Specific features of early embryogenesis in apomictic Poa pratensis L

We studied the early stages of embryo formation in apomictic Poa partensis L. It was shown that during transition to parthenogenesis, at least at the initial stages of embryogenesis, the algorithm of development of the sexual embryo is preserved. This could be due to the system of genetic control of embryogenesis, common for amphimixis and apomixis. We described asynchrony of developmental processes both within the efflorescence (asynchronous maturation of ovules) and within the ovule and even gametophyte (different timing of induction of apoarchesporic initials and oospores). This feature of pseudogamous apomicts allows them to produce simultaneously both sexual and apomictic progenies.     

Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy

Apomixis is facultative in characterized members of the genus Hieracium. The three components that comprise the apomictic mechanism include apospory followed by autonomous embryo and endosperm formation. The time of aposporous embryo sac initiation and mode of embryo sac formation are different in Hieracium piloselloides (D3) and Hieracium aurantiacum (A3.4). Genetic studies have shown that a single dominant locus encodes all three components of apomixis in both species (Bicknell et al. 2000). We histologically examined a range of related, genetically characterized apomictic Hieracium plants derived from D3 and A3.4 to assess conservation of the apomictic mechanism in different genetic backgrounds. The plants varied in ploidy, and also in the amount of DNA introduced from sexual Hieracium pilosella (P4). An apomictic hybrid from a cross between the two apomicts was also examined. The developmental processes observed in the parental apomicts were not conserved in the examined plants and alterations occurred in the components of apomixis. One plant also exhibited adventitious embryony. The results show that other genetic factors can modify apomixis with respect to time of initiation, spatial location, and mode of developmental progression. Both the apomictic locus and the modifiers are essential for efficient penetrance of the trait in Hieracium. Some of the findings in Hieracium correspond with observations in Ranunculus and this is discussed in terms of models for apomictic development and the control of apomixis in crops. Received: 21 June 1999 / Revision accepted: 17 November 1999     

Specific features of early embryogenesis in apomictic <Emphasis Type="Italic">Poa pratensis</Emphasis> L.

We studied the early stages of embryo formation in apomictic Poa pratensis L. It was shown that during transition to parthenogenesis, at least at the initial stages of embryogenesis, the algorithm of development of the sexual embryo is preserved. This could be due to the system of genetic control of embryogenesis, common for amphimixis and apomixis. We described asynchrony of developmental processes both within the efflorescence (asynchronous maturation of seed-buds) and within the seed-bud and even gametophyte (different timing of induction of apoarchesporic initials and oospores). This feature of pseudogamous apomicts allows them to produce simultaneously both sexual and apomictic progenies.     

Asynchronous expression of duplicate genes in angiosperms may cause apomixis, bispory, tetraspory, and polyembryony

Apomicts that produce unreduced parthenogenetic eggs are generally polyploid and occur in at least 33 of 460 families of angiosperms. Embryo sacs of these apomicts form precociously from ameiotic megaspore mother cells (diplospory) or adjacent somatic cells (apospory). Polysporic species (bisporic and tetrasporic) are sexual and occur in at least 88 families. Their embryo sacs also form precociously, but only non-critical portions of meiosis are affected. It is hypothesized that (i) the partial to complete replacement of meiosis by embryo sac formation in apomictic and polysporic species results from asynchronously-expressed duplicate genes that control female development, (ii) duplicate genes result from polyploidy or paleopolyploidy (diploidized polyploidy with chromatin from multiple genomes), (iii) apomixis results from competition between nearly complete sets of asynchronously-expressed duplicate genes, and (iv) polyspory and polyembryony result from competition between incomplete sets of asynchronously-expressed duplicate genes. Phylogenetic and genomic studies were conducted to evaluate this hypothesis. Apomictic, polysporic, and polyembryonic species tended to occur together in cosmopolitan families in which temporal variation in female development is expected, apomicts were generally polyploid with few chromosomes per genome (X = 9.6pL0.4 SE), and polysporic and polyembryonic species were paleopolyploid with many chromosomes per genome (x= 15.7pL0.6 and 13.2pL0.4, respectively). These findings support the proposed duplicate-gene asynchrony hypothesis and further suggest asexual reproduction in apomicts preserves primary genomes, sexual reproduction in polysporic and polyembryonic polyploids accelerates paleopolyploidization, and pa-leopolyploidization may sometimes eliminate gene duplications required for apomixis while retaining duplications required for polyspory or polyembryony. Hence, apomixis, with its long-term reproductive stability, may occasionally serve as an evolutionary springboard in the evolution of normal and developmentally-novel paleopolyploid sexual species and genera.     

