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Additionally we analysed the features of
Additionally, we analysed the features of apomict and sexual Boechera species DNMT proteins. All DNMT proteins showed highly similar theoretical pI, molecular weight and predicted sub-cellular localization (Table 2).
Discussion
Identification of the structure and expression levels of DNMTs during seed development is an important step to understand the role of epigenetic regulators of apomixis in Boechera. Our results showed that the structure of DNMT proteins and conserved domains in apomict and sexual Boechera species share common features with those characterized in other organisms (Garg et al., 2014, Ramos et al., 2013).
Our phylogenetic results in overall demonstrate that MET1, CMT3 and DRM2 genes are conserved in photosynthetic organism groups. This finding is parallel with the previous studies of those genes (Bhuiyan et al., 2015, Grossman et al., 2010, Noy-Malka et al., 2014, Pavlopoulou and Kossida, 2007, Sharma et al., 2009). According to the alignment structure of MET1 (Online Resource 1) green algae are more similar to monocotyledons than dicotyledons. Therefore, the green algae group is placed as sister group to monocotyledons. We did not detect significant similarity between green algae and other groups on DRM2 tree, which results incongruent placement of green algae taxa in DRM2 tree. The plant specific CMT3 gene alignment showed more reliable similarity among all the groups and provided better resolution and separation on the tree. Boechera DNMT sequences were grouped in dicotyledons with Brassicaceae taxa as expected. Among Boechera group, B. stricta and B. divaricarpa separated from B. holboellii by minor difference on each DNMT tree. This separation was supported by high (85–100%) bootstrap values, and explain the interspecific hybrid origin of the B. divaricarpa between sexual and apomict Boechera species (Dobes et al., 2004, Koch et al., 2003).
Previously, it was reported that a small number of LY2409881 remodelling enzymes were differentially expressed significantly during female gametophyte development in maize and its apomict relative Tripsacum (Garcia-Aguilar et al., 2010). The researchers also compared the level of DNMT mRNA of ovules dissected at the megaspore mother cell and embryo sac stages in B. holboellii with A. thaliana. They indicated that DRM2 was downregulated during gametophyte development in apomeiotic ovules but CMT3 and DDM1 had similar expression patterns in both species (Garcia-Aguilar et al., 2010). In contrast to this, we found that DRM2 is upregulated in apomictic Boechera following fertilization. But, we analysed later development stages and obtained tissues from hand-pollinated siliques at developing endosperm and embryo. Also, the expression level of MET1 and CMT3 are slightly different in both species following fertilization.
The diploid B. stricta genotype used in this work, was reported to be sexual and produces seeds composed of a 2C embryo and a 3C endosperm (Schranz et al., 2006, Aliyu et al., 2010). Diploid apomictic B. divaricarpa is pseudogamous and can produce both reduced and unreduced male gametes (Schranz et al., 2006). Since pseudogamous endosperm development was characterized by fertilization of the unreduced central cell (4m) by reduced or the unreduced pollen (1p or 2p), different endosperm ploidies were reported in various diploid Boechera species including most common 6C (4m+2p) (Aliyu et al., 2010). Our previous works on the diploid apomictic B. divaricarpa accession used in this study showed reduced pollen production (Taşkın et al., 2009). Therefore, this accession can produce pseudogamous 5C (4m+1p) or 6C (4m+2p, polyspemy) endosperm with no harmful effects on seed development. Endosperm development has been associated with genomic imprinting in sexual and apomictic Boechera species (Naumova et al., 2001, Aliyu et al., 2010), in agreement with this, our results showed that the expression levels of DNMTs are different in apomicts compared with sexual Boechera species after pollination. Also, an epigenetic control of parthenogenesis and autonomous endosperm development were suggested previously for Paspalum spp. (Podio et al., 2014) and Arabidopsis (Ohad et al., 1996). In Arabidopsis fie-1 mutant lines, an autonomous endosperm development was reported in half of the ovules (Ohad et al., 1996, Ohad et al., 1999). Later, this phenotype was promoted when an anti-sense construct succeed MET1 silencing by hypomethylation (Vinkenoog et al., 2000). Also, recently, an interaction between the polycomb group protein MEDEA and the DNMT MET1 was revealed to repress autonomous endosperm development in Arabidopsis (Schmidt et al., 2013).