons across the periplasm and outer membrane is variable. This combination of abundant cytochromes with weak sequence conservation suggests they may not be specific terminal reductases, but rather may be important in their heme-bearing capacity, as sinks for electrons between the inner-membrane electron transport chain and the extracellular acceptor.

Evolution from a respiratory ancestor to fill syntrophic and fermentative niches: comparative fenomics of six Geobacteraceae species.

BACKGROUND: The anaerobic degradation of organic matter in natural environments, and the biotechnical use of anaerobes in energy production and remediation of subsurface environments, both require the cooperative activity of a diversity of microorganisms in different metabolic niches. The Geobacteraceae family contains members with three important anaerobic metabolisms: fermentation, syntrophic degradation of fermentation intermediates, and anaerobic respiration. RESULTS: In order to learn more about the evolution of anaerobic microbial communities, the genome sequences of six Geobacteraceae species were analyzed. The results indicate that the last common Geobacteraceae ancestor contained sufficient genes for anaerobic respiration, completely oxidizing organic compounds with the reduction of external electron acceptors, features that are still retained in modern Geobacter and Desulfuromonas species. Evolution of specialization for fermentative growth arose twice, via distinct lateral gene transfer events, in Pelobacter carbinolicus and Pelobacter propionicus. Furthermore, P. carbinolicus gained hydrogenase genes and genes for ferredoxin reduction that appear to permit syntrophic growth via hydrogen production. The gain of new physiological capabilities in the Pelobacter species were accompanied by the loss of several key genes necessary for the complete oxidation of organic compounds and the genes for the c-type cytochromes required for extracellular electron transfer. CONCLUSION: The results suggest that Pelobacter species evolved parallel strategies to enhance their ability to compete in environments in which electron acceptors for anaerobic respiration were limiting. More generally, these results demonstrate how relatively few gene changes can dramatically transform metabolic capabilities and expand the range of environments in which microorganisms can compete.

Mitochondrial DNA suggests at least 11 origins of parasitism in angiosperms and reveals genomic chimerism in parasitic plants.

BACKGROUND: Some of the most difficult phylogenetic questions in evolutionary biology involve identification of the free-living relatives of parasitic organisms, particularly those of parasitic flowering plants. Consequently, the number of origins of parasitism and the phylogenetic distribution of the heterotrophic lifestyle among angiosperm lineages is unclear. RESULTS: Here we report the results of a phylogenetic analysis of 102 species of seed plants designed to infer the position of all haustorial parasitic angiosperm lineages using three mitochondrial genes: atp1, coxI, and matR. Overall, the mtDNA phylogeny agrees with independent studies in terms of non-parasitic plant relationships and reveals at least 11 independent origins of parasitism in angiosperms, eight of which consist entirely of holoparasitic species that lack photosynthetic ability. From these results, it can be inferred that modern-day parasites have disproportionately evolved in certain lineages and that the endoparasitic habit has arisen by convergence in four clades. In addition, reduced taxon, single gene analyses revealed multiple horizontal transfers of atp1 from host to parasite lineage, suggesting that parasites may be important vectors of horizontal gene transfer in angiosperms. Furthermore, in Pilostyles we show evidence for a recent host-to-parasite atp1 transfer based on a chimeric gene sequence that indicates multiple historical xenologous gene acquisitions have occurred in this endoparasite. Finally, the phylogenetic relationships inferred for parasites indicate that the origins of parasitism in angiosperms are strongly correlated with horizontal acquisitions of the invasive coxI group I intron. CONCLUSION: Collectively, these results indicate that the parasitic lifestyle has arisen repeatedly in angiosperm evolutionary history and results in increasing parasite genomic chimerism over time.

Multiple major increases and decreases in mitochondrial substitution rates in the plant family Geraniaceae.

