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Background. Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis, but the mechanisms by which E. coli K1 causes meningitis are not clear. Methods. We identified 22 E. coli RS218-derived genomic islands (RDIs), using a comparative genome analysis of meningitis-causing E. coli K1 strain RS218 (O18:K1:H7) and laboratory K-12 strain MG1655. Series of RDI deletion mutants were constructed and examined for phenotypes relevant to E. coli K1 meningitis. Results. We identified 9 RDI deletion mutants (RDI 1, 4, 7, 12, 13, 16, 20, 21, and 22) that exhibited defects in meningitis development. RDI 16 and 21 mutants had profound defects in the induction of a high level of bacteremia in neonatal rats, and RDI 4 mutants exhibited a moderate defect in the induction of bacteremia. RDI 1 and 22 mutants showed defects in the ability to invade human brain microvascular endothelial cells (HBMECs), and RDI 12 mutants were defective in the ability to bind to HBMECs. RDI 13 and 20 mutants were defective in the ability to both bind to and invade HBMECs. RDI 7 mutants were defective in the induction of bacteremia and in the ability to both bind to and invade HBMECs. Conclusions. These results provide a framework for the future discovery and analysis of bacteremia and meningitis caused by E. coli K1 strain RS218. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Abstract Background The majority of nitrogen accumulating in cereal grains originates from proteins remobilised from vegetative organs. However, interactions between grain filling and remobilisation are poorly understood. We used transcriptome large-scale pyrosequencing of flag leaves, glumes and developing grains to identify cysteine peptidase and N transporter genes playing a role in remobilisation and accumulation of nitrogen in barley. Results Combination of already known and newly derived sequence information reduced redundancy, increased contig length and identified new members of cysteine peptidase and N transporter gene families. The dataset for N transporter genes was aligned with N transporter amino acid sequences of rice and Arabidopsis derived from Aramemnon database. 57 AAT, 45 NRT1/PTR and 22 OPT unigenes identified by this approach cluster to defined subgroups in the respective phylogenetic trees, among them 25 AAT, 8 NRT1/PTR and 5 OPT full-length sequences. Besides, 59 unigenes encoding cysteine peptidases were identified and subdivided into different families of the papain cysteine peptidase clade. Expression profiling of full-length AAT genes highlighted amino acid permeases as the group showing highest transcriptional activity. HvAAP2 and HvAAP6 are highly expressed in vegetative organs whereas HvAAP3 is grain-specific. Sequence similarities cluster HvAAP2 and the putative transporter HvAAP6 together with Arabidopsis transporters, which are involved in long-distance transfer of amino acids. HvAAP3 is closely related to AtAAP1 and AtAAP8 playing a role in supplying N to developing seeds. An important role in amino acid re-translocation can be considered for HvLHT1 and HvLHT2 which are specifically expressed in glumes and flag leaves, respectively. PCA and K-means clustering of AAT transcript data revealed coordinate developmental stages in flag leaves, glumes and grains. Phloem-specific metabolic compounds are proposed that might signal high grain demands for N to distantly located plant organs. Conclusions The approach identified cysteine peptidases and specific N transporters of the AAT family as obviously relevant for grain filling and thus, grain yield and quality in barley. Up to now, information is based only on transcript data. To make it relevant for application, the role of identified candidates in sink-source communication has to be analysed in more detail.

Abstract Background Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks. Results By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme. Conclusions By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.

Abstract Background Evolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome E. coli MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential. Results We analyze an extreme case of toxic plasmid clone instability, and show that random host IS element hopping, causing inactivation of the toxic cloned sequences, followed by automatic selection of the fast-growing mutants, can prevent the maintenance of a clone developed for vaccine production. Analyzing the molecular details, we identify a hydrophobic protein as the toxic byproduct of the clone, and show that IS elements spontaneously landing in the cloned fragment relieve the cell from the stress by blocking transcription of the toxic gene. Bioinformatics analysis of sequence reads from early shotgun genome sequencing projects, where clone libraries were constructed and maintained in E. coli, suggests that such IS-mediated inactivation of ectopic genes inhibiting the growth of the E. coli cloning host might happen more frequently than generally anticipated, leading to genomic instability and selection of altered clones. Conclusions Delayed genetic adaptation of clean-genome, IS-free MDS42 host improves maintenance of unstable genetic constructs, and is suggested to be beneficial in both laboratory and industrial settings.

Abstract Background Deletion of large blocks of nonessential genes that are not needed for metabolic pathways of interest can reduce the production of unwanted by-products, increase genome stability, and streamline metabolism without physiological compromise. Researchers have recently constructed a reduced-genome Escherichia coli strain MDS42 that lacks 14.3% of its chromosome. Results Here we describe the reengineering of the MDS42 genome to increase the production of the essential amino acid L-threonine. To this end, we over-expressed a feedback-resistant threonine operon (thrA*BC), deleted the genes that encode threonine dehydrogenase (tdh) and threonine transporters (tdcC and sstT), and introduced a mutant threonine exporter (rhtA23) in MDS42. The resulting strain, MDS-205, shows an ~83% increase in L-threonine production when cells are grown by flask fermentation, compared to a wild-type E. coli strain MG1655 engineered with the same threonine-specific modifications described above. And transcriptional analysis revealed the effect of the deletion of non-essential genes on the central metabolism and threonine pathways in MDS-205. Conclusion This result demonstrates that the elimination of genes unnecessary for cell growth can increase the productivity of an industrial strain, most likely by reducing the metabolic burden and improving the metabolic efficiency of cells.

Abstract Background DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data. Results Here we examine the expression data obtained from 682 Affymetrix GeneChips® with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t-distribution. Conclusion In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the Kα coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the Kα distributions are so close to t-distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance. Reviewers This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).

