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Plant species respond to varying plant species diversity and associated changes in their abiotic and biotic environment with changes in their phenotype. However, it is not clear to what degree this phenotypic differentiation is due to genotype diversity within populations or phenotypic plasticity of plant individuals. We studied individuals of 16 populations of the clonal herb Taraxacum officinale grown in plant communities of different species richness in a 17-year-old grassland biodiversity experiment (Jena Experiment). We collected 12 individuals in each population to measure phenotypic traits and identify distinct genotypes using microsatellite DNA markers. Plant species richness did not influence population-level genotype and trait diversity. However, it affected the expression of several phenotypic traits, e.g. leaf and inflorescence number, maximum leaf length and seed mass, which increased with increasing plant species richness. Moreover, population-level trait diversity correlated positively with genotype richness for leaf dry matter content (LDMC) and negatively with inflorescence number. For several traits (i.e. seed mass, germination rate, LDMC, specific leaf area (SLA)), a larger portion of variance was explained by genotype identity, while variance in other traits (i.e. number of inflorescences, leaf nitrogen concentration, leaf number, leaf length) resided within genotypes and thus was mostly due to phenotypic plasticity. Overall, our findings show that plant species richness positively affected the population means of some traits related to whole-plant performance, whose variation was achieved through both phenotypic plasticity and genotype composition of a population.

Introducing asexual reproduction through seeds - apomixis - into crop species could revolutionize agriculture by allowing F1 hybrids with enhanced yield and stability to be clonally propagated. Engineering synthetic apomixis has proven feasible in inbred rice through the inactivation of three genes (MiMe), which results in the conversion of meiosis into mitosis in a line ectopically expressing the BABYBOOM1 (BBM1) parthenogenetic trigger in egg cells. However, only 10-30% of the seeds are clonal. Here, we show that synthetic apomixis can be achieved in an F1 hybrid of rice by inducing MiMe mutations and egg cell expression of BBM1 in a single step. We generate hybrid plants that produce more than 95% of clonal seeds across multiple generations. Clonal apomictic plants maintain the phenotype of the F1 hybrid along successive generations. Our results demonstrate that there is no barrier to almost fully penetrant synthetic apomixis in an important crop species, rendering it compatible with use in agriculture.

研究グループは、日本の山野に生える4種の雌雄異株樹種、ヤマコウバシ、アブラチャン、クロモジ、ダンコウバイのDNA情報から、これらの樹種が雌雄異株性の不利をいかに克服して集団を維持しているのか、その仕組みを探りました。その結果、アブラチャン、クロモジ、ダンコウバイの3種は、自家受精や単為生殖を行わず、必ず雌雄の交配によって近交弱勢を避けていることが示されたものの、ヤマコウバシは雌株が単独で種子を生産することで雌雄異株性の不利を克服し、さらに日本のヤマコウバシがたった1本の雌株から生じた巨大なクローンであることが分かりました。この巨大クローンの分布の範囲は距離にして1000 kmを超える、世界的にも極めて珍しい大規模なものです。, Research Highlights: genetic diversity in populations were compared among related shrub species with different reproductive systems. Background and Objectives: Lindera species are dioecious trees or shrubs that produce seeds by mating of males and females. To evaluate the importance of genetic diversity for the persistence of natural populations, we compared genetic information among four Lindera species in Japan. Three are dioecious shrubs (Lindera praecox, Lindera umbellata, and Lindera obtusiloba) that produce seeds by sexual reproduction. The remaining species, Lindera glauca, reproduces by apomixis; only female plants are found in Japan. Materials and Methods: all four species were sampled across a wide geographic area, from Tohoku to Kyushu, Japan. Single nucleotide polymorphisms (SNPs) were detected by multiplexed ISSR genotyping by sequencing (MIG-seq) and the resulting genetic diversity parameters were compared among populations. Results: in all sexually reproducing species, the values of observed heterozygosity were close to the expected ones and the inbreeding coefficients were nearly 0. These results were supposed to be caused by their obligate outcrossing. The genetic difference increased, in ascending order, between a mother plant and its seeds, within populations, and across geographic space. We observed a substantial geographic component in the genetic structure of these species. For L. glauca, the genetic difference between a mother and its seeds, within populations, and across space were not significantly different from what would be expected from PCR errors. Genetic diversity within and among populations of L. glauca was extremely low. Conclusions: apomixis has the advantage of being able to found populations from a single individual, without mating, which may outweigh the disadvantages associated with the extremely low genetic diversity of L. glauca. This may explain why this species is so widely distributed in Japan. Provided that the current ge

The origin and dispersal of cultivated and wild mandarin and related citrus are poorly understood. Here, comparative genome analysis of 69 new east Asian genomes and other mainland Asian citrus reveals a previously unrecognized wild sexual species native to the Ryukyu Islands: C. ryukyuensis sp. nov. The taxonomic complexity of east Asian mandarins then collapses to a satisfying simplicity, accounting for tachibana, shiikuwasha, and other traditional Ryukyuan mandarin types as homoploid hybrid species formed by combining C. ryukyuensis with various mainland mandarins. These hybrid species reproduce clonally by apomictic seed, a trait shared with oranges, grapefruits, lemons and many cultivated mandarins. We trace the origin of apomixis alleles in citrus to mangshanyeju wild mandarins, which played a central role in citrus domestication via adaptive wild introgression. Our results provide a coherent biogeographic framework for understanding the diversity and domestication of mandarin-type citrus through speciation, admixture, and rapid diffusion of apomictic reproduction.

