Haploid Pollen Grains Research Articles

Epigenetic Patterns in Genetically Imbalanced Polyploid Dog Rose Hybrids (Rosa L. sect. Caninae (DC.) Ser.) Revealed by cDNA–Amplified Fragment Length Polymorphisms and Methylation-Sensitive Amplified Polymorphisms

Premise of research. Spontaneous hybrids between species of Rosa sect. Caninae represent a unique example to study the genetic and epigenetic consequences of an imbalanced parental contribution to polyploid hybrids. Due to the unique canina meiosis, pentaploid or hexaploid dog rose hybrids contain a single subgenome transmitted by the haploid pollen grain of the paternal species and four to five subgenomes transmitted by the tetraploid or pentaploid egg cell of the maternal species. During this study, we investigated epigenetic changes in spontaneous reciprocal hybrids between the subsections Caninae and Rubigineae compared to their parental species in a natural population in eastern Germany.Methodology. For investigating epigenetic patterns, we applied cDNA–amplified fragment length polymorphisms (AFLPs) and methylation-sensitive amplified polymorphisms (MSAPs). Additionally, we analyzed microsatellite data and measured morphological characters.Pivotal results. In line with expectations deduced from the genetic composition of the pentaploid and hexaploid hybrids, we observed a maternal bias of DNA methylation and expression patterns. We did not detect silencing of the respective paternal subgenomes. However, the percentage of paternally expressed fragments differed between plant organs. We found a higher number of paternally expressed fragments in sepals compared to leaves, suggesting that the parental genetic information underwent a rapid subfunctionalization in recently formed hybrids. Moreover, epigenetic data obtained from cDNA-AFLPs and MSAPs correlated stronger with morphology than with genetic data, based on microsatellites.Conclusions. The genetic contribution of subgenomes is strongly biased toward the maternal parent in dog rose hybrids, leading to a nearly apomictic mode of inheritance. However, we found that the underrepresented paternal genome is not silenced but transcribed and differentially expressed between plant organs. The correlation between epigenetic data and morphological data implies that the probably fast-evolving epigenetic changes have a major impact on dog rose phenotypes and may affect the establishment of hybrids in natural populations.

To be or not to be the odd one out - Allele-specific transcription in pentaploid dogroses (Rosa L. sect. Caninae (DC.) Ser)

BackgroundMultiple hybridization events gave rise to pentaploid dogroses which can reproduce sexually despite their uneven ploidy level by the unique canina meiosis. Two homologous chromosome sets are involved in bivalent formation and are transmitted by the haploid pollen grains and the tetraploid egg cells. In addition the egg cells contain three sets of univalent chromosomes which are excluded from recombination. In this study we investigated whether differential behavior of chromosomes as bivalents or univalents is reflected by sequence divergence or transcription intensity between homeologous alleles of two single copy genes (LEAFY, cGAPDH) and one ribosomal DNA locus (nrITS).ResultsWe detected a maximum number of four different alleles of all investigated loci in pentaploid dogroses and identified the respective allele with two copies, which is presumably located on bivalent forming chromosomes. For the alleles of the ribosomal DNA locus and cGAPDH only slight, if any, differential transcription was determined, whereas the LEAFY alleles with one copy were found to be significantly stronger expressed than the LEAFY allele with two copies. Moreover, we found for the three marker genes that all alleles have been under similar regimes of purifying selection.ConclusionsAnalyses of both molecular sequence evolution and expression patterns did not support the hypothesis that unique alleles probably located on non-recombining chromosomes are less functional than duplicate alleles presumably located on recombining chromosomes.

Complex self-incompatibility systems in Ranunculus acris L. and Beta vulgaris L.

The families Ranunculaceae and Chenopodiaceae, presumably belonging to a dicot evolutionary branch with ancestral basis shared with the monocots, both show a mechanism of self- and cross-incompatibility involving more than one incompatibility locus (S-locus) and requiring that all of the S-genes acting on the haploid pollen grain be matched in the pistil. This type of incompatibility system was previously considered to be unique for the grasses. However, whereas two S-loci with such complementary interaction have been established in the grasses, there are at least three S-loci in Ranunculus acris, and in Beta vulgaris there are at least four. The observations strongly support the theory of the incompatibility genes being ancient constituents of the breeding systems of the angiosperms. Most probably a complex type of incompatibility control was already present at the presumed common ancestral basis of the monocots and the investigated dicot evolutionary branch. Self-incompatibility systems involving more than one S-locus may occur more frequently than hitherto expected. This has important implications for plant breeding which bear on the possibilities of reconstructing an incompatibility system from different forms within a normally self-fertilizing species and, thereby, to bring about obligatory cross-fertilization.