Sexual devolution in plants: apomixis uncloaked?

There are a growing number of examples where naturally occurring mutations disrupt an established physiological or developmental pathway to yield a new condition that is evolutionary favored. Asexual reproduction by seed in plants, or apomixis, occurs in a diversity of taxa and has evolved from sexual ancestors. One form of apomixis, diplospory, is a multi-step development process that is initiated when meiosis is altered to produce an unreduced rather than a reduced egg cell. Subsequent parthenogenetic development of the unreduced egg yields genetically maternal progeny. While it has long been apparent from cytological data that meiosis in apomicts was malfunctional or completely bypassed, the genetic basis of the phenomenon has been a long-standing mystery. New data from genetic analysis of Arabidopsis mutants in combination with more sophisticated molecular understanding of meiosis in plants indicate that a weak mutation of the gene SWI, called DYAD, interferes with sister chromatid cohesion in meiosis I, causes synapsis to fail in female meiosis and yields two unreduced cells. The new work shows that a low percentage of DYAD ovules produce functional unreduced egg cells (2n) that can be fertilized by haploid pollen (1n) to give rise to triploid (3n) progeny. While the DYAD mutants differ in some aspects from naturally occurring apomicts, the work establishes that mutation to a single gene can effectively initiate apomictic development and, furthermore, focuses efforts to isolate apomixis genes on a narrowed set of developmental events. Profitable manipulation of meiosis and recombination in agronomically important crops may be on the horizon.     

Sexual and apomictic reproduction in Hieracium subgenus pilosella are closely interrelated developmental pathways

Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in HIERACIUM:     

Heterochronic features of the female germline among several sexual diploid <Emphasis Type="Italic">Tripsacum</Emphasis> L. (Andropogoneae,Poaceae)

One element of gametophytic apomixis is unreduced embryo sac (ES) formation, which often occurs precociously displacing or replacing meiosis and causing apospory or diplospory, respectively. This study evaluated a premise that apomixis may evolve in hybridogenous plants that contain duplicate sets of allelically divergent ovule development heterochrony genes. The duplicate sets of genes would belong to duplicate genomic regions that are recombinationally isolated from each other (no gene flow) by allopolyploidy or paleopolyploidy, and this isolation would genetically stabilize apomixis. For apomixis to evolve, the ancestral donors of the duplicate regions must have differed from each other in timing of megasporogenesis, ES formation and embryony such that epigenetic misexpressions, or competitions in expression, of the duplicate heterochrony genes in hybridogenous derivatives would cause apomixis. Herein, we report substantial heterochrony in onset timing of germline stages among several sexual diploid Tripsacum genotypes, which may have been progenitors of apomictic polyploid Tripsacum. Tripsacum floridanum and Tripsacum zopilotense genotypes entered meiosis early. The former advanced rapidly through ES formation, but the latter entered a lengthy lag phase prior to ES formation. In two Tripsacum dactyloides var. dactyloides genotypes, meiosis occurred late and was followed by a distinct lag phase prior to ES formation. Likewise, the T. dactyloides var. meridonale genotype entered meiosis late, but the lag phase was brief. These differences appear to reflect allelic diversity at loci responsible for onset timing of different germline development stages within and across species and possibly across the recombinationally isolated duplicate chromosome regions in the Tripsacum paleopolyploid haplome (x = 18). Unique combinations of divergent alleles in hybridogenous plants coupled with polyploidy induced gene misexpressions may be required for apomixis to evolve. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Sexual and apomictic development in Hieracium

 Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis. Received: 11 February 1998 / Revision accepted: 23 July 1998     

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

. In the autonomous apomictic Taraxacum officinale (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.     