Polyploidy plays a major role in plant evolution. The establishment of new polyploids is often a consequence of a single or few successful polyploidization events occurring within a species' evolutionary trajectory. New polyploid lineages can play different roles in plant diversification and go through several evolutionary stages influenced by biotic and abiotic constraints and characterized by extensive genetic changes. The study of such changes has been crucial for understanding polyploid evolution. Here, we use the multiploid-species Paspalum intermedium to study population-level genetic and morphological variation and ecological differentiation in polyploids. Using flow cytometry, amplified fragment length polymorphism (AFLP) genetic markers, environmental variables, and morphological data, we assessed variations in ploidy, reproductive modes, and the genetic composition in 35 natural populations of P. intermedium along a latitudinal gradient in South America. Our analyses show that apomictic auto-tetraploids are of multiple independent origin. While overall genetic variation was higher in diploids, both diploids and tetraploids showed significant variation within and among populations. The spatial distribution of genetic variation provides evidence for a primary origin of the contact zone between diploids and tetraploids and further supports the hypothesis of geographic displacement between cytotypes. In addition, a strong link between the ecological differentiation of cytotypes and spatial distribution of genetic variation was observed. Overall, the results indicate that polyploidization in P. intermedium is a recurrent phenomenon associated to a shift in reproductive mode and that multiple polyploid lineages from genetically divergent diploids contributed to the successful establishment of local polyploid populations and dispersal into new environments.

Asexual reproduction has often been regarded as an evolutionary dead end, but asexual lineages (most notably those that are apomictic) are present in several angiosperm families and often comprise a large number of taxa, both widespread and endemic. Investigating correlations between genetic, environmental and taxonomic factors and the incidence of apomixis has represented a challenge for many years, with previous analyses frequently omitting one or more of these variables. Here, flow cytometric seed screening, cytological data and ecological variables have been integrated in a phylogenetic framework to create a comprehensive dataset for 229 of Asteraceae from the European Alps. Data were analysed using phylogenetically informed generalized linear mixed models (pMCMCglmm) where elevation, ploidy and phenology were assessed for their potential correlation with asexual reproduction and apomixis type. Although apomixis is not dominant among the species studied, our results confirm that an odd ploidy (e.g. 3x) and, to a lesser extent, an even polyploid level (i.e. 4x) significantly increase its probability, most probably due to chromosome misalignments during meiosis. The distribution of apomictic species does not correlate with elevation, and there is a weak correlation between early flowering initiation and aposporous apomixis. Although current and future changes in climate may severely impact the survival of the flora of the European Alps, asexual reproduction and polyploidization may prove to be, at least temporarily, lifelines for the survival of a species under the novel climatic conditions. Therefore, uncovering how apomicts and polyploids evolve and persist will be essential for understanding the ecology of the European Alps and hence informing future conservation strategies.

Razviće semena kod skrivenosemenica nastaje kao rezultat procesa dvojnog oplođenja, u kome se jedna spermatična ćelija spaja sa jajnom ćelijom, dok se druga spermatična ćelija spaja sa centralnom ćelijom. Koordinisanо razvićе semenjače, endosperma i embriona nakon oplođenja zavisi od ravnotežnog odnosa roditeljskih genoma, što je preduslov za odvijanje normalnog razvića semena. Narušavanje ravnoteže u genomskom doprinosu roditelja često dovodi do brojnih poremećaja u razviću semena i embriona, a u drastičnim slučajevima može dovesti i do potpunog prekida razvića semena. Ova pojava predstavlja jednu od glavnih prepreka za upotrebu genetičkog inženjerstva u cilju stvaranja apomiktičnih semena, kao i za prevazilaženje međuvrsnih seksualnih reproduktivnih barijera. Jedan od glavnih mehanizama koji narušava ravnotežu odnosa roditeljskih genoma u semenu model biljke Arabidopsis thaliana jeste represija sinteze auksina uz pomoć Polikomb represivnog kompleksa 2 (PRC2). Međutim, ni smanjenje nivoa ekspresiju gena PRC2 kompleksa ni povišavanje nivoa auksina nisu dovoljni da dovedu do apomiksije. Evolutivno konzervisana uloga PRC2 tokom razvića zasniva se na ulozi ovog kompleksa da postranslaciono modifikuje histon H3, a proteini koji prepoznaju posttranslacione modifikacije (PTM) histona, odnosno čitači histonskog koda, često su uključeni u regulaciju razvića u sprezi sa PRC2. Stoga čitači histonskog koda predstavljaju pogodne kandidate za dodatni uticaj na funkciju PRC2 kompleksa. Dosadašnji podaci pokazuju da su čitači histonskih modifikacija biljaka uključeni u veliki broj različitih bioloških i razvojnih procesa u ćeliji, ali da je broj opisanih čitača relativno mali. Predmet istraživanja ove doktorske disertacije jeste regulacija razvića semena kod biljke А. thaliana, sa posebnim osvrtom na ulogu EML proteina u čitanju određenih modifikacija histona, kao i na ulogu ovih proteina u kontroli endogenog nivoa auksina i njegove kompartmentizacije tokom razvića semena. Rezulta, In flowering plants, seed development is initiated by double fertilization of the female gametophyte, whereby one sperm cell fuses with the egg cell, while the second fuses with the central cell. The successful and coordinated interaction between the developing seed compartments (seed coat, endosperm and embryo) depends on the balance in parental genome contributions. Any change in the genomic contribution of one parent often leads to severe defects including seed abortion. This represents one of the main obstacles for the engineering of asexual reproduction through seeds (apomixis), and for generating new interspecies hybrids. The repression of auxin synthesis by the Polycomb Repressive Complex 2 (PRC2) is a major mechanism contributing to sensing genome balance. However, current efforts focusing on identification of regulators decreasing PRC2 or elevating auxin levels have not yet resulted in the production of apomictic seed. Evolutionary conserved role of PRC2 during developmental processes in plants is based on the role of this complex to modify histone H3. Furthermore, proteins involved in recognizing histone posttranslational modifications (PTMs) are often included in the regulation of plant development in conjunction with in PRC2. So far a relatively few plant so-called histone readers have been identified to be instrumental for many developmental processes. Results from this study show that EML1 and EML3, proteins that belong to the EMSY-Like Tudor/Agenet protein family, are H3K36me3 histone readers necessary to maintain parental genome balance in Arabidopsis. By analyzing the mutant phenotypes, our results demonstrate that both EML1 and EML3 are required to prevent seed development before fertilization, and to regulate the balance of parental contributions after fertilization. We hypothesize that EML1 and EML3 function to repress paternal gene expression by regulating auxin transport and signaling...