Occurrence of 2n gametes in the F1 hybrids of Oriental × Asiatic lilies (Lilium): Relevance to intergenomic recombination and backcrossing

Cytological modes of the origin of 2n gametes were investigated in six different genotypes of F1 hybrids between Oriental and Asiatic (OA) lilies (Lilium, 2n = 2x = 24). Chromosome pairing between the parental genomes was very low, the average frequency range from 0.3 to 1.2 bivalents per cell among the genotypes. Within a genotype the frequency of bivalents varied from 0 to 6 in some cases. The normally occurring haploid pollen grains were totally sterile. In contrast, in different genotypes, variable percentages of 2n pollen were found and shown to be fertile as estimated from pollen germination. A cytological analysis of Metaphase I and subsequent stages of meiosis using genomic in situ hybridization (GISH) revealed that there was intergenomic recombination between the alien genomes. Following Metaphase I stage, three different types of abnormal cytological events led to the formation of 2n pollen: (i) Post-Metaphase I division (PMI), (ii) Post-Metaphase II division (PMII) and (iii) Asymmetric Cytokinesis of the pollen mother cell followed by chromosome division. All three cytological events led to first division restitution (FDR) gametes. Based on in vitro pollen germination it was proved for two genotypes that 2n pollen was viable only during the first day of anthesis. It was possible to use 2n pollen successfully for backcrossing. Implications of 2n pollen for intergenomic recombination in BC1 progenies are discussed.

Evolution by Reticulation: European Dogroses Originated by Multiple Hybridization Across the Genus Rosa

The European dogroses (Rosa sect. Caninae (DC.) Ser.) are characterized by a unique meiosis system ("canina-meiosis"), which controls the heterogamous development of tetraploid egg cells and haploid pollen grains resulting in a pentaploid somatic status. This permanent anorthoploidy is supposed to have originated by a hybridization event in the postglacial period. In this study we present molecular evidence by an analysis of nuclear ribosomal DNA data that dogroses are complex allopolyploids resulting from multiple hybridization events. As previously described, the nrITS-1 region does not undergo concerted evolution in dogroses. Thus, different ITS-1 sequences persist within single individuals. Secondary structure predictions do not point to the existence of pseudogenes within these ITS-1 types. Our data suggest that the pentaploid Caninae genome originated from different members of nondogroses and the now extinct Protocaninae.

Arabidopsis hapless mutations define essential gametophytic functions.

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.

Comparative cyto-embryological investigations of sexual and apomictic dandelions ( Taraxacum ) and their apomictic hybrids

In the autonomous apomictic Taraxacum offinale (common dandelion), parthenogenetic egg cells develop into embryos and central cells into endosperm without prior fertilisation. Unreduced (2n) megaspores are formed via meiotic diplospory, a nonreductional type of meiosis. In this paper, we describe the normal developmental pathways of sexual and apomictic reproduction and compare these with the development observed in the apomictic hybrids. In sexual diploids, a standard type of megasporogenesis and embryo sac development is synchronised between florets in individual capitula. In contrast, we observed that megasporogenesis and gametogenesis proceeded asynchronously between florets within a single capitulum of natural triploid apomicts. In addition, autonomous endosperm and embryo development initiated independently within individual florets. Parthenogenetic initiation of embryo development in outdoor apomicts was found to be temperature-dependent. Egg cells produced in natural apomicts were not fertilised after pollination with haploid pollen grains although pollen tubes were observed to grow into their embryo sacs. Both reductional and diplosporous megasporogenesis were observed in individual inflorescences of triploid apomictic hybrids. Embryo and endosperm development initiated independently in natural and hybrid apomicts. [KEYWORDS: apomixis, autonomous endosperm, diplospory embryo, development, parthenogenesis]

Instability of the ploidy level in autotetraploid sorghum plants from a line with variable male fertility