Apomixis in Tripsacum: comparative mapping of a multigene phenomenon.

A relationship has been established between the expression of apomixis in natural polyploids of Tripsacum dactyloides and fertility as measured by percent seed set. Thus, fertility may be reliably used as a defining phenotype for apomixis when scoring the progeny from diploid (2n = 2x = 36) x tetraploid (2n = 4x = 72) crosses in Tripsacum. By exploiting the relationship between apomixis and fertility, as defined by seed set, analyses were performed on a set of related second-generation triploid populations segregating for apomixis. These populations were derived from sexual (diploid) x apomictic (tetraploid) crosses. Six out of 25 genome-dispersed restriction fragment length polymorphism (RFLP) markers co-segregate with fertility. Five of these markers were previously reported and include: php20855, tda48, tda53, umc62, and umc83, and are linked to Tripsacum genetic linkage groups F, I, H, L, and A, respectively. Significantly, we report here the syntenic relationships of the maize chromosome intervals to Tripsacum that segregate for numerous meiosis-specific and fertility-associated genes. Utilizing RFLP locus comparative mapping based on conservation of chromosome (genic) regions between related species, it may be concluded that the genes controlling fertility have been preserved in both Tripsacum and maize. A sixth marker, umc166, has also been shown to co-segregate with fertility and is conserved in both grass species. Specifically, umc166 is linked to Tripsacum linkage group D and, by syntenic comparison, to the short arm of maize chromosome 5. Encoded within this marked interval is the gene Ameiotic1 (Am1) whose function is required for the initiation of meiosis in both micro- and megaspore mother cells and whose absence of expression in the female is, in all likelihood, a prerequisite for the expression of apomixis.     

Developmental genetics of gametophytic apomixis

Some higher plants reproduce asexually by apomixis, a natural way of cloning through seeds. Apomictic plants produce progeny that are an exact genetic replica of the mother plant. The replication is achieved through changes in the female reproductive pathway such that female gametes develop without meiosis and embryos develop without fertilization. Although apomixis is a complex developmental process, genetic evidence suggests that it might be inherited as a simple mendelian trait - a paradox that could be explained by recent data derived from apomictic species and model sexual organisms. The data suggest that apomixis might rely more on a global deregulation of sexual reproductive development than on truly new functions, and molecular mechanisms for such a global deregulation can be proposed. This new understanding has direct consequences for the engineering of apomixis in sexual crop species, an application that could have an immense impact on agriculture.     

On the origin and evolution of apomixis in Boechera

The genetic mechanisms causing seed development by gametophytic apomixis in plants are predominantly unknown. As apomixis is consistently associated with hybridity and polyploidy, these confounding factors may either (a) be the underlying mechanism for the expression of apomixis, or (b) obscure the genetic factors which cause apomixis. To distinguish between these hypotheses, we analyzed the population genetic patterns of diploid and triploid apomictic lineages and their sexual progenitors in the genus Boechera (Brassicaceae). We find that while triploid apomixis is associated with hybridization, the majority of diploid apomictic lineages are likely the product of intra-specific crosses. We then show that these diploid apomicts are more likely to sire triploid apomictic lineages than conspecific sexuals. Combined with flow cytometric seed screen phenotyping for male and female components of apomixis, our analyses demonstrate that hybridization is an indirect correlate of apomixis in Boechera.     