BACKGROUND: Rates of synonymous nucleotide substitutions are, in general, exceptionally low in plant mitochondrial genomes, several times lower than in chloroplast genomes, 10-20 times lower than in plant nuclear genomes, and 50-100 times lower than in many animal mitochondrial genomes. Several cases of moderate variation in mitochondrial substitution rates have been reported in plants, but these mostly involve correlated changes in chloroplast and/or nuclear substitution rates and are therefore thought to reflect whole-organism forces rather than ones impinging directly on the mitochondrial mutation rate. Only a single case of extensive, mitochondrial-specific rate changes has been described, in the angiosperm genus Plantago. RESULTS: We explored a second potential case of highly accelerated mitochondrial sequence evolution in plants. This case was first suggested by relatively poor hybridization of mitochondrial gene probes to DNA of Pelargonium hortorum (the common geranium). We found that all eight mitochondrial genes sequenced from P. hortorum are exceptionally divergent, whereas chloroplast and nuclear divergence is unexceptional in P. hortorum. Two mitochondrial genes were sequenced from a broad range of taxa of variable relatedness to P. hortorum, and absolute rates of mitochondrial synonymous substitutions were calculated on each branch of a phylogenetic tree of these taxa. We infer one major, approximately 10-fold increase in the mitochondrial synonymous substitution rate at the base of the Pelargonium family Geraniaceae, and a subsequent approximately 10-fold rate increase early in the evolution of Pelargonium. We also infer several moderate to major rate decreases following these initial rate increases, such that the mitochondrial substitution rate has returned to normally low levels in many members of the Geraniaceae. Finally, we find unusually little RNA editing of Geraniaceae mitochondrial genes, suggesting high levels of retroprocessing in their history. CONCLUSION: The existence of major, mitochondrial-specific changes in rates of synonymous substitutions in the Geraniaceae implies major and reversible underlying changes in the mitochondrial mutation rate in this family. Together with the recent report of a similar pattern of rate heterogeneity in Plantago, these findings indicate that the mitochondrial mutation rate is a more plastic character in plants than previously realized. Many molecular factors could be responsible for these dramatic changes in the mitochondrial mutation rate, including nuclear gene mutations affecting the fidelity and efficacy of mitochondrial DNA replication and/or repair and--consistent with the lack of RNA editing--exceptionally high levels of "mutagenic" retroprocessing. That the mitochondrial mutation rate has returned to normally low levels in many Geraniaceae raises the possibility that, akin to the ephemerality of mutator strains in bacteria, selection favors a low mutation rate in plant mitochondria.

Rate variation in parasitic plants: correlated and uncorrelated patterns among plastid genes of different function.

BACKGROUND: The analysis of synonymous and nonsynonymous rates of DNA change can help in the choice among competing explanations for rate variation, such as differences in constraint, mutation rate, or the strength of genetic drift. Nonphotosynthetic plants of the Orobanchaceae have increased rates of DNA change. In this study 38 taxa of Orobanchaceae and relatives were used and 3 plastid genes were sequenced for each taxon. RESULTS: Phylogenetic reconstructions of relative rates of sequence evolution for three plastid genes (rbcL, matK and rps2) show significant rate heterogeneity among lineages and among genes. Many of the non-photosynthetic plants have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not always immediate upon loss of photosynthesis. Overall there is a poor correlation of synonymous and nonsynonymous rates. There is, however, a strong correlation of synonymous rates across the 3 genes studied and the lineage-speccific pattern for each gene is strikingly similar. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way. There is a weaker correlation across genes for nonsynonymous rates. Here the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene. CONCLUSIONS: The distinctive pattern of rate increases in Orobanchaceae has at least two causes. It is clear that there is a relaxation of constraint in many (though not all) non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors.

GapCoder automates the use of indel characters in phylogenetic analysis.

BACKGROUND: Several ways of incorporating indels into phylogenetic analysis have been suggested. Simple indel coding has two strengths: (1) biological realism and (2) efficiency of analysis. In the method, each indel with different start and/or end positions is considered to be a separate character. The presence/absence of these indel characters is then added to the data set. ALGORITHM: We have written a program, GapCoder to automate this procedure. The program can input PIR format aligned datasets, find the indels and add the indel-based characters. The output is a NEXUS format file, which includes a table showing what region each indel characters is based on. If regions are excluded from analysis, this table makes it easy to identify the corresponding indel characters for exclusion. DISCUSSION: Manual implementation of the simple indel coding method can be very time-consuming, especially in data sets where indels are numerous and/or overlapping. GapCoder automates this method and is therefore particularly useful during procedures where phylogenetic analyses need to be repeated many times, such as when different alignments are being explored or when various taxon or character sets are being explored. GapCoder is currently available for Windows from http://www.home.duq.edu/~youngnd/GapCoder.

THE CONSEQUENCES OF BEING DIFFERENT: SINISTRAL COILING IN CERION.

The overwhelming predominance of dextral coiling in gastropods is an outstanding and puzzling phenomenon. A few sinistral specimens (left coiling individuals) have been found in many dextral species. Only six sinistral shells have ever been found in Cerion; we base this analysis on the five available shells. We ask whether reversed symmetry is a simple either-or switch without further consequences for shell form, or whether sinistrality engenders associated effects, making left-coiling shells unlike their dextral deme-mates in other ways. All five sinistral shells differ in features of size and coiling late in growth, leading to relatively small apertures and a slight twist in the axis of coiling. We detect and measure this effect as follows: in multivariate morphospace, sinistrals occupy peripheral positions among their dextral deme-mates; in univariate analysis, sinistrals are consistently different for a set of characters involving covariance patterns never before seen in a decade of studies on ontogenetic and age-standardized variation in dextrals; a bootstrap procedure does not recover similar patterns in randomly constituted samples of dextrals matching the true sinistral distribution; direct x-ray measures of the coiling axis detect its slight twist in sinistrals. We discuss the implications of these unsuspected associations for the issues of developmental constraint upon the evolution of morphology.