Parthenogenesis is the spontaneous development of an embryo from an unfertilized egg cell. It naturally occurs in a variety of plant and animal species. In plants, parthenogenesis usually is found in combination with apomeiosis (the omission of meiosis) and pseudogamous or autonomous (with or without central cell fertilization) endosperm formation, together known as apomixis (clonal seed production). The initiation of embryogenesis in vivo and in vitro has high potential in plant breeding methods, particularly for the instant production of homozygous lines from haploid gametes [doubled haploids (DHs)], the maintenance of vigorous F1-hybrids through clonal seed production after combining it with apomeiosis, reverse breeding approaches, and for linking diploid and polyploid gene pools. Because of this large interest, efforts to identify gene(s) for parthenogenesis from natural apomicts have been undertaken by using map-based cloning strategies and comparative gene expression studies. In addition, engineering parthenogenesis in sexual model species has been investigated via mutagenesis and gain-of-function strategies. These efforts have started to pay off, particularly by the isolation of the PsASGR-BabyBoom-Like from apomictic Pennisetum, a gene proven to be transferable to and functional in sexual pearl millet, rice, and maize. This review aims to summarize the current knowledge on parthenogenesis, the possible gene candidates also outside the grasses, and the use of these genes in plant breeding protocols. It shows that parthenogenesis is able to inherit and function independently from apomeiosis and endosperm formation, is expressed and active in the egg cell, and can induce embryogenesis in polyploid, diploid as well as haploid egg cells in plants. It also shows the importance of genes involved in the suppression of transcription and modifications thereof at one hand, and in embryogenesis for which transcription is allowed or artificially overexpressed on the othe

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of apomictic species. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. Using a hybrid approach that combines homology-based and de novo methods 27,048 protein-coding genes were predicted. Also repeats, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. In addition, we explored the histidine exonuclease APOLLO locus, related to apomixis in Boechera, and proposed model of its evolution through the series of duplications. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species.

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis. For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non -model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

The success or continuous existence of species requires continuous adaptation to changes in the environment to survive and contribute offspring to the next generation. Selection acts on the phenotype, which is in turn determined by the complex interplay of genetic, epigenetic and environmental variation. (Natural) selection leads to ‘survival of the fittest’ or best-adapted individuals to their local environment, ultimately determining which individuals contribute offspring to the next generation. Understanding the mechanisms by which epigenetic and genetic variation can arise and get passed on through generations determines our understanding of inheritance and evolution. Hitherto, the mechanistic understanding of genetics has shaped the scientific view of inheritance and evolution, leading to the gene-centered paradigm of Neo-Darwinism. However, recent studies indicate that besides genetic (DNA sequence) variation, epigenetic variation can also be transmitted between generations. Further studies on the properties and transgenerational dynamics of epigenetic variation are needed to enhance our understanding of heritability and evolution. Epigenetic variation has distinct properties and different transgenerational dynamics compared to genetic variation. Epigenetic variation helps to regulate gene expression and determines the different cell types and function in eukaryotes. The main function of DNA methylation, an important part of the epigenetic code, is to prevent the spread of selfish genetic elements in the genome and to establish the different cellular profiles observed in multicellular organisms. One differentiating feature of epigenetic variation compared to genetic variation is that (specific) epigenetic variation can arise under the influence of stress. This can enable a trans-generational stress-response of organisms which can have a positive influence on the phenotype and (natural) selection on either the (enhanced level of) transgenerational phenotypic pl

Rubus subgen. Rubus (bramble) is one of the large plant genera in Europe, consisting of only a few sexual biological species and at least 700 apomictic lineages. In this thesis, it is argued that the stabilised apomict lineages should best be regarded species, even if their distribution area doesn’t meet the requirements of a regional species as defined in several publications by Weber. Included is a checklist of Dutch bramble species, comprising 191 species belonging to Rubus subgen. Rubus. In the Netherlands, 97 of the 191 species are classified as regional species, with a distribution area diameter under 500 km. On the basis of distribution data of bramble species in Ireland, the UK, Denmark, Germany, and the Netherlands, 12 phytogeographical bramble-regions are distinguished. Although ecological factors play a role in the realisation of these regions, it is argued that the found patterns are primarily the result of evolutionary processes. The density and species composition of Rubus scrubs in the Netherlands is studied using landscape transects. At landscape scale, the bramble species in the scrubs are not randomly distributed, causing a spatial clustering of floristically similar bramble scrubs. It was concluded that only a part of the diversity of Rubus scrubs was accounted for in the Dutch national vegetation classification, and a new scrub type (the Rubetum taxandriae) was described on the basis of these findings. Rubus scrubs are an important biotope for rare shrub species and endemic Rubus species. Additionally, scrubs rich in bramble species are important because they provide foraging and nesting habitats for numerous vertebrates and invertebrates. It is recommended to include the apomict Rubus species in biodiversity accounts, for instance in the national standard list of plant species, as well as the Red List.