Apomixis: Developmental Characteristics and Genetics

Asexual reproduction through seeds, or apomixis, is widespread in angiosperms, although does not happen frequently. It occurs in no major crop plant, but its deployment in major crops would afford advantages for breeding and maintenance of hybrid genotypes. Deployment is still a long-term goal, however, since the genetic mechanisms underlying apomixis in nature have not been determined nor has the isolation of apomictic mutants in sexual plants been achieved. Nevertheless, an increasing intensity of research toward these goals over the last decade has greatly expanded our knowledge of genome structure and gene expression in naturally occurring apomicts and female gametophyte development in sexual plants. A common working hypothesis is that apomixis is a “deregulation” of sexual processes and is increasingly supported by gene expression data. Nevertheless, the search for a unique trigger that initiates apomictic development still cannot be disqualified. Further characterization of female gametophyte-related genes and genomes of apomicts and model sexual plants will be fruitful for identifying overlaps in developmental networks.

     

Copy Number Variation in Transcriptionally Active Regions of Sexual and Apomictic Boechera Demonstrates Independently Derived Apomictic Lineages

In asexual (apomictic) plants, the absence of meiosis and sex is expected to lead to mutation accumulation. To compare mutation accumulation in the transcribed genomic regions of sexual and apomictic plants, we performed a double-validated analysis of copy number variation (CNV) on 10 biological replicates each of diploid sexual and diploid apomictic Boechera, using a high-density (>700 K) custom microarray. The Boechera genome demonstrated higher levels of depleted CNV, compared with enriched CNV, irrespective of reproductive mode. Genome-wide patterns of CNV revealed four divergent lineages, three of which contain both sexual and apomictic genotypes. Hence genome-wide CNV reflects at least three independent origins (i.e., expression) of apomixis from different sexual genetic backgrounds. CNV distributions for different families of transposable elements were lineage specific, and the enrichment of LINE/L1 and long term repeat/Copia elements in lineage 3 apomicts is consistent with sex and meiosis being mechanisms for purging genomic parasites. We hypothesize that significant overrepresentation of specific gene ontology classes (e.g., pollen–pistil interaction) in apomicts implies that gene enrichment could be an adaptive mechanism for genome stability in diploid apomicts by providing a polyploid-like system for buffering the effects of deleterious mutations.     

Altering sexual development inArabidopsis

The reproductive system determines the way in which gametes develop and interact to form a new organism. Therefore, it exerts the primary level of control of genotypic frequencies in plant populations, and plays a fundamental role in plant breeding. A basic understanding of plant reproductive development will completely transform current breeding strategies used for seed production. Apomixis is an asexual form of reproduction in which embryogenesis occurs in a cell lineage lacking both meiosis and fertilization, and that culminates in the formation of viable progeny genetically identical to the mother plant. The transfer of apomixis into sexual crops will allow the production of self-perpetuating improved hybrids, and the fixation of any desired heterozygous genotype. The initiation of apomictic development invariably takes place at early stages of ovule ontogeny, before the establishment of the megagametophytic phase. The developmental versatility associated with megagametophyte formation suggests that the genetic and molecular regulation of apomixis is intimately related to the regulation of sexuality. Differences between the initiation of sexual and apomictic development may be determined by regulatory genes that act during megasporogenesis, and that control events leading to the formation of unreduced female gametophytes. To test this hypothesis, we are isolating and characterizing genes that act during megasporogenesis inArabidopsis thaliana and investigating their potential role in the induction of apomixis. We are using a recently established transposon-based enhancer detection and gene trap insertional mutagenesis system that allows the identification of genes based on their expression patterns. An initial screen of transposants has yielded over 20 lines conferring restricted GUS expression during early ovule development. We have obtained the sequence of genomic fragments flanking the transposon insertion. Several have homology to genes playing important roles in plant and animal development. They include cell cycle regulators, enzymes involved in callose hydrolysis, leucine-rich repeat protein kinase receptors, and expressed sequence tags (ESTs) of unknown function. Independently, a genetic screen allows the identification of female sterile mutants defective in megasporogenesis. Results from these experiments will improve our basic understanding of reproductive development in plants, and will set the basis for a sustained effort in plant germ line biotechnology, a first step toward a flexible transfer of apomixis into a large variety of sexual crops.     

Sexual reproduction is the default mode in apomictic Hieracium subgenus Pilosella, in which two dominant loci function to enable apomixis

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis.