Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis.

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.

Reproductive development in apomictic populations of Arabis holboellii (Brassicaceae).

Megasporogenesis, megagametogenesis and seed formation were analyzed cytologically in populations of Arabis holboellii originating from North America (Colorado) and Greenland. The Colorado population contained only triploid plants, while the Greenland population consisted of diploid and triploid plants. The penetrance of the apomictic trait was assessed at the level of embryo sac development. All populations showed facultative apomeiotic embryo sac development; however the penetrance of this trait differed between the populations. Apomeiotic and meiotic embryo sac development were characterized by diplosporous dyad formation (Taraxacum-type) and meiotic tetrad formation (Polygonum-type), respectively. Flow cytometric analyses of single mature seeds from all three populations suggest that only unreduced gametes participate in viable seed development. Pseudogamy was the predominant mode of endosperm formation; however, autonomous endosperm development was also observed. The fertilization of unreduced egg cells with unreduced pollen was observed at a low frequency in the Greenland populations. The mechanisms of apomictic reproduction in A. holboellii are discussed.

Apomixis and sexuality in Paspalum simplex: characterization of the mode of reproduction in segregating progenies by different methods.

Segregating progenies of crosses between sexual and apomictic genotypes of Paspalum simplex were analysed for the formation of meiotic versus aposporous embryo sacs, zygotic versus parthenogenetic embryos, and autonomous versus pseudogamous endosperms by using cytoembryological and flow cytometric analyses. Reduced and unreduced 8-nucleated embryo sacs were the final product of female gametophyte development in sexual and aposporous genotypes, respectively. An incomplete penetrance of parthenogenesis was detected in aposporous genotypes. The relative DNA content of endosperm nuclei revealed the normal 2:1 maternal to paternal ratio in sexuals and a 4:1 ratio in apomicts, indicating insensitivity of the apomictic genotypes to endosperm imprinting. Apospory, parthenogenesis and pseudogamy are located on a relatively large linkage group and are inherited together with previously developed molecular markers as a single genetic unit in segregating progenies.

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis.

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.35× larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (BIII hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin:: ß -glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.

Parthenogenetic egg cells of wheat: cellular and molecular studies.

The 'Salmon' system of wheat comprises three isogenic alloplasmic lines with either zygotic (aS) or autonomous, fertilisation-independent (cS kS) embryo development. While the initiation of embryogenesis from the isolated sexual egg cell depends on in vitro fertilisation, the corresponding parthenogenetic egg cell develops into an early embryo without fertilisation. This demonstrates that parthenogenesis is an inherent feature of the isolated egg cell. Based on this observation, we have constructed egg-cell-specific cDNA libraries and report first results of a sequencing project aimed at the isolation of putative egg-cell-specific and parthenogenesis-related genes.

An efficient screen for reproductive pathways using mature seeds of monocots and dicots.

Seed samples of 32 species (obligate and facultative sexuals and apomicts of monocots and dicots) were investigated by flow cytometry to reveal the pathway of reproduction. Ten different pathways of seed formation could be reconstructed considering whether the female and/or male gametes were reduced or unreduced, the embryos arose via the zygotic or parthenogenetic route and the endosperm via the pseudogamous or autonomous route. The screen is suited to select sporophytic or gametophytic mutants in sexual species, to identify pure sexual or obligate apomictic genotypes from facultative apomictic species, and to analyze the inheritance of the individual reproductive processes. Corresponding unique results are presented for Arabidopsis, Arabis, Hypericum and Poa. The screen of mature seeds by flow cytometry yielded more information about the reproductive behavior of individual plants than any other available test, and is very useful both in basic research and plant breeding.

A specific alpha-tubulin is associated with the initiation of parthenogenesis in 'Salmon' wheat lines.

The 'Salmon system' consists of isogenic but alloplasmic wheat lines with either sexual or autonomous embryo development. Using two-dimensional gel electrophoresis these lines have been screened for proteins potentially involved in the initiation of parthenogenesis. A temporally altered expression of the polypeptide 'P 115.1' in the sexual and parthenogenetic 'Salmon' lines seems to be related with the autonomous embryo formation. Around anthesis when most of the egg cells begin the parthenogenetic development, the polypeptide 'P 115.1' was present in ovaries of the parthenogenetic lines but not in ovaries of the sexual line. Moreover, this polypeptide is only expressed in the ovaries of amphidiploid parthenogenetic plants containing differentiated embryo sacs. It is absent from ovaries of the analogous polyhaploid plants, which lack any embryo sac structure within their ovules. Furthermore, the polypeptide was neither detectable in meristematic tissue of root tips nor in leaves. N-terminal amino acid sequencing identified 'P 115.1' as an alpha-tubulin. Thus, 'P 115.1' apparently represents an embryo sac-specific isoform of alpha-tubulin involved in the initiation of embryo development.

A pollen allergen-encoding gene is expressed in wheat ovaries.

To isolate genes specifically expressed at the initiation of plant embryo development we have applied a sensitive subtractive hybridization technique for three isogenic wheat lines of the so-called 'Salmon system' with either zygotic or autonomous embryo development. Here we present a gene sequence showing a high homology to grass pollen allergens of type II/III thought to be expressed in pollen tissue only. Surprisingly, the pollen allergen-like sequence, designated Tri a III, is also expressed in gynoecia of the sexual, male fertile wheat line '(aestivum)-Salmon', whereas the two parthenogenetic and male sterile wheat lines '(caudata)-Salmon' and '(kotschyi)-Salmon' completely lack any Tri a III transcript. Our data suggest a positive correlation between the expression of this clone and the manifestation of male fertility. Northern and in situ hybridization analysis revealed that, in addition to its presence in pollen, Tri a III is expressed in the parenchymatous tissue of '(aestivum)-Salmon' ovaries exclusively at the day of anthesis. This precise temporal and spatial expression pattern suggests a more general function of the pollen allergen-like sequence Tri a III not limited to the exhibition of allergens in pollen grains.

Successful crosses between Festuca arundinacea Schreb. and Dactylis glomerata L.

Five F1 plants have been obtained after extensive crossing between different ecotypes or varieties of Festuca arundinacea Schreb. and Dactylis glomerata L. The success did not appear to depend on specific treatments (spraying with ε-aminocaproic acid or gibberellic acid or pre-pollination with killed pollen from the seed parent), but the crossability is limited to exceptional plants.F1 hybrids showed characteristics of both the parents. In four hybrids various developmental disturbances were observed (low viability, aneusomaty, absence of development of inflorescences). Only one hybrid consistently showed 2n=35 chromosomes, good viability and growth, however, it was sterile. After clonal propagation, attempts for polyploidization were started.