Papaver bracteatum, known for its high thebaine content and absence of morphine, has emerged as a promising alternative to opium poppy for codeine production. In this study, our objective was to create a diverse panel representing the natural variation of this species in Iran. To achieve this, we employed genotyping-by-sequencing to obtain genome-wide distributed single-nucleotide polymorphisms (SNPs) for phylogeographic analysis, population structure assessment, and evaluation of genetic diversity within P. bracteatum populations. A total of 244 P. bracteatum individuals from 13 distinct populations formed seven genetic groups, along with one highly admixed population. We observed a clear split between the populations inhabiting the Alborz Mts. in the east and Zagros Mts. in the west. In between these mountain ranges, the population of Kachal Mangan exhibited a high degree of genetic admixture between both genetic groups. At or after the end of the last glacial maximum, when climate conditions rapidly changed, all P. bracteatum populations experienced a strong demographic bottleneck reducing the already small effective population sizes further before they increased to their recent strengths. Our results suggest that the ongoing climate change together with human pressure on the species' habitats and limited seed-dispersal ability are potential factors contributing today to rising genetic isolation of P. bracteatum populations. Our results provide genetic data that can be used for conservation measures to safeguard the species' genetic diversity as a resource for future breeding approaches in this medicinally important species., (© 2024. The Author(s).)

Background: The increasing demand for saffron metabolites in various commercial industries, including medicine, food, cosmetics, and dyeing, is driven by the discovery of their diverse applications. Saffron, derived from Crocus sativus stigmas, is the most expensive spice, and there is a need to explore additional sources to meet global consumption demands. In this study, we focused on yellow-flowering crocuses and examined their tepals to identify saffron-like compounds., Results: Through metabolomic and transcriptomic approaches, our investigation provides valuable insights into the biosynthesis of compounds in yellow-tepal crocuses that are similar to those found in saffron. The results of our study support the potential use of yellow-tepal crocuses as a source of various crocins (crocetin glycosylated derivatives) and flavonoids., Conclusions: Our findings suggest that yellow-tepal crocuses have the potential to serve as a viable excessive source of some saffron metabolites. The identification of crocins and flavonoids in these crocuses highlights their suitability for meeting the demands of various industries that utilize saffron compounds. Further exploration and utilization of yellow-tepal crocuses could contribute to addressing the growing global demand for saffron-related products., (© 2024. The Author(s).)

Safflower (Carthamus tinctorius, Asteraceae) is a source of high-quality edible oil growing in moisture-limited environments. Despite its economic importance, the relationships to close wild species in Carthamus and the presence and relationships of ecotypes within safflower are still not fully clarified. Here we use genotyping-by-sequencing to identify the wild progenitor of C. tinctorius, infer phylogenetic relationship within the series Carthamus and identify groups of closely related lineages within cultivated safflower. Phylogenetic and population genomic analyses found C. palaestinus to be the closest relative and single progenitor of C. tinctorius, which confirms the Levant as the area of domestication of the crop. Flow cytometry showed all analyzed samples of C. oxyacantha, C. palaestinus and C. tinctorius to be diploid (2n = 2x = 24) with 2C genome sizes of 2.4-2.7 pg. Analyses of a set of 114 worldwide distributed safflower accessions arrived at two to five genetic groups, which showed, however, no correlation with the geographic origins of these accessions. From this, we conclude that the trade of safflower seeds resulted in multiple introductions of genotypes from the Levant into other areas with suitable climate conditions for the plant, as well as exchange of genotypes among these areas., (© 2023. The Author(s).)

Spring crocuses, the eleven species within Crocus series Verni (Iridaceae), consist of di- and tetraploid cytotypes. Among them is a group of polyploids from southeastern Europe with yet-unclear taxonomic affiliation. Crocuses are generally characterized by complex dysploid chromosome number changes, preventing a clear correlation between these numbers and ploidy levels. To reconstruct the evolutionary history of series Verni and particularly its polyploid lineages associated with C. heuffelianus , we used an approach combining phylogenetic analyses of two chloroplast regions, 14 nuclear single-copy genes plus rDNA spacers, genome-wide genotyping-by-sequencing (GBS) data, and morphometry with ploidy estimations through genome size measurements, analysis of genomic heterozygosity frequencies and co-ancestry, and chromosome number counts. Chromosome numbers varied widely in diploids with 2 n = 8, 10, 12, 14, 16, and 28 and tetraploid species or cytotypes with 2 n = 16, 18, 20, and 22 chromosomes. Crocus longiflorus , the diploid with the highest chromosome number, possesses the smallest genome (2C = 3.21 pg), while the largest diploid genomes are in a range of 2C = 7-8 pg. Tetraploid genomes have 2C values between 10.88 pg and 12.84 pg. Heterozygosity distribution correlates strongly with genome size classes and allows discernment of di- and tetraploid cytotypes. Our phylogenetic analyses showed that polyploids in the C. heuffelianus group are allotetraploids derived from multiple and partly reciprocal crosses involving different genotypes of diploid C. heuffelianus (2 n = 10) and C. vernus (2 n = 8). Dysploid karyotype changes after polyploidization resulted in the tetraploid cytotypes with 20 and 22 chromosomes. The multi-data approach we used here for series Verni , combining evidence from nuclear and chloroplast phylogenies, genome sizes, chromosome numbers, and genomic heterozygosity for ploidy estimations, provides a way to disentangle the evolution of plant taxa with complex karyotype changes that can be used for the analysis of other groups within Crocus and beyond. Comparing these results with morphometric analysis results in characters that can discern the different taxa currently subsumed under C. heuffelianus.

The Astragalus subgenus Hypoglottis Bunge, which consists of several sections, is one of the taxonomically most complicated groups in the genus. The Astragalus section Stereothrix Bunge belongs to this subgenus and is a significant element of the Irano-Turanian floristic region. A molecular phylogenetic analysis of this section and its closely related taxa using nuclear ribosomal DNA internal transcribed spacers (ITS) and external transcribed spacer (ETS) regions as well as plastid mat K sequences were conducted. Parsimony analyses and Bayesian phylogenetic inference revealed that the section is not monophyletic in its current form, as some taxa belonging to closely related sections such as Hypoglottidei DC. and the Malacothrix Bunge group within the sect. Stereothrix render it paraphyletic. Moreover, species groups belonging to sect. Stereothrix are placed in different clades within the phylogenetic tree of subgenus Hypoglottis , which indicates polyphyly, i.e., multiple independent origins of taxa placed in the sect. Stereothrix . Molecular dating of the group estimated an age of 3.62 (1.73-5.62) My for this assemblage with the major diversification events happening during the last 2 My. Many species groups separated only within the last 0.5 to 1 My. Based on morphological and molecular data, we discuss the phylogenetic relationships of the groups and synonymy of species. In addition, the included taxa of sect. Hypoglottidei are not monophyletic and include species belonging to sects. Hololeuce , Koelziana , Malacothrix , Onobrychoideae, and Ornithodpodium group within the sect. Stereothrix taxa. We conclude that only an analysis including all groups and nearly all species of the sections within the Hypoglottis clade can finally result in an new evolutionary-based system for these taxa.

