Eragrostis curvula includes biotypes reproducing through obligate and facultative apomixis or, rarely, full sexuality. We previously generated a “tetraploid-dihaploid-tetraploid” series of plants consisting of a tetraploid apomictic plant (T), a sexual dihaploid plant (D) and a tetraploid artificial colchiploid (C). Initially, plant C was nearly 100% sexual. However, its capacity to form non-reduced embryo sacs dramatically increased over a four year period (2003–2007) to reach levels of 85–90%. Here, we confirmed high rates of apomixis in plant C, and used AFLPs and MSAPs to characterize the genetic and epigenetic variation observed in this plant in 2007 as compared to 2003. Of the polymorphic sequences, some had no coding potential whereas others were homologous to retrotransposons and/or protein-coding-like sequences. Our results suggest that in this particular plant system increased apomixis expression is concurrent with genetic and epigenetic modifications, possibly involving transposable elements.

Weeping lovegrass is a forage grass cultivated in semiarid regions of the world that reproduces mainly by apomixis (diplospory), a process that involves the formation of asexual seeds and bypasses the processes of meiosis and fertilisation. The aim of this work was to evaluate and compare different techniques (cytoembryology, callose deposition, flow cytometry and progeny tests) to determine the reproductive mode of weeping lovegrass. Typical sexual and apomictic processes were clearly differentiated using cytoembryology, and different callose deposition patterns were observed in sexual and apomictic genotypes. Previous studies indicated that presence of callose on the cell wall of the megaspore mother cell is associated only with sexual processes. Nevertheless, our results also found callose deposition in apomictic genotypes, although clearly different from the pattern found in sexual processes, allowing discrimination between sexual and apomictic plants. Flow cytometry seed screening using individual seeds did not differentiate between sexual and apomictic plants as the embryo : endosperm DNA content ratio was similar in sexual and apomictic plants. Progeny tests using molecular markers showed uniform patterns in offspring from apomictic plants and variable patterns among the progeny of sexual plants. The results obtained from cytological studies and progeny tests were similar, indicating that both methods provide useful tools for determination of reproductive mode. However, the callose test with aniline blue was faster and easier to use than other techniques.

Eragrostis curvula (Schrad.) Nees (weeping lovegrass) is an apomictic species native to Southern Africa that is used as forage grass in semiarid regions of Argentina. Apomixis is a mechanism for clonal propagation through seeds that involves the avoidance of meiosis to generate an unreduced embryo sac (apomeiosis), parthenogenesis, and viable endosperm formation in a fertilization-dependent or -independent manner. Here, we constructed the first saturated linkage map of tetraploid E. curvula using both traditional (AFLP and SSR) and high-throughput molecular markers (GBS-SNP) and identified the locus controlling diplospory. We also identified putative regulatory regions affecting the expressivity of this trait and syntenic relationships with genomes of other grass species. We obtained a tetraploid mapping population from a cross between a full sexual genotype (OTA-S) with a facultative apomictic individual of cv. Don Walter. Phenotypic characterization of F 1 hybrids by cytoembryological analysis yielded a 1:1 ratio of apomictic vs. sexual plants (34:27, X 2 = 0.37), which agrees with the model of inheritance of a single dominant genetic factor. The final number of markers was 1,114 for OTA-S and 2,019 for Don Walter. These markers were distributed into 40 linkage groups per parental genotype, which is consistent with the number of E. curvula chromosomes (containing 2 to 123 markers per linkage group). The total length of the OTA-S map was 1,335 cM, with an average marker density of 1.22 cM per marker. The Don Walter map was 1,976.2 cM, with an average marker density of 0.98 cM/marker. The locus responsible for diplospory was mapped on Don Walter linkage group 3, with other 65 markers. QTL analyses of the expressivity of diplospory in the F 1 hybrids revealed the presence of two main QTLs, located 3.27 and 15 cM from the diplospory locus. Both QTLs explained 28.6% of phenotypic variation. Syntenic analysis allowed us to establish the groups of homologs/homeologs for each linkage map. The genetic linkage map reported in this study, the first such map for E. curvula , is the most saturated map for the genus Eragrostis and one of the most saturated maps for a polyploid forage grass species.

This work was aimed at identifying genes that show altered expression profiles in response to changes in ploidy and/or reproductive mode (from sexual to apomictic) in the African grass Eragrostis curvula. A differential display analysis was performed on leaf and flower transcriptomes from a series of genetically related euploid plants, including tetraploid apomictic, diploid sexual, and tetraploid sexual plants. More than 100 primer combinations were used to generate 11,864 total markers, yielding 1293 differential bands. Of these bands, 11.84% to 6.74% were related to ploidy and 0.71% to 2.17% to the reproductive mode, depending on the tissue. A small percentage of bands showed similar expressions between the tetraploid apomictic and the diploid sexual plants. Expression-based similarity dendrograms were constructed. Our data suggested that ploidy is more decisive than tissue type in defining the transcriptome structure. Out of 102 fragments sequenced, 50 showed strong homology to known genes. The differentially expressed genes were mapped in silico onto maize chromosomes. Several candidates mapped within the linkage group syntenic to the Tripsacum dactyloides diplospory-governing region. The evidence indicates that expression of genes located around the diplospory-associated region may be strongly influenced by ploidy and may be silenced in the apomictic genotype. These findings are discussed in the context of diplospory molecular control and its connection with ploidy.