Currently, Brazil is the largest producer and exporter of tropical forage seeds in Latin America, where the demand for seeds quality and quantity has been leading the entire production chain. This study aimed at evaluating sexual interspecific hybrid populations in order to select genitors to assist in cultivated tropical pastures of Brachiaria genus diversification. The experiment was conducted at Embrapa Beef Cattle in Campo Grande-MS, during the January-August 2013 period. Half-brothers progenies test was performed, for the selection between and within families, and both the best genitors and the best individuals within progenies were selected on the basis of progeny. The experimental design was a randomized block with ten replicates and five individually analyzed plants for total and leaf dry matter production, leaf percentage, leaf:stem ratio, and sprouting capacity. Considering a selection intensity of 1 % of the selected hybrids, 100 % of the products originate from the best progenitor cross, with selection gains ranging from 11.07 % to 30.39 %. These results allow us to affirm that it is possible to make a selection of the best individuals with significant gains in the breeding program., Atualmente o Brasil é o maior produtor e exportador de sementes forrageiras tropicais da América Latina, em que as demandas por qualidade e quantidade de sementes vêm impulsionando toda a cadeia produtiva. Com este trabalho objetivou-se avaliar populações híbridas interespecíficas sexuais visando a selecionar genitoras para colaborar na diversificação de pastagens tropicais cultivadas do gênero Brachiaria. O experimento foi realizado na Embrapa Gado de Corte em Campo Grande-MS, no período de janeiro a agosto de 2013. Foi realizado teste de progênies de meio irmãos, visando a seleção entre e dentro de famílias, sendo selecionados os melhores genitores com base na progênie e, também, os melhores indivíduos dentro das progênies. O delineamento experimental foi em blocos ao acaso, com dez repetições e parcelas com cinco plantas, avaliadas individualmente para produção de massa seca total e foliar, porcentagem de lâminas foliares, relação lâmina foliar:pseudocolmo e capacidade de rebrota. Adotando-se a intensidade de seleção de 1 % dos híbridos selecionados, 100 % são produtos do cruzamento das melhores progenitoras, com ganhos de seleção variando de 11,07 a 30,39 %. Estes resultados permitem afirmar que é possível fazer a seleção dos melhores indivíduos com ganhos expressivos ao programa de melhoramento.

Epigenetic variation, such as changes in DNA methylations, regulatory small RNAs (sRNAs) and chromatin modifications can be induced by environmental stress. There is increasing information that such induced epigenetic modifications can be transmitted to offspring, potentially mediating adaptive transgenerational responses to environmental changes. However, it is unclear if this phenomenon is common and relevant for adaptation under natural conditions. My thesis study aimed to examine epigenetic inheritance in common and widespread apomictic dandelions (Taraxacum officinale Wig.). Due to their asexual reproduction mode by producing clonal seeds offspring from seeds are genetically uniform and thus suitable to investigate epigenetic effects that are not confounded with genetic variation. I exposed apomictic dandelion lineages to drought and salicylic acid (SA) stress, which induces plant defense responses following pathogen attack, and found effects on patterns of DNA methylation up to two stress-free offspring generations after exposure. However, a heritable stress signal was not present in all tests and was stress- and lineage-dependent. Drought stress triggered a weak and lineage-dependent signal that was lost again in the second offspring generation. SA treatment revealed a stress-related increased rate of DNA methylation changes in the two offspring generations, but no stress signal was found in the stressed generation itself. I also observed changes in small RNA production due the drought and SA stress experienced two generations ago. These transgenerational sRNA effects showed association with gene functions related to grandparental drought and SA stress, which suggests functional relevance of the transgenerational effects. I used a reciprocal transplantation field experiment to investigate whether exposing dandelions to natural field stresses also triggers DNA methylation changes. The experiment revealed evidence of adaptive divergence between the populations

Background: Asexual reproduction has the potential to enhance deleterious mutation accumulation and to constrain adaptive evolution. One source of mutations that can be especially relevant in recent asexuals is activity of transposable elements (TEs), which may have experienced selection for high transposition rates in sexual ancestor populations. Predictions of genomic divergence under asexual reproduction therefore likely include a large contribution of transposable elements but limited adaptive divergence. For plants empirical insight into genome divergence under asexual reproduction remains limited. Here, we characterize expression divergence between clone members of a single apomictic lineage of the common dandelion (Taraxacum officinale) to contribute to our knowledge of genome evolution under asexuality. Results: Using RNA-Seq, we show that about one third of heritable divergence within the apomictic lineage is driven by TEs and TE-related gene activity. In addition, we identify non-random transcriptional differences in pathways related to acyl-lipid and abscisic acid metabolisms which might reflect functional divergence within the apomictic lineage. We analyze SNPs in the transcriptome to assess genetic divergence between the apomictic clone members and reveal that heritable expression differences between the accessions are not explained simply by genome-wide genetic divergence. Conclusion: The present study depicts a first effort towards a more complete understanding of apomictic plant genome evolution. We identify abundant TE activity and ecologically relevant functional genes and pathways affecting heritable within-lineage expression divergence. These findings offer valuable resources for future work looking at epigenetic silencing and Cis-regulation of gene expression with particular emphasis on the effects of TE activity on asexual species' genome.

Illumina sequencing Trinity assembly

In this review, we report on the most important ideas and features in the field of fern evolution, with particular interest into the main evolutionary mechanisms that shaped the evolutionary history of their diversification. An exhaustive account for the bibliographical corpus on the matter falls beyond the space and extent of this work; instead, we will try to focus the different issues by including featured examples from both historical and recent approaches to the study of the evolutionary mechanisms in ferns., En esta revisión, informamos sobre las ideas y características más importantes en el campo de la evolución de los helechos, con un interés particular en los principales mecanismos evolutivos que dieron forma a la historia evolutiva de su diversificación. Una relación exhaustiva de toda la bibliografía de este asunto va más allá del alcance de este trabajo, en su lugar, vamos a tratar de enfocar los diferentes temas con la inclusión de ejemplos destacados, tanto históricos como recientes, en el estudio de los mecanismos evolutivos de los helechos.

Limonium quinnii and L. rhodense (Plumbaginaceae) are here described from the maritime cliffs of Rhodes Island (Greece). These new species can be included in the Limonium palmare aggregate, a group highly diversified in the eastern Mediterranean but still poorly known. The differences among L. quinnii and L. rhodense with the related taxa are provided and discussed. Data on the breeding systems, ecology and distribution of both species are also given.

The region coding for the second largest subunit of RNA polymerase II (RPB2) was explored for resolving interspecific relationships in Arnica and lower level taxa in general. The region between exons 17 and 23 was cloned and sequenced for 33 accessions of Arnica and four outgroup taxa. Three paralogues of the RPB2-d copy (RPB2-dA, B and C) were detected in Arnica and outgroup taxa, indicating that the duplications must have occurred before the divergence of Arnica. Parsimony and Bayesian analyses of separate alignments of the three copies reveal complex patterns in Arnica, likely reflecting a history of lineage sorting in combination with apomixis, polyploidization, and possibly hybridization. Cloned sequences of some taxa do not form monophyletic clades within paralogues, but form multiple strongly supported clades with sequences of other taxa. Some well supported groups are present in more than one paralogue and many groups are in line with earlier hypotheses regarding interspecific relationships within the genus. Low levels of homoplasy in combination with relatively high sequence variation indicates that the introns of the RPB2 region could be suitable for phylogenetic studies in low level taxonomy.

• Background and Aims Large scale ploidy surveys using flow cytometry have become an essential tool to study plant genome dynamics and to get insights into the mechanisms and genetic barriers framing ploidy diversity. Being confirmed as an ideal complement to traditional techniques such as chromosome counting, the analysis of cytotype diversity in plant systems such as Sorbus will provide primary investigation into the potential patterns and the evolutionary implications of hybrid speciation. • Methods Ploidy levels were assessed by means of the relative nuclear DNA content using propidium iodide flow cytometry in 474 Sorbus samples collected from 65 populations in Southern Wales and South-West England. Statistical tests were applied to evaluate the utility of this technique to confidently discriminate ploidy in the genus. • Key results Flow cytometric profiles revealed the presence of five cytotypes (2x, 3x, 4x and 5x), confirming in many cases chromosome counts previously reported and demonstrating cytotype heterogeneity within specific Sorbus aggregates. Diploid cytotypes were restricted to the potential parental species and homoploid hybrids. Most of the samples processed were polyploid. The occurrence of the pentaploid cytotype had previously only been reported from a single specimen; it is now confirmed for two pentaploid taxa occurring at different sites. • Conclusions FCM has been proved a useful method to carry out population surveys to study ploidy diversity, revealing some cytoypes which occur at very low frequencies. The noteworthy coexistence of several cytotypes in Sorbus populations has been probably facilitated by the overlapping distribution of many of the species studied, which might explain as well the high incidence of potential hybrid apomictic polyploids. These results will provide a solid baseline for molecular research aiming to better understand the genetic pathways controlling the formation and establishment of polyploid Sorbus.

¿ Background and Aims: Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation. ¿ Methods: Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of 'Tardivo di Ciaculli' mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype ('Carrizo' citrange; Citrus sinensis ¿ Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries. ¿ Key Results: Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area. ¿ Conclusions: Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression o

After a transition from sexuality to asexuality, the evolutionary dynamics in apomictic lineages will largely depend on the frequency of recombination. We evaluated the presence and extent of asexuality and recombination within populations of the Easter daisy, Townsendia hookeri, from the Yukon Territory, Canada. Amplified fragment-length polymorphism (AFLP) fingerprints were used to genotype 78 individuals from four populations. Multilocus AFLP genotypes from each population were subjected to four tests for deviations from free recombination among loci, and the long-term frequency of sexuality was estimated for each population with a novel procedure. In addition, a sample of individuals was surveyed for genome size using flow cytometry, and pollen was assayed for male fertility. One male-fertile, diploid population showed evidence of rampant recombination. Two male-sterile populations (i.e., with aborted anthers) were tetraploid and asexual. The remaining population was male-sterile and included both triploids and tetraploids. Evidence of both sexuality and asexuality was uncovered in this mixed-ploidy population, at an equilibrium rate of approximately three sexual events every two generations. The presence and extent of sexuality differed with ploidy, while cryptic sex was uncovered within a morphologically asexual population, thus reinforcing the power of genome surveys to assess reproductive dynamics at the limit of a plant's geographical range.

Alchemilla (the lady’s mantles) is a well known but inconspicuous group in the Rosaceae, notable for its ornamental leaves and pharmaceutical properties. The systematics of Alchemilla has remained poorly understood, most likely due to confusion resulting from apomixis, polyploidisation and hybridisation, which are frequently observed in the group, and which have led to the description of a large number of (micro-) species. A molecular phylogeny of the genus, including all sections of Alchemilla and Lachemilla as well as five representatives of Aphanes, based on the analysis of the chloroplast trnL–trnF and the nuclear ITS regions is presented here. Gene phylogenies reconstructed from the nuclear and chloroplast sequence data were largely congruent. Limited conflict between the data partitions was observed with respect to a small number of taxa. This is likely to be the result of hybridisation/introgression or incomplete lineage sorting. Four distinct clades were resolved, corresponding to major geographical division and life forms: Eurasian Alchemilla, annual Aphanes, South American Lachemilla and African Alchemilla. We argue for a wider circumscription of the genus Alchemilla, including Lachemilla and Aphanes, based on the morphology and the phylogenetic relationships between the different clades.

This report was commissioned by the COGEM in order to reassess the knowledge basis on outcrossing in the plant species presently categorised as basically having a selfing breeding system in the so-called “Lijst van inhullingsverplichtingen” (“ list of inflorescence bagging regulations”), formerly called “Appendix C”

We conducted a cytogenetic study of sexual lines of Boechera stricta and Boechera holboellii (2n = 14) and seven diploid apomictic accessions of their interspecific hybrid Boechera divaricarpa and B. holboellii (2n = 14 or 15). By studying chromosome morphology, rDNA repeats, genome painting, male meiosis, pollen morphology, and flow-cytometry seed screens, we revealed an unexpected plethora of chromosome forms, pairing behavior, and hybrid composition in all apomictic lines. Genome painting demonstrated that the apomicts are alloploid with variable numbers of B. stricta and B. holboellii-like chromosomes. We assume that large-scale homeologous chromosome substitutions took place in the apomictic hybrids that resulted from recurrent diploid-polyploid transitions through restitutional meiosis and polyploicly-diploid transitions through reductional meiosis. A second peculiarity was the presence of a largely heterochromatic chromosome (Het) in all apomictic accessions (2n = 14 and 15) and an additional smaller chromosome (Den in the aneuploids (2n = 15). Both chromosomes share repetitive pericentromere repeats with those from the sexual B. stricta, suggesting that they originated from this species. Pairing and behavior at meiosis I of the Het share features with both Y and B chromosomes and suggest that the Del arose from a translocation event or homeologous recombination between a B. holboellii (or related taxon) and a B. stricta chromosome. Based on its presence exclusively in apomictic accessions, we propose that the Het chromosome plays a role in the genetic control of apomixis.

Microsatellite markers were used to evaluate the genetic diversity and population genetic structure in the critically endangered Limonium dufourii(Plumbaginaceae), a highly endemic triploid species from the coasts of eastern Spain. Sixty-five alleles from 13 microsatellite regions were amplified in a sample of 122 individuals collected from the six extant populations. Microsatellite patterns were consistent with the triploid nature of L. dufourii. Alleles were unambiguously assigned to two different parental subgenomes in this hybrid species and the greater contribution of the diploid parental subgenome was confirmed. Eleven, 25 and 26 multilocus genotypes were recorded from the haploid, diploid and from the combined information of both subgenomes, respectively. Genetic diversity was mostly distributed among populations (72.06% of the total genetic variation). Genotypes from Marjal del Moro populations grouped into two highly structured clusters (88.41% of the total variance). The observed patterns of distribution of genetic diversity are interpreted to result from multiple hybridization events and isolation between populations. Threats to this species are mainly anthropogenic (urbanization and tourism pressure), although stochastic risks cannot be ignored. Therefore, in order to preserve extant genetic variation of L. dufourii, in situ strategies such as the preservation of its habitat are a high priority. Several recommendations in order to assist ex situ measures to guarantee the success of conservation strategies and maintain the relationships between individuals and populations are proposed.

Geographic patterns of parthenogenesis and the number of transitions from sexual diploidy to asexual (apomictic) autopolyploidy were examined for 40 populations of the Easter daisy, Townsendia hookeri. Analyses of pollen diameter and stainability characterized 15 sexual diploid and 25 apomictic polyploid populations from throughout the plant's western North American range. Sexual diploids were restricted to two Wisconsin refugia: Colorado/Wyoming, south of the ice sheets, and northern Yukon/Beringia. Chloroplast DNA sequencing uncovered 17 polymorphisms within the ndhF gene and trnK intron, yielding 10 haplotypes. Phylogenetic analyses indicated that five exclusively polyploid haplotypes were derived from four haplotypes that are shared among ploidies, conservatively inferring a minimum of four origins of apomictic polyploidy. Three of these apomictic polyploid origins were derived from southern sexual diploids, while the fourth origin was derived from northern sexual diploids. Analyses of regional diversity were suggestive of a formerly broad distribution for sexual diploids that has become subsequently fragmented, possibly due to the last round of glaciation. As sexual diploids were exclusively found north and south of the glacial maximum, while formerly glaciated areas were exclusively inhabited by asexual polyploids derived from both northern and southern sexual lineages, it is more likely that patterns of glaciation, as opposed to a particular latitudinal trend, played a causal role in the establishment of the observed pattern of geographic parthenogenesis in Easter daisies.