The Euro-Siberian steppe flora consists of warm- and cold-adapted species, which may have responded differently to Pleistocene glacials and interglacials. Genotyping-by-sequencing individuals from across the distribution range of the pheasant's eye (Adonis vernalis), we aimed to gain insight into steppe florogenesis based on the species' evolutionary history. Although the primary area of origin of the species group comprising A. vernalis, A. villosa and A. volgensis is in Asia, our results indicate that recent populations of A. vernalis are not of Asian origin but evolved in the southern part of Europe during the Pleistocene, with Spanish populations clearly genetically distinct from the Southeastern European populations. We inferred that A. vernalis migrated eastwards from the sub-Mediterranean forest-steppes of Southeastern Europe into the continental forest-steppe zone. Eastern European populations had the highest private allelic richness, indicating long-term large population sizes in this region. As a thermophilic species, A. vernalis seems unlikely to have survived in the cold deserts of the Last Glacial Maximum in Western Siberia, so this region was likely (re)colonized postglacially. Overall, our results reinforce the importance of identifying the area of origin and the corresponding ecological requirements of steppe plants in order to understand the composition of today's steppe flora., (© 2022. The Author(s).)

The post-Miocene climatic histories of arid environments have been identified as key drivers of dispersal and diversification. Here, we investigate how climatic history correlates with the historical biogeography of the Atacama Desert genus Cristaria (Malvaceae). We analyze phylogenetic relationships and historical biogeography by using next-generation sequencing (NGS), molecular clock dating, Dispersal Extinction Cladogenesis and Bayesian sampling approaches. We employ a novel way to identify biogeographically meaningful regions as well as a rarely utilized program permitting the use of dozens of ancestral areas. Partial incongruence between the established taxonomy and our phylogenetic data argue for a complex historical biogeography with repeated introgression and incomplete lineage sorting. Cristaria originated in the central southern part of the Atacama Desert, from there the genus colonized other areas from the late Miocene onwards. The more recently diverged lineages appear to have colonized different habitats in the Atacama Desert during pluvial phases of the Pliocene and early Pleistocene. We show that NGS combined with near-comprehensive sampling can provide an unprecedented degree of phylogenetic resolution and help to correlate the historical biogeography of plant communities with cycles of arid and pluvial phases., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Astragalus section Hymenostegis is one of the important characteristic elements of thorn-cushion formations in the Irano-Turanian floristic region. In this paper, we examined the chromosome number of 17 species (15 new reports) and provide estimates of genome size for 62 individuals belonging to 38 taxa of A. sect. Hymenostegis , some species outside this section, plus two Oxytropis species. Based on chromosome counts 11 species were found to be diploid (2 n = 16), four species tetraploid (2 n = 32) and two taxa hexaploid (2 n = 48). From genome size measurements on silica-gel dried material, three ploidy levels (2 x , 4 x and 6 x ) were inferred, with a majority of species being diploid. The 2C values reach from 2.07 pg in diploid Astragalus   zohrabi to 7.16 pg in hexaploid A . rubrostriatus . We found indications that species might occur with different cytotypes. A phylogenetic framework using nrDNA ITS sequences was constructed to understand the evolution of ploidy changes and genome sizes. It showed that genome size values among the studied taxa differ only slightly within ploidy levels and are nearly constant within most species and groups of closely related taxa within the genus Astragalus . The results of this study show that there is a rather strong correlation between genome sizes and chromosome numbers in sect. Hymenostegis . The resolution of the ITS-based phylogenetic tree is too low to infer evolutionary or environmental correlations of genome size differences. Polyploidization seems to contribute to the high species number in Astragalus , however, in sect. Hymenostegis it is not the main driver of speciation.

The cultivation and domestication of plants are human-driven processes that change the biology and attributes of a plant. Ipheion uniflorum is a bulbous geophyte known as Spring Starflower whose cultivation dates back to the first half of the 19th century. At least seven cultivars have been developed from natural stands. However, comparative analyses of wild and cultivated materials are largely missing. In the present study, we provide a morphological evaluation and analyses of the cytological and genetic variability of I. uniflorum that reveal significant levels of differentiation and evidence of artificial selection in the Spring Starflower. Distinctive phenotypic characters in cultivated materials that are rarely found or lacking in wild plants and natural populations, such as pink or violet flowers, together with its reduced heterozygosity and starting genetic differentiation support the view of early mechanisms of domestication acting upon Spring Starflower plants. The probable geographic origin of the cultivated forms is discussed together with perspectives for plant breeding.

