Background: Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur.

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants’ ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.

Apomixis, an asexual mode of reproduction through seeds, holds much promise for agricultural advances. However, the molecular mechanisms underlying this trait are still poorly understood. We previously isolated several transcripts representing novel sequences differentially expressed in reproductive tissues of sexual and apomictic plants. Here, we report the characterization of two of these unknown RNA transcripts (experimental codes N17 and N22). Since original fragments showed no significant homologies to sequences at databases, preliminary genomic PCR experiments were carried out to discard possible contaminations. RACE extension on flanking regions provided longer sequences for the candidates and additional related transcripts, which revealed similarity to LTR retrotransposons carrying short transduplicated segments of protein-coding genes. Interestingly, some transduplicated segments corresponded to genes previously associated with apomictic development. Gene copy number estimations revealed a moderate representation of the elements in the genome, with significantly increased numbers in a sexual genotype with respect to an apomictic one. Genetic mapping of N17 showed that a copy of this particular element was located onto Paspalum notatum linkage group F3c, at a central non-recombinant region resembling a centromere. Expression analysis showed an increased activity of N17 and N22 sense strands in ovules of the sexual genotypes. A retrotransposon-specific differential display analysis aimed at detecting related sequences allowed the identification of a complex family, with the majority of its members represented in the sexual genotype. Our results suggest that these elements could be participating in regulatory pathways related to apomixis and sexuality.

BbrizGID1 is expressed in the nucellus of apomictic Brachiaria brizantha, previous to aposporous initial differentiation. AtGID1a overexpression triggers differentiation of Arabidopsis thaliana MMC-like cells, suggesting its involvement in ovule development. GIBBERELLIN-INSENSITIVE DWARF1 (GID1) is a gibberellin receptor previously identified in plants and associated with reproductive development, including ovule formation. In this work, we characterized the Brachiaria brizantha GID1 gene (BbrizGID1). BbrizGID1 showed up to 92% similarity to GID1-like gibberellin receptors of other plants of the Poaceae family and around 58% to GID1-like gibberellin receptors of Arabidopsis thaliana. BbrizGID1 was more expressed in ovaries at megasporogenesis than in ovaries at megagametogenesis of both sexual and apomictic plants. In ovules, BbrizGID1 transcripts were detected in the megaspore mother cell (MMC) of sexual and apomictic B. brizantha. Only in the apomictic plants, expression was also observed in the surrounding nucellar cells, a region in which aposporous initial cells differentiate to form the aposporic embryo sac. AtGID1a ectopic expression in Arabidopsis determines the formation of MMC-like cells in the nucellus, close to the MMC, that did not own MMC identity. Our results suggest that GID1 might be involved in the proper differentiation of a single MMC during ovule development and provide valuable information on the role of GID1 in sexual and apomictic reproduction.