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Introduction: Persian walnut (Juglans regia) is an economically important nut fruit species cultivated worldwide for its nutritious kernel and timber quality wood. Walnut trees are mostly hetero-dichogamous and, depending on the genotype, some cultivars are protogynous, while others are protandrous. Although selfing is possible when male and female blooms overlap, the dichogamy of the species promotes outcrossing. In addition to sexual reproduction, some reports indicate that elements of apomixis may occur in commercial orchards of walnut varieties and in the last two decades, nut production by apomixis has been reported in walnut. However, there are no reliable studies on the occurrence of apomictic reproduction based on cytoembryological observations and/or molecular marker-progeny tests. This study addresses the combined use of molecular and cytological analyses to gain new insights into the population genetics and reproduction systems of J. regia. Methods: We systematically analyzed the reproductive origin of individual progeny plants from 8 different cultivated walnut genotypes using microsatellite genotyping and carried out cytohistological investigations of 5 cultivated walnut genotypes arising seed sets from isolated flowers, to shed light on the mode of reproduction. Results and discussion: These cytometric and genotyping analyses did not support any asexual mode of reproduction or asexual propagation by seed and all individuals studied were identified as zygotic plants produced by crossing. Likewise, the cytological findings did not confirm completely the first component of apomixis, namely apomeiosis. On the other hand, according to histological evidence, adventitious embryony seems to take place at low frequency. Overall, our findings suggest that the occurrence of gametophytic apomixis is unlikely in J. regia, but sporophytic apomixis cannot be completely ruled out. [ABSTRACT FROM AUTHOR]

Introduction: Persian walnut (Juglans regia) is an economically important nut fruit species cultivated worldwide for its nutritious kernel and timber quality wood. Walnut trees are mostly hetero-dichogamous and, depending on the genotype, some cultivars are protogynous, while others are protandrous. Although selfing is possible when male and female blooms overlap, the dichogamy of the species promotes outcrossing. In addition to sexual reproduction, some reports indicate that elements of apomixis may occur in commercial orchards of walnut varieties and in the last two decades, nut production by apomixis has been reported in walnut. However, there are no reliable studies on the occurrence of apomictic reproduction based on cytoembryological observations and/or molecular marker-progeny tests. This study addresses the combined use of molecular and cytological analyses to gain new insights into the population genetics and reproduction systems of J. regia. Methods: We systematically analyzed the reproductive origin of individual progeny plants from 8 different cultivated walnut genotypes using microsatellite genotyping and carried out cytohistological investigations of 5 cultivated walnut genotypes arising seed sets from isolated flowers, to shed light on the mode of reproduction. Results and discussion: These cytometric and genotyping analyses did not support any asexual mode of reproduction or asexual propagation by seed and all individuals studied were identified as zygotic plants produced by crossing. Likewise, the cytological findings did not confirm completely the first component of apomixis, namely apomeiosis. On the other hand, according to histological evidence, adventitious embryony seems to take place at low frequency. Overall, our findings suggest that the occurrence of gametophytic apomixis is unlikely in J. regia, but sporophytic apomixis cannot be completely ruled out. [ABSTRACT FROM AUTHOR]

In angiosperms, there are two pathways of reproduction through seeds: sexual, or amphimictic, and asexual, or apomictic. The essential feature of apomixis is that an embryo in an ovule is formed autonomously. It may form from a cell of the nucellus or integuments in an otherwise sexual ovule, a process referred to as adventitious embryony. Alternatively, the embryo may form by parthenogenesis from an unreduced egg that forms in an unreduced embryo sac. The latter may form from an ameiotic megasporocyte, in which case it is referred to as diplospory, or from a cell of the nucellus or integument, in which case it is referred to as apospory. Progeny of apomictic plants are generally identical to the mother plant. Apomixis has been seen over the years as either a gain- or loss-of-function over sexuality, implying that the latter is the default condition. Here, we consider an additional point of view, that apomixis may be anciently polyphenic with sex and that both reproductive phenisms involve anciently canalized components of complex molecular processes. This polyphenism viewpoint suggests that apomixis fails to occur in obligately sexual eukaryotes because genetic or epigenetic modifications have silenced the primitive sex apomixis switch and/or disrupted molecular capacities for apomixis. In eukaryotes where sex and apomixis are clearly polyphenic, apomixis exponentially drives clonal fecundity during reproductively favorable conditions, while stress induces sex for stress-tolerant spore or egg formation. The latter often guarantees species survival during environmentally harsh seasons. [ABSTRACT FROM AUTHOR]

Key message: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Abstract: Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 ( RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog ( N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types. [ABSTRACT FROM AUTHOR]

The identification and characterization of transcripts involved in flower organ development, plant reproduction and metabolism represent key steps in plant phenotypic and physiological pathways, and may generate high-quality transcript variants useful for the development of functional markers. This study was aimed at obtaining an extensive characterization of the olive flower transcripts, by providing sound information on the candidate MADS-box genes related to the ABC model of flower development and on the putative genetic and molecular determinants of ovary abortion and pollen-pistil interaction. The overall sequence data, obtained by pyrosequencing of four cDNA libraries from flowers at different developmental stages of three olive varieties with distinct reproductive features (Leccino, Frantoio and Dolce Agogia), included approximately 465,000 ESTs, which gave rise to more than 14,600 contigs and approximately 92,000 singletons. As many as 56,700 unigenes were successfully annotated and provided gene ontology insights into the structural organization and putative molecular function of sequenced transcripts and deduced proteins in the context of their corresponding biological processes. Differentially expressed genes with potential regulatory roles in biosynthetic pathways and metabolic networks during flower development were identified. The gene expression studies allowed us to select the candidate genes that play well-known molecular functions in a number of biosynthetic pathways and specific biological processes that affect olive reproduction. A sound understanding of gene functions and regulatory networks that characterize the olive flower is provided. [ABSTRACT FROM AUTHOR]

Background: St. John's wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Recently gained biological information has shown that this species is also an attractive model system for the study of a naturally occurring form of asexual reproduction called apomixis, which allows cloning plants through seeds. In aposporic gametogenesis, one or multiple somatic cells belonging to the ovule nucellus change their fate by dividing mitotically and developing functionally unreduced embryo sacs by mimicking sexual gametogenesis. Although the introduction of apomixis into agronomically important crops could have revolutionary implications for plant breeding, the genetic control of this mechanism of seed formation is still not well understood for most ofthe model species investigated so far. We used Roche 454 technology to sequence the entire H. perforatum flower transcriptome of whole flower buds and single flower verticils collected from obligately sexual and unrelated highly or facultatively apomictic genotypes, which enabled us to identify RNAs that are likely exclusive to flower organs (i.e., sepals, petals, stamens and carpels) or reproductive strategies (i.e., sexual vs. apomictic). Results: Here we sequenced and annotated the flower transcriptome of H. perforatum with particular reference to reproductive organs and processes. In particular, in our study we characterized approximately 37,000 transcripts found expressed in male and/or female reproductive organs, including tissues or cells of sexual and apomictic flower buds. Ontological annotation was applied to identify major biological processes and molecular functions involved in flower development and plant reproduction. Starting from this dataset, we were able to recover and annotate a large number of transcripts related to meiosis, gametophyte/gamete formation, and embryogenesis, as well as genes that are exclusively or preferentially expressed in sexual or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. Conclusions: Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity ofthe flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding ofthe molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression in plants. Many miRNA families and their targets have been extensively studied in model species and major crops. We have characterized mature miRNAs along with their precursors and potential targets in Hypericum to generate a comprehensive list of conserved miRNA families and to investigate the regulatory role of selected miRNAs in biological processes that occur in the flower. St. John's wort ( Hypericum perforatum L., 2 n = 4 x = 32), a medicinal plant that produces pharmaceutically important metabolites with therapeutic activities, was chosen because it is regarded as an attractive model system for the study of apomixis. A computational in silico prediction of structure, in combination with an in vitro validation, allowed us to identify 7 pre-miRNAs, including miR156, miR166, miR390, miR394, miR396, and miR414. We demonstrated that H. perforatum flowers share highly conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide range of molecular functions, including metabolism, response to stress, flower development, and plant reproduction. Our analysis paves the way toward identifying flower-specific miRNAs that may differentiate the sexual and apomictic reproductive pathways. [ABSTRACT FROM AUTHOR]

Abstract: Apomixis is desirable in agriculture as a reproductive strategy for cloning plants by seeds. Because embryos derive from the parthenogenic development of apomeiotic egg cells, apomixis excludes fertilization in addition to meiotic segregation and recombination, resulting in offspring that are exact replicas of the parent. Introgression of apomixis from wild relatives to crop species and transformation of sexual genotypes into apomictically reproducing ones are long-held goals of plant breeding. In fact, it is generally accepted that the introduction of apomixis into agronomically important crops will have revolutionary implications for agriculture. This review deals with the current genetic and molecular findings that have been collected from model species to elucidate the mechanisms of apomeiosis, parthenogenesis and apomixis as a whole. Our goal is to critically determine whether biotechnology can combine key genes known to control the expression of the processes miming the main components of apomixis in plants. Two natural apomicts, as the eudicot Hypericum perforatum L. (St. John''s wort) and the monocot Paspalum spp. (crowngrass), and the sexual model species Arabidopsis thaliana are ideally suited for such investigations at the genomic and biotechnological levels. Some novel views and original concepts have been faced on this review, including (i) the parallel between Y-chromosome and apomixis-bearing chromosome (e.g., comparative genomic analyses revealed common features as repression of recombination events, accumulation of transposable elements and degeneration of genes) from the most primitive (Hypericum-type) to the most advanced (Paspalum-type) in evolutionary terms, and (ii) the link between apomixis and gene-specific silencing mechanisms (i.e., likely based on chromatin remodelling factors), with merging lines of evidence regarding the role of auxin in cell fate specification of embryo sac and egg cell development in Arabidopsis. The production of engineered plants exhibiting apomictic-like phenotypes is critically reviewed and discussed. [Copyright &y& Elsevier]

The introduction of apomixis – seed formation without fertilization – into crop plants is a long-held goal of breeding research, since it would allow for the ready fixation of heterozygosity. The genetic basis of apomixis, whether of the aposporous or the diplosporous type, is still only poorly understood. Hypericum perforatum (St John’s wort), a plant with a small genome and a short generation time, can be aposporous and/or parthenogenetic, and so represents an interesting model dicot for apomixis research. Here we describe a genetic analysis which first defined and then isolated a locus (designated HAPPY for ypericum OS OR ) associated with apospory . Amplified fragment length polymorphism (AFLP) profiling was used to generate a cleaved amplified polymorphic sequence (CAPS) marker for HAPPY which co-segregated with apospory but not with parthenogenesis, showing that these two components of apomixis are independently controlled. Apospory was inherited as a dominant simplex gene at the tetraploid level. Part of the HAPPY sequence is homologous to the Arabidopsis thaliana gene ARI7 encoding the ring finger protein ARIADNE7. This protein is predicted to be involved in various regulatory processes, including ubiquitin-mediated protein degradation. While the aposporous and sexual alleles of the HAPPY component HpARI were co-expressed in many parts of the plant, the gene product of the apomict’s allele is truncated. Cloning HpARI represents the first step towards the full characterization of HAPPY and the elucidation of the molecular mechanisms underlying apomixis in H. perforatum. [ABSTRACT FROM AUTHOR]