Capsella is a model plant genus of the Brassicaceae closely related to Arabidopsis . To disentangle its biogeographical history and intrageneric phylogenetic relationships, 282 individuals of all five currently recognized Capsella species were genotyped using a restriction digest-based next-generation sequencing method. Our analysis retrieved two main lineages within Capsella that split c . one million years ago, with western C. grandiflora and C. rubella forming a sister lineage to the eastern lineage consisting of C. orientalis . The split was attributed to continuous latitudinal displacements of the Eurasian steppe belt to the south during Early Pleistocene glacial cycles. During the interglacial cycles of the Late Pleistocene, hybridization of the two lineages took place in the southwestern East European Plain, leading to the allotetraploid C. bursa-pastoris . Extant genetic variation within C. orientalis postdated any extensive glacial events. Ecological niche modeling showed that suitable habitat for C. orientalis existed during the Last Glacial Maximum around the north coast of the Black Sea and in southern Kazakhstan. Such a scenario is also supported by population genomic data that uncovered the highest genetic diversity in the south Kazakhstan cluster, suggesting that C. orientalis originated in continental Asia and migrated north- and possibly eastwards after the last ice age. Post-glacial hybridization events between C. bursa-pastoris and C. grandiflora/rubella in the southwestern East European Plain and the Mediterranean gave rise to C. thracica . Introgression of C. grandiflora / rubella into C. bursa-pastoris resulted in a new Mediterranean cluster within the already existing Eurasian C. bursa-pastoris cluster. This study shows that the continuous displacement and disruption of the Eurasian steppe belt during the Pleistocene was the driving force in the evolution of Capsella ., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Speciation proceeds through mechanisms that promote reproductive isolation and shape the extent of genetic variation in natural populations, and thus its study is essential to understand the evolutionary processes leading to increased biodiversity. Chromosomal rearrangements are known to facilitate reproductive isolation by hybrid sterility and favour speciation events. The genus Ipheion (Amaryllidaceae, Allioideae) is unique as its species exhibit a remarkable karyological variability but lack population-level genetic data. To unveil the diversification processes acting upon the formation of new lineages within Ipheion in the Pampas of South America, we combined morphology and karyology approaches with genotyping-by-sequencing. Our phylogenomic and population genomics results supported the taxonomic division of Ipheion into three morphological and genetically well-differentiated groups. The origin of Ipheion uniflorum was traced back to its current southern distribution area in the southern Pampean region (in Argentina), from where it had expanded to the north reaching Uruguay. Our results further suggested that chromosome rearrangements and ploidy shifts had triggered speciation events, first during the origin of I. uniflorum and later during its subsequent diversification into I. recurvifolium and I. tweedieanum, in both cases reinforced by extrinsic factors and biogeographical settings. The current study illustrates the analytical power of multidisciplinary approaches integrating phylo- and population genomics with classic analyses to reveal evolutionary processes in plants., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Nuclear ribosomal DNA (nrDNA) has displayed extraordinary dynamics during the evolution of plant species. However, the patterns and evolutionary significance of nrDNA array expansion or contraction are still relatively unknown. Moreover, only little is known of the fate of minority nrDNA copies acquired between species via horizontal transfer. The barley genus Hordeum (Poaceae) represents a good model for such a study, as species of section Stenostachys acquired nrDNA via horizontal transfer from at least five different panicoid genera, causing long-term co-existence of native ( Hordeum -like) and non-native (panicoid) nrDNAs. Using quantitative PCR, we investigated copy number variation (CNV) of nrDNA in the diploid representatives of the genus Hordeum . We estimated the copy number of the foreign, as well as of the native ITS types (ribotypes), and followed the pattern of their CNV in relation to the genus' phylogeny, species' genomes size and the number of nrDNA loci. For the native ribotype, we encountered an almost 19-fold variation in the mean copy number among the taxa analysed, ranging from 1689 copies (per 2C content) in H. patagonicum subsp. mustersii to 31342 copies in H. murinum subsp. glaucum . The copy numbers did not correlate with any of the genus' phylogeny, the species' genome size or the number of nrDNA loci. The CNV was high within the recognised groups (up to 13.2 × in the American I-genome species) as well as between accessions of the same species (up to 4×). Foreign ribotypes represent only a small fraction of the total number of nrDNA copies. Their copy numbers ranged from single units to tens and rarely hundreds of copies. They amounted, on average, to between 0.1% ( Setaria ribotype) and 1.9% ( Euclasta ribotype) of total nrDNA. None of the foreign ribotypes showed significant differences with respect to phylogenetic groups recognised within the sect. Stenostachys . Overall, no correlation was found between copy numbers of native and foreign nrDNAs suggesting the sequestration and independent evolution of native and non-native nrDNA arrays. Therefore, foreign nrDNA in Hordeum likely poses a dead-end by-product of horizontal gene transfer events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krak, Caklová, Kopecký, Blattner and Mahelka.)

Constituting one of Earth's major biomes, steppes are characterised by naturally treeless extra-tropical vegetation. The formation of the Eurasian steppe belt, the largest steppe region in the world, began in Central Asia during the Neogene. In the glacial stages of the Pleistocene, steppe displaced forest vegetation, which in turn recolonised the area during the warmer interglacial periods, thus affecting the distribution of plants adapted to these habitats. Krascheninnikovia ceratoides (Chenopodiaceae) is a plant characteristic of dry steppe and semi-desert formations. Earlier studies showed that the ancestor of this autochthonous steppe element originated in Central Asia during the Miocene/Pliocene, i.e., in the same region and at the same time as the first appearance of steppe vegetation. However, as the extant lineages of Krascheninnikovia ceratoides diversified only 2.2 ± 0.9 Mya, it may represent a modern element of current dry steppe and semi-desert formations, rather than a component of the first steppe precursors of the Miocene. As such, it may have capitalised on the climatic conditions of the cold stages of the Quaternary to expand its range and colonise suitable habitats outside of its area of origin. To test this hypothesis, phylogeographic methods were applied to high-resolution genotyping-by-sequencing data. Our results indicate that Krascheninnikovia originated in western Central Asia and the Russian Altai, then spread to Europe in the West, and reached North America in the East. The populations of eastern Central Asia and North America belong to the same clade and are genetically clearly distinct from the Euro-Siberian populations. Among the populations west of the Altai Mountains, the European populations are genetically distinct from all others, which could be the result of the separation of populations east and west of the Urals caused by the Pleistocene transgressions of the Caspian Sea.

The geographical origin of watermelon ( Citrullus lanatus ) remains debated. While a first hypothesis suggests the center of origin to be West Africa, where the endemic sister species C. mucosospermus thrives, a second hypothesis suggests northeastern Africa where the white-fleshed Sudanese Kordophan melon is cultivated. In this study, we infer biogeographical and haplotype genealogy for C. lanatus, C. mucosospermus, C. amarus , and C. colocynthis using noncoding cpDNA sequences ( trn T- trn L and ndh F -rpl 32 regions) from a global collection of 135 accessions. In total, we identified 38 haplotypes in C. lanatus , C. mucosospermus , C. amarus , and C. colocynthis ; of these, 21 were found in Africa and 17 appear endemic to the continent. The least diverse species was C. mucosospermus (5 haplotypes) and the most diverse was C. colocynthis (16 haplotypes). Some haplotypes of C. mucosospermus were nearly exclusive to West Africa, and C. lanatus and C. mucosospermus shared haplotypes that were distinct from those of both C. amarus and C. colocynthis . The results support previous findings that revealed C. mucosospermus to be the closest relative to C. lanatus (including subsp. cordophanus ). West Africa, as a center of endemism of C. mucosospermus, is an area of interest in the search of the origin of C. lanatus . This calls for further historical and phylogeographical investigations and wider collection of samples in West and northeastern Africa., Competing Interests: The authors declare that they have no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome-wide sequence information, independent data were obtained from genotyping-by-sequencing and a target-enrichment experiment that returned 244 low-copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent-based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat-group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat-group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae., (© 2019 The Authors. The Plant Journal © 2019 John Wiley & Sons Ltd.)