It was found that, in some of the plants in generations C1-C5 of induced tetraploids in the semisterile sorghum line AS-1-30, more than 30% of pollen grains (PGs) have sizes typical of haploid PGs. Pollen of these plants was used to pollinate different lines of sorghum with cytoplasmic male sterility (CMS). Diploid hybrids were obtained, which confirms the presence of haploid PGs in the tetraploids studied. When tetraploid plants with an increased frequency of haploid PGs were pollinated with pollen of plants from fertile diploid lines, diploid hybrids were also obtained. This demonstrates that the tetraploids studied had haploid ovicells. In generation C4, a chimeric plant was found; one of its shoots was diploid (judging from morphological characters) and produced as many as 99% of haploid PGs. It is assumed that haploid gametes in autotetraploids are formed through somatic reduction of chromosomes at different ontogenetic stages.

Regulation and Function of a Pollen-specific ACC Synthase Gene from Petunia

At anthesis, petunia pollen contains large amounts of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This ACC is thought to contribute to the rapid burst of ethylene produced by the pistil following pollination. An analysis of ACC content in developing anthers revealed that ACC began to accumulate the day before anthesis, indicating its synthesis was a late event in pollen development. We employed degenerate DNA primers to conserved amino acid sequences of ACC synthesis to amplify a cDNA from anther mRNA by RT-PCR. The resulting cDNA (pACS2) was sequenced and found to represent ACC synthase. Use of pACS2 as a hybridization probe revealed an increase in ACC synthase mRNA concomitant with the increase in ACC content. Further analysis indicated the ACC synthase mRNA was localized specifically to the haploid pollen grain. In an attempt to determine the function of ACC in pollen maturation or pollen–pistil interactions, we have generated a series of transgenic petunias designed to inhibit the accumulation of ACC in pollen. For these experiments, we have employed a pollen-specific promoter (LAT52) from tomato to drive the expression of antisense pACS2 or the coding region of ACC deaminase. The results of the experiments will be discussed.

Regulation and Function of a Pollen-specific ACC Synthase Gene from Petunia

At anthesis, petunia pollen contains large amounts of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This ACC is thought to contribute to the rapid burst of ethylene produced by the pistil following pollination. An analysis of ACC content in developing anthers revealed that ACC began to accumulate the day before anthesis, indicating its synthesis was a late event in pollen development. We employed degenerate DNA primers to conserved amino acid sequences of ACC synthesis to amplify a cDNA from anther mRNA by RT-PCR. The resulting cDNA (pACS2) was sequenced and found to represent ACC synthase. Use of pACS2 as a hybridization probe revealed an increase in ACC synthase mRNA concomitant with the increase in ACC content. Further analysis indicated the ACC synthase mRNA was localized specifically to the haploid pollen grain. In an attempt to determine the function of ACC in pollen maturation or pollen–pistil interactions, we have generated a series of transgenic petunias designed to inhibit the accumulation of ACC in pollen. For these experiments, we have employed a pollen-specific promoter (LAT52) from tomato to drive the expression of antisense pACS2 or the coding region of ACC deaminase. The results of the experiments will be discussed.

A statistical mixture model for estimating the proportion of unreduced pollen grains in perennial ryegrass (Lolium perenne L.) via the size of pollen grains

The size of pollen grains is commonly used to indicate the ploidy level of pollen grains. In this paper observations of the diameter of pollen grains are evaluated from one diploid accession of perennial ryegrass (Lolium perenne L.), which was expected to produce diploid (unreduced) pollen grains in addition to haploid pollen grains. The considerable overlap of the diameter distributions of haploid and diploid pollen grains severely hampers the accurate estimation of the proportion of diploid pollen grains. To overcome this problem we develop in this paper a statistical normal mixture model and we describe a method to test for the production of diploid pollen grains from a diploid parent, and to estimate the proportion of diploid pollen grains.

Cell volume ratios of natural and of induced tetraploid and diploid flowering plants.

Many volume ratios between tetraploid and diploid guard cells of stomata and between diploid and haploid pollen grains were compiled from the literature and from own unpublished results. The sources of possible errors are discussed. No difference was found between induced auto- and allotetraploids. Some of the ratios with tetraploids of the C0 generation, but less so of later generations, were extremely high. The medians of the ratios were highest in the C0 generation (pollen grains 2.0-2.1, guard cells 2.4) and smaller in later generations (pollen grains 1.9, guard cells 2.1). The ratios of tetraploids from nature were 1.5-1.8 and thus significantly smaller than 2. The reduction may be a result of a selection for cell size during evolution, or of a physiological self-adjustment.The length/width ratios of guard cells were on average greater, but those of anisodiametric pollen grains smaller in induced tetraploids than in diploids, with a broad range of variation. The finding for guard cells is at variance with a widely held opinion.

