Temporal and spatial expression of genes involved in DNA methylation during reproductive development of sexual and apomictic Eragrostis curvula.

Recent reports in model plant species have highlighted a role for DNA methylation pathways in the regulation of the somatic-to-reproductive transition in the ovule, suggesting that apomixis (asexual reproduction through seeds) likely relies on RdDM downregulation. Our aim was therefore to explore this hypothesis by characterizing genes involved in DNA methylation in the apomictic grass Eragrostis curvula. We explored floral transcriptomes to identify homologs of three candidate genes, for which mutations in Arabidopsis and maize mimic apomixis (AtAGO9/ZmAGO104, AtCMT3/ZmDMT102/ZmDMT105, and AtDDM1/ZmCHR106), and compared both their spatial and temporal expression patterns during reproduction in sexual and apomictic genotypes. Quantitative expression analyses revealed contrasting expression patterns for the three genes in apomictic vs sexual plants. In situ hybridization corroborated these results for two candidates, EcAGO104 and EcDMT102, and revealed an unexpected ectopic pattern for the AGO gene during germ line differentiation in apomicts. Although our data partially support previous results obtained in sexual plant models, they suggest that rather than an RdDM breakdown in the ovule, altered localization of AtAGO9/ZmAGO104 expression is required for achieving diplospory in E. curvula. The differences in the RdDM machinery acquired during plant evolution might have promoted the emergence of the numerous apomictic paths observed in plants.

Tetraploid races of Paspalum notatum show polysomic inheritance and preferential chromosome pairing around the apospory-controlling locus.

The objective of this work was to determine the type of inheritance (disomic/polysomic) in tetraploid (2 n=4 x=40) Paspalum notatum and investigate the transmission pattern of the chromosome region associated with apospory. An F(1) family segregating for the reproductive mode (aposporous vs non-aposporous) was generated by crossing a tetraploid sexual plant as female parent with an apomictic individual as pollen donor. Pollen mother cells from both parental plants were examined to ascertain chromosome-pairing behavior at meiosis. The high rate of quadrivalent chromosome associations indicated an autotetraploid origin of the species, although bivalent pairing and occasional univalents were detected. The observation of a lagging bivalent, a bridge of chromatin, or two aligned laggards in the aposporous parent suggested a chromosome inversion in this strain. Segregation ratios of AFLP markers and the proportion of linkages in repulsion versus coupling phase denoted tetrasomic inheritance, but markers displaying disomic ratios were also observed. Preferential chromosome pairing (disomic inheritance) in the chromosome segment related to apospory was detected. The possible relationship between a chromosome rearrangement and the inheritance of apospory is discussed.

Effect of pollination timing on the rate of apomictic reproduction revealed by RAPD markers in paspalum notatum.

Progeny tests employing molecular markers allow the identification of individuals originated by sexual means among the offspring of a facultative apomict. The objective of this work was to evaluate the effect of the pollination timing on the proportion of sexually formed individuals in progenies of a facultative apomictic Paspalum notatum genotype. Progeny families of approx. 30 plants each were generated at five different pollination times: 1-3 d pre-anthesis; at anthesis; and 2, 4 and 6 d post-anthesis. Cytoembryological analyses indicated that approx. 17% of the ovules carried a meiotic cytologically reduced embryo sac in florets formed simultaneously with those used for crosses. The parental plants and the five F1 families were analysed using RAPD molecular markers. Ninety-five oligonucleotides were assayed on the progenitors in order to search for male-specific bands. Eight primers presenting clear polymorphic bands were selected for use in the progeny tests. The proportion of sexually produced progeny reached 3.4% before anthesis and 20 % at anthesis, while pollination after anthesis generated only maternal plants. A second progeny of 97 plants obtained from pollination at anthesis produced 16 off-type plants (16.5%), of which only one was a B(III) hybrid (2n + n). Our results indicate that pollination at anthesis allows the greatest potential for sexuality to be expressed in this facultative apomictic genotype. When pollination is delayed as soon as 2 d after anthesis, only the aposporous sacs develop endosperm through pseudogamy to set seed.

Isolation of cDNA clones differentially expressed in flowers of apomictic and sexual Paspalum notatum.

Paspalum notatum is a subtropical forage grass, which reproduces by either sexuality or aposporous gametophytic apomixis. The objective of this work was to identify and isolate mRNA transcripts differentially expressed during the development of the megagametophyte from spikelets of apomictic and sexual P. notatum. Crossing of a sexual mother plant with an apomictic pollen donor generated a progeny family segregating for reproductive mode. Individuals from this F1 family were cytoembryologically classified as sexual or apomictic. Spikelet mRNA compositions from both groups of plants were compared by differential display using an RNA-bulked procedure. Fifty primer combinations were assayed to generate nearly 2,500 total bands in the fingerprints. Three transcripts expressed at higher levels in apomictic plants (apo417, apo398, and apo396) were identified, isolated and cloned. Sequencing showed a high level of homology among the isolated clones. Analysis by RT-PCR Southern blots followed by densitometric studies confirmed that expression reached a level around 30 times higher in apomictic than in sexual individuals and was probably induced at early stages of the megagametophyte development. Genomic DNA from the parental and the F1 progeny plants showed 4-5 bands when hybridised with apo417 in Southern blots. Comparisons to the sequence data banks revealed no identities to genes of known function. However, a putative deduced 3' protein fragment showed homology to the well-characterised KSP multi-phosphorylation domain previously detected in several cdc2-regulated cytoskeletal proteins.