Poor morphological and molecular differentiation in recently diversified lineages is a widespread phenomenon in plants. Phylogenetic relationships within such species complexes are often difficult to resolve because of the low variability in traditional molecular loci. Furthermore, biological phenomena responsible for topological incongruence such as Incomplete Lineage Sorting (ILS) and hybridisation complicate the resolution of phylogenetic relationships among closely related taxa. In this study, we employ a Genotyping-by-sequencing (GBS) approach to disentangle evolutionary relationships within a species complex belonging to the Neotropical orchid genus Cycnoches. This complex includes seven taxa distributed through Central America and the Colombian Chocó, and is nested within a clade estimated to have first diversified in the early Quaternary. Previous phylogenies inferred from few loci failed to provide support for internal relationships within the complex. Our Neighbour-net and coalescent-based analyses inferred from ca. 13,000 GBS loci obtained from 31 individuals belonging to six of the seven traditionally accepted Cycnoches taxa provided a robust phylogeny for this group. The genus Cycnoches includes three main clades that are further supported by morphological traits and geographic distributions. Similarly, a topology reconstructed through maximum likelihood (ML) inference of concatenated GBS loci produced results that are comparable with those reconstructed through coalescence and network-based methods. Our comparative phylogenetic informativeness analyses suggest that the low support evident in the ML phylogeny might be attributed to the abundance of uninformative GBS loci, which can account for up to 50% of the total number of loci recovered. The phylogenomic framework provided here, as well as morphological evidence and geographical patterns, suggest that the six entities previously thought to be different species or subspecies might actually represent only three distinct segregates. We further discuss the limited phylogenetic informativeness found in our GBS approach and its utility to disentangle relationships within recent and rapidly evolving species complexes. Our study is the first to demonstrate the utility of GBS data to reconstruct relationships within young (~2 Ma) Neotropical plant clades, opening new avenues for studies of species complexes that populate the species-rich orchid family., (Copyright © 2019. Published by Elsevier Inc.)

Crocus sativus, the saffron crocus, is the source of saffron, which is made from the dried stigmas of the plant. It is a male-sterile triploid lineage that ever since its origin has been propagated vegetatively. Its mode of evolution and area of origin are matters of long-lasting debates. Here we analyzed chloroplast genomes and genome-wide DNA polymorphisms obtained through genotyping-by-sequencing (GBS) to infer the parent and area of origin of C. sativus. These data were complemented by genome size measurements and analyses of nuclear single-copy genes. We could place 99.3% of saffron GBS alleles in Crocus cartwrightianus, a species occurring in southeastern mainland Greece and on Aegean islands, identifying it as the sole progenitor of the saffron crocus. Phylogenetic and population assignment analyses together with chloroplast polymorphisms indicated the C. cartwrightianus population in the vicinity of Athens as most similar to C. sativus. We conclude that the crop is an autotriploid that evolved in Attica by combining two different genotypes of C. cartwrightianus. Triploid sterility and vegetative propagation prevented afterwards segregation of the favorable traits of saffron, resulting in worldwide cultivation of a unique clonal lineage., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.

Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next-generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom-designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single-nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%-63.5% (mean over all loci) in the three study species. Thus, the information obtained by next-generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.

Phylogenetic relationships among the taxa of Crocus series Crocus are still unclear, preventing the understanding of species diversity and the evolution of the important spice saffron (Crocus sativus). Therefore, we analyzed sequences of two chloroplast (trnL-trnF, matK-trnK) and three nuclear (TOPO6, ribosomal DNA ETS and ITS) marker regions to infer phylogenetic relationships among all species belonging to series Crocus. Our phylogenetic analyses resolved the relationships among all taxa of the series. Crocus hadriaticus and the former C. pallasii subspecies appeared polyphyletic. The latter deserve elevating the subspecies to species rank, while for C. hadriaticus a detailed study of species boundaries is necessary. Multi-locus and also genome-wide single nucleotide polymorphism data obtained through genotyping-by-sequencing placed C. sativus within C. cartwrightianus with no indication that other Crocus species contributed to the evolution of the triploid. Our analyses thus made an autotriploid origin of C. sativus from C. cartwrightianus very likely., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Wild barley forms a two-rowed spike with a brittle rachis whereas domesticated barley has two- or six-rowed spikes with a tough rachis. Like domesticated barley, 'agriocrithon' forms a six-rowed spike; however, the spike is brittle as in wild barley, which makes the origin of agriocrithon obscure. Haplotype analysis of the Six-rowed spike 1 (vrs1) and Non-brittle rachis 1 (btr1) and 2 (btr2) genes was conducted to infer the origin of agriocrithon barley. Some agriocrithon barley accessions (eu-agriocrithon) carried Btr1 and Btr2 haplotypes that are not found in any cultivars, implying that they are directly derived from wild barley through a mutation at the vrs1 locus. Other agriocrithon barley accessions (pseudo-agriocrithon) carried Btr1 or Btr2 from cultivated barley, thus implying that they originated from hybridization between six-rowed landraces carrying btr1Btr2 and Btr1btr2 genotypes followed by recombination to produce Btr1Btr2. All materials we collected from Tibet belong to pseudo-agriocrithon and thus do not support the Tibetan Plateau as being a center of barley domestication. Tracing the evolutionary history of these allelic variants revealed that eu-agriocrithon represents six-rowed barley lineages that were selected by early farmers, once in south-eastern Turkmenistan (vrs1.a1) and again in the eastern part of Uzbekistan (vrs1.a4)., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

The taxa of Astragalus section Hymenostegis are an important element of mountainous and steppe habitats in Southwest Asia. A phylogenetic hypothesis of sect. Hymenostegis has been obtained from nuclear ribosomal DNA internal transcribed spacer (ITS) and plastid ycf1 sequences of up to 303 individuals from 106 species, including all 89 taxa currently assigned to sect. Hymenostegis, 14 species of other Astragalus sections, and two species of Oxytropis and one Biserrula designated as outgroups. Bayesian phylogenetic inference and parsimony analyses reveal that three species from two other closely related sections group within sect. Hymenostegis, making the section paraphyletic. DNA sequence diversity is generally very low among Hymenostegis taxa, which is consistent with recent diversification of the section. We estimate that diversification in sect. Hymenostegis occurred in the middle to late Pleistocene, with many species arising only during the last one million years, when environmental conditions in the mountain regions of Southwest and Central Asia cycled repeatedly between dry and more humid conditions.