Random arrangement of chromosomes inUvularia (Liliaceae)

Chromosome arrangement in interphase has been inferred from an analysis of the relative positions of the chromosomes and the chromosome arms in untreated haploid pollen grain metaphases ofUvularia grandiflora. The distances between centromeres forming the smallest possible circle were measured in 43 metaphases. The relative positions of the chromosomes did not differ significantly from randomness. Neither did similar-sized chromosome arms show any tendency to be next to each other. The results thus disagree both with the hypothesis ofComings (1968) that each chromosome occupies a definite position in the interphase nucleus and with the claim ofBennett (1982) that similar-sized chromosome arms lie next to each other.

Recent advances in anther culture of Hevea brasiliensis (Muell.-Arg.)

The yield of pollen embryoids from cultured Hevea anthers was increased 4 fold by optimizing the proportion of ammonium nitrate to potassium nitrate in the dedifferentiation medium. For optimal differentiation of pollen embryoids, kinetin, 2,4-D and α-naphtalene acetic acid are required. Anther culture for 50 days on the dedifferentiation medium is a prerequisite for the selective development of calli and embryoids from microspores.The determination of chromosome numbers in embryoids, plantlets and regenerated trees reveals that they originate from (poly)haploid pollen grains (n=2x=18). Aneuploid, triploid (3x=27) and tetraploid (4x=36) cells were encountered in increasing frequencies as the embryoids and plants developed. A few haploid cells with 9 chromosomes were consistently observed. Buds from shoots with mixoploid chromosome numbers can be grafted and the change in the chromosome constitution of the developing new shoots followed.

Maize pollen test systems to detect nondisjunction.

Three pollen test systems to detect the induction of nondisjunction in maize are actively being explored. (1) Each member of a tetrad of haploid microspores produced by meiosis contains a single chromosome 6 which carries the only nucleolar organizing region in the maize genome. Thus, each member of a normal tetrad contains one nucleolus. If nondisjunction took place at the first or second meiotic division, 2:2:0:0 or 2:1:1:0 tetrads would be produced respectively. (2) If a male parent carrying a dominant endosperm marker is crossed by a female carrying a recessive allele of this gene, all normal kernels would be heterozygous and would express the dominant phenotype in their endosperm. If nondisjunction of the chromosome carrying this gene took place at the second microspore division and the nullisomic and disomic sperm fertilized the polar nuclei and egg of a given embryo sac respectively, an exceptional kernel is produced which would express the recessive endosperm phenotype and contain a trisomic embryo. (3) Complementing null mutations of genes expressed in individual pollen grains can be utilized to detect nondisjunction. Normal haploid pollen grains from plants heterozygous for two complementing null alleles of a locus would each express the recessive phenotype. If nondisjunction took place during either meiotic division, disomic pollen grains containing both alleles could be produced expressing the dominant phenotype due to complementation. We are exploring this test system utilizing appropriate alcohol dehydrogenase mutants.

Maize Pollen Test Systems to Detect Nondisjunction

Three pollen test systems to detect the induction of nondisjunction in maize are actively being explored. (1) Each member of a tetrad of haploid microspores produced by meiosis contains a single chromosome 6 which carries the only nucleolar organizing region in the maize genome. Thus, each member of a normal tetrad contains one nucleolus. If nondisjunction took place at the first or second meiotic division, 2:2:0:0 or 2:1:1:0 tetrads would be produced respectively. (2) If a male parent carrying a dominant endosperm marker is crossed by a female carrying a recessive allele of this gene, all normal kernels would be heterozygous and would express the dominant phenotype in their endosperm. If nondisjunction of the chromosome carrying this gene took place at the second microspore division and the nullisomic and disomic sperm fertilized the polar nuclei and egg of a given embryo sac respectively, an exceptional kernel is produced which would express the recessive endosperm phenotype and contain a trisomic embryo. (3) Complementing null mutations of genes expressed in individual pollen grains can be utilized to detect nondisjunction. Normal haploid pollen grains from plants heterozygous for two complementing null alleles of a locus would each express the recessive phenotype. If nondisjunction took place during either meiotic division, disomic pollen grains containing both alleles could be produced expressing the dominant phenotype due to complementation. We are exploring this test system utilizing appropriate alcohol dehydrogenase mutants.