We have developed a new model for mating system analysis, which attempts to distinguish among alternative modes of self-oriented mating within populations. This model jointly estimates the rates of outcrossing, selfing, automixis and apomixis, through the use of information in the family structure given by dominant genetic marker data. The method is presented, its statistical properties evaluated, and is applied to three arctic Easter daisy populations, one consisting of diploids, the other two of tetraploids. The tetraploids are predominantly male sterile and reported to be apomictic while the diploids are male fertile. In each Easter daisy population, 10 maternal arrays of six progeny were assayed for amplified fragment length polymorphism markers. Estimates, confirmed with likelihood ratio tests of mating hypotheses, showed apomixis to be predominant in all populations (ca. 70%), but selfing or automixis was moderate (ca. 25%) in tetraploids. It was difficult to distinguish selfing from automixis, and simulations confirm that with even very large sample sizes, the estimates have a very strong negative statistical correlation, for example, they are not independent. No selfing or automixis was apparent in the diploid population, instead, moderate levels of outcrossing were detected (23%). Low but significant levels of outcrossing (2-4%) seemed to occur in the male-sterile tetraploid populations; this may be due to genotyping error of this level. Overall, this study shows apomixis can be partial, and provides evidence for higher levels of inbreeding in polyploids compared to diploids and for significant levels of apomixis in a diploid plant population.

Some plants have gained the ability to produce seed without fertilisation. This alternative to sexual reproduction, known as apomixis occurs most frequently in species of the families of the grasses, roses and composites, and mostly in polyploids and is considered one of the ways to escape from hybrid sterility. An impressive number of apomictic mechanisms have so far been described; most of them with different developmental modes of embryo and endosperm. The trait is potential very promising for producing uniform seeds at high efficiency and low cost, and belongs therefore since long as one to the most wanted grails of the plant breeding community. This PhD thesis points at several fascinating genetical and chromosomal aspects of apomixis using the model species Arabidopsis thaliana and the apomicts of the closely related Boechera holboellii complex. Focus is on two different studies in apomixis research: 1) unravelling chromosome organisation and genomic composition of some of the natural diploid apomicts of Boechera and 2) genetic analysis of AtSERK 1 transformed Arabidopsis for engineering apomictic elements in this sexually propagating diploid.Boechera's unsurpassed suitability for research of natural apomicts is its impressive genomic plasticity, large geographical distribution and an endless source of different accessions. Quite exceptional is that apomixis also can occur at the diploid level. The chromosome study involving the two sexual diploid species, B. holboellii and B. stricta (2n=14), and six apomictic accessions of B. holboellii and B. X divaricarpa (2n=14 and 15) demonstrated obvious differences between the sexual and apomictic taxa. DAPI fluorescence and FISH with rDNA probes on mitotic cell spreads clearly uncovered two different aberrantchromosome, including a largely heterochromatic chromosome ( Het ) and one much smaller chromosome (Del). Moreover, striking variation between the assumed homologues and great differences between the chromosome po

Apomixis is asexual plant reproduction through seeds that are produced with no prior genome reduction or fertilisation. The use of apomixis holds enormous potential for future plant breeding and production. The outcome of the research described in this thesis suggests that the genetic basis for apomixis in Taraxacum (dandelions) comprises three major dominant genes whose timely expression may be influenced by modifier genes. Its also suggests that genome interactions effect successful establishment of apomixis in polyploid hybrids of sexual x apomictic dandelions. The work consisted of cytogenetic and embryological studies performed at the Laboratory of Genetics, Wageningen University, and the analysis of inheritance of apomixis in sexual x apomict crossings performed at the Netherlands Institute of Ecology, Centre for Terrestrial Ecology, (NIOO-CTO), Heteren, the Netherlands. A theoretical model for the Taraxacum type of apomixis is described. As a conclusion, we discuss several aspects of the evolution of apomixis in Taraxacum which serve to explain why Taraxacum is a very successful genus.

Apomixis is asexual reproduction leading to a seed. It is the predominant mode of reproduction in grasses of the genus Brachiaria.Brachiaria decumbens is one of the widely cultivated species of grasses in the tropical areas. The knowledge about the mechanisms of apomixis and the possibility of controlling this system will extend the possibilities of introducing variability in Brachiaria and will maintain the apomictic character desirable for its propagation.A comparison of gametophyte development between a sexual diploid (D4) and an apomictic tetraploid (D58) genotype of Brachiaria decumbens was made.A calendar made based on the development in time show the differences between sexual and apomictic development. The aposporic embryo sac of the Panicum type grows faster than the Polygonum meiotic one. The apospore initials in Brachiaria decumbens were observed very early in the ovule, next to archespore or to the meiocyte. During microspore and ovule development, the pattern of callose deposition was different in sexual and apomictic plants.The distribution of the nuclei, positioned in one pole in the aposporic embryo sac allowed to differentiate them from the bipolar distribution of the nuclei observed in meiotic embryo sacs of the sexual plants. Cellularisation took place early in aposporic embryo sac and by the time of anthesis embryogenesis had already started in the apomict. In ovules of apomictic plants many embryo sacs developed. Apomixis expressed in the female side, had also an influence on the male side.Histochemical tests developed to localise the in situ sucrose synthase or invertase activity were used during ovule development in an apomictic and a sexual genotype of Brachiaria decumbens . A delay of activity of both enzymes was observed in apomictic compared to the sexual development. This delay was also reflected by the amount of carbohydrates detected in a HPLC assay. Furthermore antibodies were used to localise the enzyme in the cell. The higher level o