Background: Triticeae, the tribe of wheat grasses, harbours the cereals barley, rye and wheat and their wild relatives. Although economically important, relationships within the tribe are still not understood. We analysed the phylogeny of chloroplast lineages among nearly all monogenomic Triticeae taxa and polyploid wheat species aiming at a deeper understanding of the tribe's evolution. We used on- and off-target reads of a target-enrichment experiment followed by Illumina sequencing., Results: The read data was used to assemble the plastid locus ndhF for 194 individuals and the whole chloroplast genome for 183 individuals, representing 53 Triticeae species and 15 genera. We conducted Bayesian and multispecies coalescent analyses to infer relationships and estimate divergence times of the taxa. We present the most comprehensive dated Triticeae chloroplast phylogeny and review previous hypotheses in the framework of our results. Monophyly of Triticeae chloroplasts could not be confirmed, as either Bromus or Psathyrostachys captured a chloroplast from a lineage closely related to a Bromus-Triticeae ancestor. The most recent common ancestor of Triticeae occurred approximately between ten and 19 million years ago., Conclusions: The comparison of the chloroplast phylogeny with available nuclear data in several cases revealed incongruences indicating past hybridizations. Recent events of chloroplast capture were detected as individuals grouped apart from con-specific accessions in otherwise monopyhletic groups.

Cistanthe longiscapa is an endemic annual herb and characteristic element of the Chilean Atacama Desert. Principal threats are the destruction of its seed deposits by human activities and reduced germination rates due to the decreasing occurrence of precipitation events. To enable population genetic and phylogeographic analyses in this species we performed paired-end shotgun sequencing (2x100 bp) of genomic DNA on the Illumina HiSeq platform and identified microsatellite (SSR) loci in the resulting sequences. From 29 million quality-filtered read pairs we obtained 549,174 contigs (average length 614 bp; N50 = 904). Searching for SSRs revealed 10,336 loci with microsatellite motifs. Initially, we designed primers for 96 loci, which were tested for PCR amplification on three C. longiscapa individuals. Successfully amplifying loci were further tested on eight individuals to screen for length variation in the resulting amplicons, and the alleles were exemplarily sequenced to infer the basis for the observed length variation. Finally we arrived at 26 validated SSR loci for population studies in C. longiscapa, which resulted in 146 bi-allelic SSR markers in our test sample of eight individuals. The genomic sequences were also used to assemble the plastid genome of C. longiscapa, which provides an additional set of maternally inherited genetic markers.

The movement of nuclear DNA from one vascular plant species to another in the absence of fertilization is thought to be rare. Here, nonnative rRNA gene [ribosomal DNA (rDNA)] copies were identified in a set of 16 diploid barley ( Hordeum ) species; their origin was traceable via their internal transcribed spacer (ITS) sequence to five distinct Panicoideae genera, a lineage that split from the Pooideae about 60 Mya. Phylogenetic, cytogenetic, and genomic analyses implied that the nonnative sequences were acquired between 1 and 5 Mya after a series of multiple events, with the result that some current Hordeum sp. individuals harbor up to five different panicoid rDNA units in addition to the native Hordeum rDNA copies. There was no evidence that any of the nonnative rDNA units were transcribed; some showed indications of having been silenced via pseudogenization. A single copy of a Panicum sp. rDNA unit present in H. bogdanii had been interrupted by a native transposable element and was surrounded by about 70 kbp of mostly noncoding sequence of panicoid origin. The data suggest that horizontal gene transfer between vascular plants is not a rare event, that it is not necessarily restricted to one or a few genes only, and that it can be selectively neutral.

Why are certain bacterial genomes so small and compact? The adaptive genome streamlining hypothesis posits that selection acts to reduce genome size because of the metabolic burden of replicating DNA. To reveal the impact of genome streamlining on cellular traits, we reduced the Escherichia coli genome by up to 20% by deleting regions which have been repeatedly subjects of horizontal transfer in nature. Unexpectedly, horizontally transferred genes not only confer utilization of specific nutrients and elevate tolerance to stresses, but also allow efficient usage of resources to build new cells, and hence influence fitness in routine and stressful environments alike. Genome reduction affected fitness not only by gene loss, but also by induction of a general stress response. Finally, we failed to find evidence that the advantage of smaller genomes would be due to a reduced metabolic burden of replicating DNA or a link with smaller cell size. We conclude that as the potential energetic benefit gained by deletion of short genomic segments is vanishingly small compared with the deleterious side effects of these deletions, selection for reduced DNA synthesis costs is unlikely to shape the evolution of small genomes., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As a result of a taxonomic and phylogenetic revision of Astragalus section Hymenostegis we identified a new species of Astragalus from northwestern Iran, namely A. remotispicatus spec. nov., which is described and illustrated here. It is morphologically similar to A. karl-heinzii in possessing a lax inflorescence. Phylogenetic inference of the nuclear ribosomal DNA internal transcribed spacer (ITS) region support A. remotispicatus as a clearly distinct species within the lax-inflorescence group of this section. Also the placement of A. sciureus var. subsessilis was found to be wrong and this taxon should be treated as a synonym within A. kohrudicus.