Jatropha curcas L. (2n = 2x = 22) is becoming a popular non-food oleaginous crop in several developed countries due to its proposed value in the biopharmaceutical industry. Despite the potentials of its oil-rich seeds as a renewable source of biodiesel and an interest in large-scale cultivation, relatively little is known with respect to plant reproduction strategies and population dynamics. Here, genomic DNA markers and FCSS analyses were performed to gain insights into ploidy variation and heterozygosity levels of multiple accessions, and genomic relationships among commercial varieties of Jatropha grown in different geographical areas. The determination of ploidy and the differentiation of either pseudogamous or autonomous apomixis from sexuality were based on the seed DNA contents of embryo and endosperm. The presence of only a high 2C embryo peak and a smaller 3C endosperm peak (ratio 2:3) is consistent with an obligate sexual reproductive system. Because of the lack of either 4C or 5C endosperm DNA estimates, the occurrence of gametophytic apomixis seems unlikely in this species but adventitious embryony cannot be ruled out. The investigation of genetic variation within and between cultivated populations was carried out using dominant RAPD and Inter-SSR markers, and codominant SSR markers. Nei's genetic diversity, corresponding to the expected heterozygosity, was equal to He = 0.3491 and the fixation index as low as Fst = 0.2042. The main finding is that seeds commercialized worldwide include a few closely related genotypes, which are not representative of the original Mexican gene pool, revealing high degrees of homozygosity for single varieties and very low genetic diversity between varieties. [ABSTRACT FROM AUTHOR]

Apomixis, as it exists in Kentucky bluegrass (Poa pratensis L.), is a nearly ideal way of maintaining superior hybrids and complex traits. It combines the genetic advantages of vegetative propagation (maintenance of the genotype) with the agronomic advantages of sexual reproduction (use of the seed). Lack of knowledge on its inheritance at present hinders attempts to manipulate and transfer the apomictic trait beyond natural sexual barriers. We analyzed the occurrence of meiotic (haploid) and aposporic (diploid) parthenogenesis in P. pratensis genotypes with contrasting degrees of apomixis using embryo rescue and in vitro culture techniques. The in vitro rescue of autonomous embryos obtained after auxin-induced parthenocarpy permitted us to investigate and establish the embryological derivation of progeny plants of certain parthenogenetic origin. All recovered plants were characterized both for chromosome number and DNA fingerprint to ascertain their genetic origin. Our experiments indicated that parthenogenetic capacity is preferentially expressed by aposporic egg cells and so the genetic determinants for apomixis seem to be closely linked and inherited together. We also documented parthenogenetic development of meiotic egg cells. In a highly sexual genotype that when auxin tested showed considerable parthenogenetic capacity, the egg cells appeared to exert this potential irrespective of their cytological origin. Thus, events of segregation of apospory from parthenogenesis are rare, but possible. Although the question remains open, apomixis seems to be the result of a few tightly clustered genes rather than the combination of two processes, apospory and parthenogenesis, determined by independent genetic factors. [ABSTRACT FROM PUBLISHER]

Apomixis sensu stricto (agamospermy) is asexual reproduction by seed. In angiosperms it represents an easy byway of life cycle renewal through gamete-like cells that give rise to maternal embryos without ploidy reduction (meiosis) and ploidy restitution (syngamy). The origin of apomixis still represents an unsolved problem, as it may be either evolved from sex or the other way around. This review deals with a reappraisal of the origin of apomixis in order to deepen knowledge on such asexual mode of reproduction which seems mainly lacking in the most basal angiosperm orders (i.e., Amborellales, Nymphaeales and Austrobaileyales, also known as ANA-grade), while it clearly occurs in different forms and variants in many unrelated families of monocots and eudicots. Overall findings strengthen the hypothesis that apomixis as a whole may have evolved multiple times in angiosperm evolution following different developmental pathways deviating to different extents from sexuality. Recent developments on the genetic control of apomixis in model species are also presented and adequately discussed in order to shed additional light on the antagonist theories of gain- and loss-of-function over sexuality. [ABSTRACT FROM AUTHOR]

Abstract: Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle. [Copyright &y& Elsevier]