Highly efficient sulfimidation of 1,3-dithianes by Cu(I) complexes

A series of four Cu(I) complexes were tested for sulfimidation of 1,3-dithianes in the presence of [N-(p-tolysulfonyl)imino]phenyliodinane (PhI=NTs) as the nitrene-transfer agent. Cu(TMPhen)(PPh3)Br is an efficient catalyst with more than 90% yield of the corresponding product with less reaction time as compared to the literature copper(I) complexes

A Continental-Wide Perspective: The Genepool of Nuclear Encoded Ribosomal DNA and Single-Copy Gene Sequences in North American Boechera (Brassicaceae)

74 of the currently accepted 111 taxa of the North American genus Boechera (Brassicaceae) were subject to pyhlogenetic reconstruction and network analysis. The dataset comprised 911 accessions for which ITS sequences were analyzed. Phylogenetic analyses yielded largely unresolved trees. Together with the network analysis confirming this result this can be interpreted as an indication for multiple, independent, and rapid diversification events. Network analyses were superimposed with datasets describing i) geographical distribution, ii) taxonomy, iii) reproductive mode, and iv) distribution history based on phylogeographic evidence. Our results provide first direct evidence for enormous reticulate evolution in the entire genus and give further insights into the evolutionary history of this complex genus on a continental scale. In addition two novel single-copy gene markers, orthologues of the Arabidopsis thaliana genes At2g25920 and At3g18900, were analyzed for subsets of taxa and confirmed the findings obtained through the ITS data

Chromosome studies and genetic analysis of natural and synthetic apomictic model species

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 portraits of the accessions was observed, suggesting structural karyotype heterozygosity in this material. Additionalstudieson male meiosis of the same sexual diploids and apomicts demonstrated a plethora of meiotic variants, pollen size, seed germination and nuclear contents of the seeds. Some of the apomictic accessions displayed full chromosome pairing and recombination, and reductional meiosis, whereas others demonstrated pairing failure, and skewed chromosome segregations. The course of meiosis and pollen size indicated that both n and 2n gametes were formed. In three accessions we observed chiasmate bonds between the Het chromosome and one of the autosomes; whereas theDelchromosome was associated with two other chromosomes.Genome in situ hybridization was carried out to establish the assumed hybrid composition of the apomictic B. holboellii and B. X divaricarpa accessions. In the first part we performed hybridisations with total genomic DNA of the diploid sexual B. stricta as probe and blocked with genomic DNA from B. holboellii . Chromosome complements showed fluorescent signals on the pericentromere regions of only the B. stricta chromosomes so that the parental chromosomes could be distinguished in the hybrid. An additional two-colour genome painting was developed with the simultaneous hybridisation with both B. stricta andB. holboellii probes and blocking with total genomic DNA of Arabidopsis thaliana in order to further improve the discrimination of the parental chromosomes. This genome painting study confirmed that chromosomes have evolved repeat differentiation at the pericentromere regions enabling a clear distinction of the B. holboellii and B. stricta chromosomes in the hybrids. This result has made clear that Boechera apomicts are alloploid, with different balanced or unbalanced combinations of B. holboellii and B. stricta chromosomes. Most likely such differences in genomic constitutions result from recurrent diploid-polyploid and polyploid-diploid conversions, the latter with reductional meiosis generating haploid gametes with novel combinations of homoeologous chromosomes.This genome in situ hybridisation technique revealed that B. holboellii and B. stricta have undergone dramatic evolutionary changes in the repetitive sequences in the pericentromere regions of their chromosomes, producing species-specific FISH banding. The genome painting also revealed that the Het chromosome has B. stricta repeats suggesting that this highly heterochromatic chromosome originated from a B. stricta autosome, which shows that Het resembles more a Y chromosome than a B chromosome. The Del chromosome demonstrated only a small stricta segment, which at metaphase I was found associated with two B. holboe­llii chromosomes, thus suggesting that this chromosome is likely a holboellii / stricta translocation or recombinant chromosome. The outcome of this molecular cytogenetic study has enabled us to formulate a new model on the origin or apomictic accessions and evolutionary processes of the aberrant chromosomes. Essential in the hypothesis is the assumption of an epigenetic modification in the newborn (allo)polyploidhybrid. This modification is accompanied or resulted from chromosome pairing impairments at meiotic prophase, and in the longer term led to isolation from crossover recombination, accumulation of repetitive sequences and heterochromatinization of a part of that chromosome involved. Looking at only these few apomicts it is tempting to believe that the Het chromosome in all accessions originated from the same ancestral pro- Het chromosome and that this chromosome plays a key role in the genetic elements required for the apomictic pathway.In the second part of the thesis a screening method is presented to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of Single Sequence Length Polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 ( AtSERK 1) gene expressed under the control of two different promoters: the AtLTP1 and the AtDMC1 in sexual Arabidopsis plants. In only one of the four tested F2 transgenic populations, i.e. , expressing the AtLTP1::AtSERK1 construct we observed two plants (1.8%) with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for number of heterozygous loci, it was concluded that these two putative apomictic plants resulted from either incidental recombination events displaying the genotype of the parent, or that they lost their clonal ability in the next generation. Although this genotype screening in the AtSERK 1 transformant offspring not yet resulted in potential apomicts, further refinement and automation are expected to produce an engineered apomictic system that enables identification of a small number of plants exhibiting heterozygosity fixation in an otherwise fully sexual population. Ultimately, if genes that show the potential to confer apomixis need to be introduced in crop species one will need such screening systems operative in the normal sexual background of the crop. Future experiments of AtSERK 1 may also focus on target genes under control of AtSERK 1 -mediated signalling or in combination with other genes involved in somatic embryogenesis

Asexual reproduction in a close relative of Arabidopsis: a genetic investigation of apomixis in Boechera (Brassicaceae)

Understanding apomixis (asexual reproduction through seeds) is of great interest to both plant breeders and evolutionary biologists. The genus Boechera is an excellent system for studying apomixis because of its close relationship to Arabidopsis, the o

Epigenetic changes and transposon reactivation in Thai rice hybrids. Molecular Breeding

Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we present an analysis of epigenome changes in rice hybrids that were obtained by crossing rice cultivars, most of them of indica type and Thai origin. Comparing amplified fragment length polymorphism (AFLP) fingerprints of twenty-four cultivars, we calculated Nei’s indexes for measuring genetic relationships. Epigenetic changes in their hybrids were established using methylation-sensitive AFLP fingerprinting and transposon display of the rice transposable elements (TEs) Stowaway Os-1 and Mashu, leading to the question whether the relationship between parental genomes is a predictor of epigenome changes, TE reactivation and changes in TE methylation. Our study now reveals that the genetic relationship between the parents and DNA methylation changes in their hybrids is not significantly correlated. Moreover, genetic distance correlates only weakly with Mashu reactivation, whereas it does not correlate with Stowaway Os-1 reactivation. Our observations also suggest that epigenome changes in the hybrids are localized events affecting specific chromosomal regions and transposons rather than affecting the genomic methylation landscape as a whole. The weak correlation between genetic distance and Mashu methylation and reactivation points at only limited influence of genetic background on the epigenetic status of the transposon. Our study further demonstrates that hybridizations between and among specific japonica and indica cultivars induce both genomic DNA methylation and reactivation/methylation change in the Stowaway Os-1 and Mashu transposons. The observed epigenetic changes seem to affect the transposons in a clear manner, partly driven by stochastic processes, which may account for a broader phenotypic plasticity of the hybrids. A better understanding of the epigenome changes leading to such transposon activation can lead to the development of novel tools for more variability in future rice breedin

Biogeographic distribution of polyploidy and B chromosomes in the apomictic Boechera holboellii complex

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploid individuals, whereas apomictic individuals are diploid or triploid and may additionally have B chromosomes. Using flow cytometry and karyotype analysis, we have shown that B chromosomes (a) occur in both diploid and triploid apomictic B. holboellii, (b) may occur in triploid B. drummondii, and (c) are dispensable for the plant. Both diploid and triploid karyotypes are found in multiple chloroplast haplotypes of both species, suggesting that triploid forms have originated multiple times during the evolution of this complex. B chromosome carriers are found in geographically and genetically distinct popu-lations, but it is unknown whether the extra chromosomes are shared by common descent (single origin) or have originated via introgressive hybridization and repeated transitions from diploidy to triploidy. Diploid plants containing the Bs reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. Finally, our analyses of pollen size and viability suggest that irregular chromosome segregation in some triploid lineages may lead to the generation of diploid individuals which carry the B chromosomes

Is the aneuploid chromosome in an apomictic Boechera holboellii a genuine B chromosome?

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploidy, whereas apomictic individuals can either be diploid, aneuploid or triploid. Aneuploid individuals are found in geographically and genetically distinct populations and contain a single extra chromosome. It is unknown whether the supernumerary chromosomes are shared by common descent (single origin) or have originated via introgressive hybridizations associated with the repeated transition from diploidy to triploidy. Diploid plants containing the extra chromosome(s) reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. In this study we compared flow cytometry data, chromosome morphology, and DNA sequences of sexual diploid and apomictic aneuploids in order to establish whether the extra chromosome fits the classical concept of a B chromosome. Karyotype analyses revealed that the supernumerary chromosome in the metaphase complement is heterochromatic and often smaller than the A chromosomes, and differs in length between apomictic plants from different populations. DNA sequence analyses furthermore demonstrated elevated levels of non-synonymous substitutions in one of the alleles, likely that on the aneuploid chromosome. Although the extra chromosome in apomictic Boechera does not go through normal reductional meiosis, in which it may get eliminated or accumulated by a B-chromosome-specific process, its variable size and heterochromatic nature does meet the remaining criteria for a genuine B chromosome in other species. Its prevalence and conserved genetic composition nonetheless implies that this chromosome, if truly a B, may be atypical with respect to its influence on its carriers. Copyright (C) 2004 S. Karger AG, Basel