Analysis of variation for apomictic reproduction in diploid Paspalum rufum.

BACKGROUND AND AIMS: The diploid cytotype of Paspalum rufum (Poaceae) reproduces sexually and is self-sterile; however, recurrent autopolyploidization through 2n + n fertilization and the ability for reproduction via apomixis have been documented in one genotype of the species. The objectives of this work were to analyse the variation in the functionality of apomixis components in diploid genotypes of P. rufum and to identify individuals with contrasting reproductive behaviours. METHODS: Samples of five individuals from each of three natural populations of P. rufum (designated R2, R5 and R6) were used. Seeds were obtained after open pollination, selfing, conspecific interploidy crosses and interspecific interploidy self-pollination induction. The reproductive behaviour of each plant was determined by using the flow cytometric seed screen (FCSS) method. Embryo sacs were cleared using a series of ethanol and methyl salicylate solutions and observed microscopically. KEY RESULTS: In open pollination, all genotypes formed seeds by sexual means and no evidence of apomeiotic reproduction was detected. However, in conspecific interploidy crosses and interspecific interploidy self-pollination induction, variations in the reproductive pathways were observed. While all plants from populations R2 and R6 formed seeds exclusively by sexual means, three genotypes from the R5 population developed seeds from both meiotic and aposporous embryo sacs, and one of them (R5#49) through the complete apomictic pathway (apospory + parthenogenesis + pseudogamy). Cytoembryological observations revealed the presence of both meiotic and aposporous embryo sacs in all the genotypes analysed, suggesting that parthenogenesis could be uncoupled from apospory in some genotypes. CONCLUSIONS: The results presented demonstrate the existence of variation in the functionality of apomixis components in natural diploid genotypes of P. rufum and have identified individuals with contrasting reproductive behaviours. Genotypes identified here can be crossed to generate segregating populations in order to study apomixis determinants at the diploid level. Moreover, analysis of their expression patterns, quantification of their transcript levels and an understanding of their regulation mechanisms could help to design new strategies for recreating apomixis in a diploid genome environment.

Competition between meiotic and apomictic pathways during ovule and seed development results in clonality.

Meiotic and apomictic reproductive pathways develop simultaneously in facultative aposporous species, and compete to form a seed as a final goal. This developmental competition was evaluated in tetraploid genotypes of Paspalum malacophyllum in order to understand the low level of sexuality in facultative apomictic populations. Cyto-embryology on ovules, flow cytometry on seeds and progeny tests by DNA fingerprinting were used to measure the relative incidence of each meiotic or apomictic pathway along four different stages of the plant's life cycle, namely the beginning and end of gametogenesis, seed formation and adult offspring. A high variation in the frequencies of sexual and apomictic pathways occurred at the first two stages. A trend of radical decline in realized sexuality was then observed. Sexual and apomictic seeds were produced, but the efficiency of the sexual pathway dropped drastically, and exclusively clonal offspring remained. Both reproductive pathways are unstable at the beginning of development, and only the apomictic one remains functional. Key factors reducing sexuality are the faster growth and parthenogenetic development in the aposporous pathway, and an (epi)genetically negative background related to the extensive gene de-regulation pattern responsible for apomixis. The effects of inbreeding depression during post-fertilization development may further decrease the frequency of effective sexuality.

Harnessing apomictic reproduction in grasses: what we have learned from Paspalum.

BACKGROUND: Apomixis is an alternative route of plant reproduction that produces individuals genetically identical to the mother plant through seeds. Apomixis is desirable in agriculture, because it guarantees the perpetuation of superior genotypes (i.e. heterotic hybrid seeds) by self-seeding without loss of hybrid vigour. The Paspalum genus, an archetypal model system for mining apomixis gene(s), is composed of about 370 species that have extremely diverse reproductive systems, including self-incompatibility, self-fertility, full sexual reproduction, and facultative or obligate apomixis. Barriers to interspecific hybridization are relaxed in this genus, allowing the production of new hybrids from many different parental combinations. Paspalum is also tolerant to various parental genome contributions to the endosperm, allowing analyses of how sexually reproducing crop species might escape from dosage effects in the endosperm. SCOPE: In this article, the available literature characterizing apomixis in Paspalum spp. and its use in breeding is critically reviewed. In particular, a comparison is made across species of the structure and function of the genomic region controlling apomixis in order to identify a common core region shared by all apomictic Paspalum species and where apomixis genes are likely to be localized. Candidate genes are discussed, either as possible genetic determinants (including homologs to signal transduction and RNA methylation genes) or as downstream factors (such as cell-to-cell signalling and auxin response genes) depending, respectively, on their co-segregation with apomixis or less. Strategies to validate the role of candidate genes in apomictic process are also discussed, with special emphasis on plant transformation in natural apomictic species.

Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum.

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

Reproductive Systems in Paspalum: Relevance for Germplasm Collection and Conservation, Breeding Techniques, and Adoption of Released Cultivars.

The objective of this review is to analyze and describe the impact that mode of reproduction in Paspalum has on germplasm conservation, genetic improvement, and commercialization of cultivars. Germplasm collection and conservation can now be rethought considering the newly available information related to how diversity is allocated in nature and how it can be transferred between the sexual and apomictic germplasm using novel breeding approaches. An inventory of species and accessions conserved around the world is analyzed in relation to the main germplasm banks. Because of the importance of apomixis in Paspalum species different breeding approaches have been used and tested. Knowledge related to the inheritance of apomixis, variable expressivity of the trait and techniques for early identification of apomicts has helped to improve the efficiency of the breeding methods. Novel breeding techniques are also being developed and are described regarding its advantages and limitations. Finally, the impact of reproductive mode on the adoption of the released cultivars is discussed.

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants.

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.

Gene expression analysis at the onset of aposporous apomixis in Paspalum notatum.

Apomixis is a route of asexual reproduction through seeds, that progresses in the absence of meiosis and fertilization to generate maternal clonal progenies. Gametophytic apomicts are usually polyploid and probably arose from sexual ancestors through a limited number of mutations in the female reproductive pathway. A differential display analysis was carried out on immature inflorescences of sexual and apomictic tetraploid genotypes of Paspalum notatum, in order to identify genes associated with the emergence of apospory. Analysis of approximately 10,000 transcripts led to the identification of 94 high-quality differentially expressed sequences. Assembling analysis, plus validation, rendered 65 candidate unigenes, organized as 14 contigs and 51 singletons. Thirty-four unigenes were isolated from apomictic plants and 31 from sexual ones. A total of 45 (69.2%) unigenes were functionally categorized. While several of the differentially expressed sequences appeared to be components of an extracellular receptor kinase (ERK) signal transduction cascade, others seemed to participate in a variety of central cellular processes like cell-cycle control, protein turnover, intercellular signalling, transposon activity, transcriptional regulation and endoplasmic reticulum-mediated biosynthesis. In silico mapping revealed that a particular group of five genes silenced in apomictic plants clustered in a rice genomic area syntenic with the region governing apospory in Paspalum notatum and Brachiaria brizantha. Two of these genes mapped within the set of apo-homologues in P. notatum. Four genes previously reported to be controlled by ploidy were identified among those expressed differentially between apomictic and sexual plants. In situ hybridization experiments were performed for selected clones.