The C-value paradox remains incompletely resolved after >40 yr and is exemplified by 2,350-fold variation in genome sizes of flowering plants. The carnivorous Lentibulariaceae genus Genlisea, displaying a 25-fold range of genome sizes, is a promising subject to study mechanisms and consequences of evolutionary genome size variation. Applying genomic, phylogenetic, and cytogenetic approaches, we uncovered bidirectional genome size evolution within the genus Genlisea. The Genlisea nigrocaulis Steyerm. genome (86 Mbp) has probably shrunk by retroelement silencing and deletion-biased double-strand break (DSB) repair, from an ancestral size of 400 to 800 Mbp to become one of the smallest among flowering plants. The G. hispidula Stapf genome has expanded by whole-genome duplication (WGD) and retrotransposition to 1550 Mbp. Genlisea hispidula became allotetraploid after the split from the G. nigrocaulis clade ∼29 Ma. Genlisea pygmaea A. St.-Hil. (179 Mbp), a close relative of G. nigrocaulis, proved to be a recent (auto)tetraploid. Our analyses suggest a common ancestor of the genus Genlisea with an intermediate 1C value (400-800 Mbp) and subsequent rapid genome size evolution in opposite directions. Many abundant repeats of the larger genome are absent in the smaller, casting doubt on their functionality for the organism, while recurrent WGD seems to safeguard against the loss of essential elements in the face of genome shrinkage. We cannot identify any consistent differences in habitat or life strategy that correlate with genome size changes, raising the possibility that these changes may be selectively neutral., (© 2015 The Authors.)

Polyploidization is an important speciation mechanism in the barley genus Hordeum. To analyze evolutionary changes after allopolyploidization, knowledge of parental relationships is essential. One chloroplast and 12 nuclear single-copy loci were amplified by polymerase chain reaction (PCR) in all Hordeum plus six out-group species. Amplicons from each of 96 individuals were pooled, sheared, labeled with individual-specific barcodes and sequenced in a single run on a 454 platform. Reference sequences were obtained by cloning and Sanger sequencing of all loci for nine supplementary individuals. The 454 reads were assembled into contigs representing the 13 loci and, for polyploids, also homoeologues. Phylogenetic analyses were conducted for all loci separately and for a concatenated data matrix of all loci. For diploid taxa, a Bayesian concordance analysis and a coalescent-based dated species tree was inferred from all gene trees. Chloroplast matK was used to determine the maternal parent in allopolyploid taxa. The relative performance of different multilocus analyses in the presence of incomplete lineage sorting and hybridization was also assessed. The resulting multilocus phylogeny reveals for the first time species phylogeny and progenitor-derivative relationships of all di- and polyploid Hordeum taxa within a single analysis. Our study proves that it is possible to obtain a multilocus species-level phylogeny for di- and polyploid taxa by combining PCR with next-generation sequencing, without cloning and without creating a heavy load of sequence data., (© The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

Background: Our aim is to understand the evolution of species-rich plant groups that shifted from tropical into cold/temperate biomes. It is well known that climate affects evolutionary processes, such as how fast species diversify, species range shifts, and species distributions. Many plant lineages may have gone extinct in the Northern Hemisphere due to Late Eocene climate cooling, while some tropical lineages may have adapted to temperate conditions and radiated; the hyper-diverse and geographically widespread genus Hypericum is one of these., Results: To investigate the effect of macroecological niche shifts on evolutionary success we combine historical biogeography with analyses of diversification dynamics and climatic niche shifts in a phylogenetic framework. Hypericum evolved cold tolerance c. 30 million years ago, and successfully colonized all ice-free continents, where today ~500 species exist. The other members of Hypericaceae stayed in their tropical habitats and evolved into ~120 species. We identified a 15-20 million year lag between the initial change in temperature preference in Hypericum and subsequent diversification rate shifts in the Miocene., Conclusions: Contrary to the dramatic niche shift early in the evolution of Hypericum most extant species occur in temperate climates including high elevations in the tropics. These cold/temperate niches are a distinctive characteristic of Hypericum. We conclude that the initial release from an evolutionary constraint (from tropical to temperate climates) is an important novelty in Hypericum. However, the initial shift in the adaptive landscape into colder climates appears to be a precondition, and may not be directly related to increased diversification rates. Instead, subsequent events of mountain formation and further climate cooling may better explain distribution patterns and species-richness in Hypericum. These findings exemplify important macroevolutionary patterns of plant diversification during large-scale global climate change.

DNA double-strand break (DSB) repair mechanisms differ in their requirements for a homologous repair template and in the accuracy of the result. We aimed to quantify the outcome of repair of a single targeted DSB in somatic cells of young barley (Hordeum vulgare) plants. Amplicon sequencing of three reporter constructs revealed 47 to 58% of reads as repaired via nonhomologous end-joining (NHEJ) with deletions and/or small (1 to 3 bp) insertions. Alternative NHEJ revealed 2 to 5 bp microhomology (15.7% of cases) or new replication-mediated short duplications at sealed breaks. Although deletions outweigh insertions in barley, this bias was less pronounced and deleted sequences were shorter than in Arabidopsis thaliana. Between 17 and 33% of reads likely represent restoration of the original sequence. Depending on the construct, 20 to 33% of reads arose via gene conversion (homologous recombination). Remarkably, <1 to >8% of reads apparently display synthesis-dependent strand annealing linked with NHEJ, inserting 4 to 61 bp, mostly originating from the surrounding of breakpoints. Positional coincidence of >81% of sister chromatid exchanges with target loci is unprecedented for higher eukaryotes and indicates that most repair events for staggered DSBs, at least in barley, involve the sister chromatid and occur during S or G2 phase of the cell cycle., (© 2014 American Society of Plant Biologists. All rights reserved.)

Studies of Hordeum vulgare subsp. spontaneum, the wild progenitor of cultivated barley, have mostly relied on materials collected decades ago and maintained since then ex situ in germplasm repositories. We analyzed spatial genetic variation in wild barley populations collected rather recently, exploring sequence variations at seven single-copy nuclear loci, and inferred the relationships among these populations and toward the genepool of the crop. The wild barley collection covers the whole natural distribution area from the Mediterranean to Middle Asia. In contrast to earlier studies, Bayesian assignment analyses revealed three population clusters, in the Levant, Turkey, and east of Turkey, respectively. Genetic diversity was exceptionally high in the Levant, while eastern populations were depleted of private alleles. Species distribution modeling based on climate parameters and extant occurrence points of the taxon inferred suitable habitat conditions during the ice-age, particularly in the Levant and Turkey. Together with the ecologically wide range of habitats, they might contribute to structured but long-term stable populations in this region and their high genetic diversity. For recently collected individuals, Bayesian assignment to geographic clusters was generally unambiguous, but materials from genebanks often showed accessions that were not placed according to their assumed geographic origin or showed traces of introgression from cultivated barley. We assign this to gene flow among accessions during ex situ maintenance. Evolutionary studies based on such materials might therefore result in wrong conclusions regarding the history of the species or the origin and mode of domestication of the crop, depending on the accessions included.

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Background: Interleukin-10 homologues encoded by Herpes viruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) hold interesting structural and biological characteristics compared to human interleukin-10 (hIL-10) that render these proteins promising candidates for therapeutic application in inflammatory bowel disease (IBD). Intestinal delivery of cytokines using bacterial carriers as chassis represents a novel approach for treatment of IBD patients. For proof of concept, a Sec-dependent transporter construct was designed for secretory expression of recombinant viral IL-10 proteins in the periplasm of Escherichia coli laboratory strain BL21 (DE3), which might serve as part of a prospective lysis based delivery and containment system., Results: The signal peptide of E. coli outer membrane protein F fused to the mature form of the viral IL-10 proteins enabled successful transport into the periplasm, a compartment which seems crucial for proper assembly of the dimeric configuration of the cytokines. Cytokine concentrations in different bacterial compartments were determined by ELISA and achieved yields of 67.8 ng/ml ± 24.9 ng/ml for HCMV IL-10 and 1.5 μg/ml ± 841.4 ng/ml for EBV IL-10 in the periplasm. Immunoblot analysis was used to confirm the correct size of the E. coli-derived recombinant cytokines. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the signal transduction cascade after IL-10 receptor interaction, as well as suppression of tumor necrosis factor α (TNF-α) release of lipopolysaccharide-stimulated mouse macrophages were used as read-out assays for proving in vitro biological activity of the E. coli derived, recombinant viral IL-10 counterparts., Conclusions: In this study, proof of principle is provided that E. coli cells are a suitable chassis for secretory expression of viral IL-10 cytokines encoded by codon-optimized synthetic genes fused to the E. coli ompF signal sequence. In vitro biological activity evidenced by activation of transcription factor STAT3 and suppression of TNF-α in mammalian cell lines was shown to be strictly dependent on export of viral IL-10 proteins into the periplasmic compartment. E. coli might serve as carrier system for in situ delivery of therapeutic molecules in the gut, thus representing a further step in the development of novel approaches for treatment of IBD.

Background and Aims: Supernumerary B chromosomes (Bs) represent a specific type of selfish genetic element. As Bs are dispensable for normal growth, it is expected to observe B polymorphisms among populations. To address whether Bs maintained in geographically distinct populations of cultivated and weedy rye are polymorphic, the distribution patterns and the transcriptional activity of different B-located repeats were analysed., Methods: Bs of cultivated and weedy rye from seven origins were analysed by fluorescence in situ hybridization (FISH) with probes specific for the pericentromeric and interstitial regions as well as the B-specific non-disjunction control region. The DNA replication, chromatin composition and transcription behaviour of the non-disjunction regions were determined. To address whether the B-marker repeats E3900 and D1100 have diverged genotypes of different origin at the sequence level, the genomic sequences of both repeats were compared between cultivated rye and weedy rye from five different origins., Key Results: B chromosomes in cultivated and weedy rye have maintained a similar molecular structure at the level of subspecies. The high degree of conservation of the non-disjunction control region regarding its transcription activity, histone composition and replication underlines the functional importance of this chromosome region for the maintenance of Bs. The conserved chromosome structure suggests a monophyletic origin of the rye B. As Bs were found in different countries, it is likely that Bs were frequently present in the seed material used in early agriculture., Conclusions: The surprisingly conserved chromosome structure suggests that although the rye Bs experienced rapid evolution including multiple rearrangements at the early evolutionary stages, this process has slowed significantly and may have even ceased during its recent evolution.

Crocus consists of about 100 species distributed from western Europe and northern Africa to western China, with the center of diversity on the Balkan Peninsula and in Asia Minor. Our study focuses on clarifying phylogenetic relationships and chromosome number evolution within the genus using sequences of the chloroplast trnL-F region, the nuclear ribosomal DNA internal transcribed spacer (ITS) region, and a part of the nuclear single-copy gene pCOSAt103. In a combined dataset of ITS and trnL-F sequences, 115 individuals representing 110 taxa from both subgenera and all sections and series of Crocus were analyzed with Bayesian phylogenetic inference. For pCOSAt103 79 individuals representing 74 Crocus taxa were included, and for the majority of them PCR amplicons were cloned and up to eight clones per individual were sequenced to detect allopolyploidization events. Romulea species were included as outgroup in both analyses. Characteristics of seed surface structures were evaluated by scanning electron microscopy. Phylogenetic analysis of ITS/trnL-F data resulted in a monophyletic genus Crocus, probably monophyletic sections Crocus and Nudiscapus, and inferred monophyly for eight of the 15 series of the genus. The C. biflorus aggregate, thought to be consisting of closely related subspecies, was found to be polyphyletic, the taxa occurring within three major clades in the phylogenetic tree. Cloning of pCOSAt103 resulted in the detection of homoeologous copies in about one third of the taxa of section Nudiscapus, indicating an allotetraploid origin of this section. Reconstruction of chromosome number evolution along the phylogenetic tree using a probabilistic and a parsimony approach arrived at partly contradictory results. Both analyses agreed however on the occurrence of multiple polyploidization and dysploidy events. B chromosomes evolved at least five times independently within the genus, preferentially in clades characterized by karyotype changes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John's wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

Bacteriophages, the historic model organisms facilitating the initiation of molecular biology, are still important candidates of numerous useful or promising biotechnological applications. Development of generally applicable, simple and rapid techniques for their genetic engineering is therefore a validated goal. In this article, we report the use of bacteriophage recombineering with electroporated DNA (BRED), for the first time in a coliphage. With the help of BRED, we removed a copy of mobile element IS1, shown to be active, from the genome of P1vir, a coliphage frequently used in genome engineering procedures. The engineered, IS-free coliphage, P1virdeltaIS, displayed normal plaque morphology, phage titre, burst size and capacity for generalized transduction. When performing head-to-head competition experiments, P1vir could not outperform P1virdeltaIS, further indicating that the specific copy of IS1 plays no direct role in lytic replication. Overall, P1virdeltaIS provides a genome engineering vehicle free of IS contamination, and BRED is likely to serve as a generally applicable tool for engineering bacteriophage genomes in a wide range of taxa., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)