TELOMERIC ASSOCIATIONS OF GAMETIC AND SOMATIC CHROMOSOMES IN DIPLOID AND AUTOTETRAPLOIDORNITHOGALUM VIRENS

In acetocarmine root-tip squashes, diploid cells of Ornithogalum virens in prophase exhibit configurations resulting from end-to-end associations of the six chromosomes. Homologues lie opposite one another in a ring. Prophase chromosomes of the autotretraploid cells likewise associate end-to-end; however, four homogues instead of two generally lie adjacent to one another and four (or eight) ends are often connected instead of the two (or four) found in diploid cells. Prophase chromosomes in a haploid pollen grain of a diploid form an open chain of three chromosomes, whereas in pollen from the autotetraploid balanced gametes form a configuration in which homologous pairs lie adjacent to one another and are attached end-to-end to other homologous pairs of nonhomologous chromosomes to form a chain. These observations are discussed in terms of the role telomeric associations may play in recognition and pairing of homologous chromosomes during meiotic prophase. This recognition of homologues may occur as early as syngamy.

The role of nuclear fusion in pollen embryogenesis of Datura innoxia Mill.

If anthers of Datura innoxia containing microspores are cultured at 28° C for 24 h, two forms of haploid pollen grains are produced; one with typical unequal generative and vegetative nuclei (A), and the other with equal vegetativetype nuclei (B). The A form predominates. After 24 to 72 h in culture, three embryogenic events ensue; 1) division of the vegetative cell in A grains accompanied by rapid degeneration of the generative cell, 2) simultaneous or independent mitosis of the nuclei in B grains, and 3) simultaneous mitosis of both the generative and vegetative nuclei in A grains following two successive cycles of DNA synthesis in the generative nucleus, but only one in the vegetative nucleus (C). The endoreduplicated generative nucleus gives rise to diplochromosomes which it is postulated divide on a common spindle together with the vegetative chromosomes to yield a 3n proembryoid. In other C grains, simultaneous mitosis of an endoreduplicated generative nucleus and two vegetative daughter nuclei can occur. Nuclear fusion in these grains is envisaged as leading to a 4n proembryoid. Diploid proembryoids probably arise by nuclear fusion in A or B grains. At plantlet emergence, mainly n and 2n plantlets are obtained. Higher ploidy proembryoids fail to develop probably as a result of competitive influences.Similar embryogenic events occur in cultures initiated at the stages of the first pollen mitosis and young pollen grains, but C embryogenesis predominates. Competition from lower ploidy embryoids is minimised and 3n, 4n and even 6n plantlets are obtained.

Germination in vitro of ryegrass pollen grains

Pollen grains of diploid and tetraploid Italian, perennial and hybrid ryegrass (Lolium spp.) germinated on 20% sucrose solution with 10 ppm each of CaCl2, H3BO3 and EDTA. Pollen tubes emerged within 2–3 minutes after sowing and completed full growth within 1 1/2 hours. The tube elongation showed the expected linear growth rate. The tubes of the haploid pollen grains grew faster (3.9 μm/minute) than that of the diploid (3.3 μm/minute). Diploid pollen grains germinated better on 25% than on 20% sucrose solution. Up to 25% of the diploid pollen produced two tubes.

THE WAXY LOCUS IN MAIZE. I. INTRALOCUS RECOMBINATION FREQUENCY ESTIMATES BY POLLEN AND BY CONVENTIONAL ANALYSES

THE waxy (wx) locus in maize conditions the type of starch which is synthesized in the triploid endosperm and in the haploid pollen grain. Further, the phenotype of a pollen grain is determined by its own genotype and not by that of the plant which produces the pollen grain (BRINK and MACGILLIVRAY 1924) and (DEMEREC 1924). This makes it possible to use the pollen grain as the unit of observation in genetical investigations on the wx locus. The Wx pollen grains stain blue with an iodine-potassium iodide stain while wx pollen grains stain a reddish-brown color. Since a maize plant produces millions of pollen grains, large populations can be obtained and scored easily, thus markedly enhancing genetic resolution. The investigation of low frequency events is more easily carried out with this system than with any other locus in a higher organism