The genus Nigritella (Orchidaceae-Orchidinae) is a complex of species at four different ploidy levels. Fourteen taxa at species or subspecies level have been distinguished. The triploid N. nigra ssp. nigra is endemic to Scandinavia, whereas the remaining taxa are confined to different mountain regions in Central and Southern Europe. The polyploids are of hybrid origin and have arisen from taxa at lower ploidy levels by allopolyploidization. By using a recently developed PCR based DNA fingerprinting technique (AFLP), I have investigated the evolutionary history of the genus. I have also considered the genetic relationship between Nigritella and the closely related genus Gymnadenia. Tetraploid members of Nigritella are supposed to be the result of hybridization between diploids and triploids. The latter may have contributed unreduced gametes. Three different ancient triploid taxa would explain the origin of all present-day tetraploids. Nigritella nigra ssp. nigra and the other – today probably extinct – triploids may have survived the last glacial maximum in different Central and Southern European refugia. They may then have mixed with populations of diploids, thereby giving rise to new, tetraploid taxa. All the polyploids displayed several DNA bands that were rare or absent in the extant diploids. It is therefore reasonable to assume that ancestral diploids, somewhat different from present-day taxa, have been involved – at least in the formation of the intermediate triploid forms. The diploid samples only partly clustered in accordance with morphologically defined entities. Plants with the same geographic origin tended to cluster together, regardless of taxonomic belonging. The polyploids are mainly apomicts, but in spite of this fact, certain taxa varied in a complex way. This pattern could partly be explained by recurrent polyploidization, by which a given taxon may have several independent origins. The number of origins was estimated for different taxa, and varied

A calendar of female sporogenesis and gametogenesis was made for both apomictic tetraploid (2n = 4x = 36) Brachiaria brizantha and Brachiaria decumbens and their apomictic Fl hybrids with sexual tetraploid (2n = 4x = 36) Brachiaria ruziziensis. Microgametogenesis was used as a reference. Apospory was facultative in both species and hybrids. Environmental conditions had variable effects on the level of apomixis according to each genotype. Ratios of segregation into sexuals and apomicts in the interspecific hybrids suggest an oligogenic determinism with dominant apomixis in the genus Brachiaria. Highly apomictic and partially male fertile hybrids were identified and will be used in an improvement program to transfer genes for apomixis into the sexual species B. ruziziensis.

Apomixie bij Sorbus, algemene kenmerken van het subgenus, natuurlijk areaal, vermeerdering en beschrijving van geschikte Sorbus-cultivars voor de boomkwekerij

Several embryological and cytological techniques were developed to determine the degree of aposporous parthenogenesis vs. sexuality in different genotypes of the facultative apomictic grass Poa pratensis L. Fixed embryo sacs were mechanically isolated and individually examined with phase-contrast and interference-contrast light microscopes. The degree of apospory was calculated as the ratio of embryo sacs with an embryo but without endosperm and the total number of embryo sacs studied per plant. The degree of apospory varied between 9 and 76% in nine plants. Photocytometry and flow cytometry were used to determine ploidy levels in embryo and endosperm of individual embryo sacs in order to determine their origin, asposporous vs. sexual. Results based on the calculated degree of apospory agreed with the degree of apomixis deduced from homogeneity scores in field progeny tests. Callose deposition on the megaspore mother cell during megasporogenesis was studied in the same set of genotypes and the percentage of abnormal, micropylar callose deposition closely agreed with results both of the embryo sac analysis and the progeny tests. Combined use of these techniques yields quantitative data on the aposporous tendency of individual genotypes of Poa pratensis.

Colchicine induced tetraploid (2n = 4x = 36) Brachiaria ruziziensis were used as female parent in crosses with apomictic tetraploid species (2n = 4x = 36) Brachiaria decumbens and Brachiaria brizantha. Tetraploid B. ruziziensis pollinated with B. decumbens set significantly more seed than selfed or crossed with B. brizantha. The crossability between B. ruziziensis and B. decumbens is also better than between B. ruziziensis and B. brizantha. In addition, hybrid seedlings obtained in crosses involving B. brizantha are more frequently lethal. All the viable F, hybrids are tetraploid with 36 chromosomes. Meiotic chromosome behaviour suggest that the three species belong to the same genomic group and therefore the same agamic complex. Chromosome associations at metaphase I do not allow to identify fertile and sterile hybrids. The interspecific hybrids averaged a lower fertility than their female parent, but some hybrids were more fertile than their apomictic male parent.

The cultivated potato, Solanum tuberosum L., is a highly heterozygous autotetraploid (2n=4x=48) plant species, which after its introduction into Europe in the 16th century has become one of the world's major food crops. The potato has traditionally been grown from tubers. However, as tubers are an excellent substrate for many pathogens and parasites, it is extremely difficult and expensive to produce healthy seed tubers. Most developing countries lack both the knowledge and infrastructure required for the efficient production of healthy seed tubers and the currency for its importation and distribution. As a consequence small farmers in developing countries are generally forced to use diseased tubers from a previous harvest, which may result in dramatic yield losses. In response to the urgent need of cheap but healthy plant material the International Potato Center (CIP) in Peru has, since the Planning Conference on this subject in 1979, propagated the new technology of growing potatoes from true seeds, True potato seeds are relatively easy and cheap to produce and even when harvested from heavily diseased plants generally do not carry any diseases. However one of the major problems in breeding potato varieties that can be maintained and grown from true seeds is the lack of uniformity. True seed progeny of existing varieties or inter-varietal hybrids is mostly highly heterogeneous due to the extreme heterozygosity of the potato.Several methods to synthesize sufficiently uniform true potato seed varieties have been proposed. One of these methods takes advantage of the frequent occurrence of numerically unreduced (2n) gametes in wild and cultivated diploid potato species, which enables the production of hybrid tetraploid progeny from tetraploid-diploid (unilateral sexual polyploidization) or diploid-diploid (bilateral sexual polyploidization) matings. The vigour and uniformity of tetraploid populations produced in this way, largely depends on the mode of 2n-gamete forma