Karyotype and nuclear DNA content of hexa-, octo-, and duodecaploid lines of Bromus subgen. Ceratochloa

The subgenus Ceratochloa of the genus Bromus includes a number of closely related allopolyploid forms or species that present a difficult taxonomic problem. The present work combines data concerning chromosome length, heterochromatin distribution and nuclear genome size of different 6x, 8x and 12x accessions in this subgenus. Special attention is paid to the karyotype structure and genomic constitution of duodecaploid plants recently found in South America. Hexaploid lineages possess six almost indistinguishable genomes and a nuclear DNA content between 12.72 pg and 15.10 pg (mean 1Cx value = 2.32 pg), whereas octoploid lineages contain the same six genomes (AABBCC) plus two that are characterized by longer chromosomes and a greater DNA content (1Cx = 4.47 pg). Two duodecaploid accessions found in South America resemble each other and apparently differ from the North American duodecaploid B. arizonicus as regards chromosome size and nuclear DNA content (40.00 and 40.50 pg vs. 27.59 pg). These observations suggest that the South American duodecaploids represent a separate evolutionary lineage of the B. subgenus Ceratochloa, unrecognized heretofore.     

Translocations of Chromosome End-Segments and Facultative Heterochromatin Promote Meiotic Ring Formation in Evening Primroses

Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.     

Chloroplast nucleoids are highly dynamic in ploidy,number, and structure during angiosperm leaf development

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post‐meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI‐based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring‐shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20–750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20‐fold even within individual organelles, with average values between 2.6‐fold and 6.7‐fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70–130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600–3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed‐field electrophoresis, restriction of high‐molecular‐weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single‐stranded and double‐stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.     

Breakdown of the balanced lethals in Rhoeo: the structure of the Alethal Renner complex of the homozygotic stock of Rhoeo

Fluorescence in situ hybridization, base-specific fluorescence, C-banding and silver-staining were performed to reveal the cyto-molecular constitution of the karyotype in the bivalent-forming Rhoeo spathacea concolor. It was shown that the genome of this form is almost identical to the β-complex of the ring-forming rhoeos. In spite of some modifications in the arrangement of a few distal rDNA sites and in the amount of pericentromeric AT-rich heterochromatin, the alethal genome of the bivalent-forming Rhoeo seems segmentally unaltered. Thus, the breakdown of balanced lethals in Rhoeo is most likely uncoupled from creating new chromosome arm combinations.     

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.     

Tissue culture triggers chromosome alterations, amplification, and transposition of repeat sequences in Allium fistulosum.

Structural alterations in nuclei and chromosomes of cells derived from callus culture of Allium fistulosum have been studied with fluorescent in situ hybridization (FISH) using 5S ribosomal DNA (rDNA), 45S rDNA, and 375-bp repeat probes. A high frequency of chromosome abnormalities was found to be caused by the loss of telomere-located 375-bp repeats, chromosome fusion, and subsequent breakage-fusion-bridge cycles. Products of chromosome fusions and monocentric and regularly shaped chromosomes showed additional 375-bp repeat and 45S rDNA clusters at unusual sites, suggesting dynamic copy-number changes and transposition of these repeats. Southern hybridization revealed no differences in the 375-bp repeat and 45S rDNA repeat array order or the degree of methylation between DNA isolated from leaves or tissue-culture cells. In addition, protruding, spike-like structures positive for 375-bp repeats were identified on the surface of different-sized nuclei. Transmission electron microscopy analysis revealed the accumulation of densely packed chromatin within spike-like structures. Because root calyptra cells showed similar structures, it is likely that heterochromatic spike-like structures are a feature of nondividing cells at the onset of programmed cell death.     

Structural heterozygosity, duplication of telomeric (TTTAGGG)(n) clusters and B chromosome architecture in Tradescantia virginiana L

Fluorescent in situ hybridization, C-banding/DAPI, and CMA(3)-fluorescence were performed to reveal the cytomolecular constitution of the standard (A) and supernumerary (B) chromosomes of an autotetraploid Tradescantia virginiana L. The analyses show that translocations and/or inversions have occurred during the evolution of the T. virginiana karyotype, generating a significant level of structural heterozygosity. Regarding the structural level, the present paper confirms the occurrence of small subterminal duplications and/or inversions in T. virginiana already suggested by previous authors. Interestingly, many of the distal chromosome segments in T. virginiana possess duplicated subterminal telomere clusters, heterochromatin, 5S and 45S rDNAs orderly intermixed and share this complex cytomolecular architecture with the common type of a B chromosome. Based on the obtained results, it is proposed that in T. virginiana the B chromosome may have arisen via excision from the distal region of an A chromosome. The nascent B could have retained much of the ancestral sequence arrangement, including duplicated telomeric cluster(s), heterochromatin and rDNA, but developed a new centromere/kinetochore to successfully propagate through the cell cycle.     

Molecular marker systems for Oenothera genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.