Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha.

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. RESULTS: Eight genes were selected from a cDNA library of ovaries of B. brizantha considering the similarity to reference genes: EF1 (elongation factor 1 alpha), E1F4A (eukaryotic initiation factor 4A), GAPDH (glucose-6-phosphate dehydrogenase), GDP (glyceroldehyde-3-phosphate dehydrogenase), SUCOA (succinyl-CoA ligase), TUB (tubulin), UBCE (ubiquitin conjugating enzyme), UBI (ubiquitin). For the analysis, total RNA was extracted from 22 samples and raw Ct data after qRT-PCR reaction was analyzed for primer efficiency and for an overall analysis of Ct range among the different samples. Elongation factor 1 alpha showed the highest expression levels, whereas succinyl-CoA ligase showed the lowest within the chosen set of samples. GeNorm application was used for evaluation of the best reference genes, and according to that, the least stable genes, with the highest M values were tubulin and succinyl-CoA ligase and the most stable ones, with the lowest M values were elongation factor 1 alpha and ubiquitin conjugating enzyme, when both reproductive and vegetative samples were tested. For ovaries and spikelets of both sexual and apomictic B. brizantha the genes with the lowest M values were BbrizUBCE, BbrizE1F4A and BbrizEF1. CONCLUSION: In total, eight genes belonging to different cellular processes were tested. Out of them, BbrizTUB was the less stable while BbrizEF1 followed by BbrizUBCE were the more stable genes considering male and female reproductive tissues, spikelets, roots and leaves. Regarding the best reference genes for ovary tissues, where apomictic and sexual reproduction must occur, the best reference genes were BbrizUBCE, BbrizE1F4A and BbrizEF1. Our results provide crucial information for transcriptional analysis in the Brachiaria ssp, helping to improve the quality of gene expression data in these species, which constitute an excellent plant system for the study of apomixis.

Expression analyses of Brachiaria brizantha genes encoding ribosomal proteins BbrizRPS8, BbrizRPS15a, and BbrizRPL41 during development of ovaries and anthers.

Brachiaria brizantha is a forage grass of the Poaceae family. Introduced from Africa, it is largely used for beef cattle production in Brazil. Brachiaria reproduces sexually or asexually by apomixis, and development of biotechnological tools for gene transfer is being researched to support the breeding programs. The molecular bases of reproduction have not yet been fully elucidated; it is known that gametophyte formation and main reproductive events occur inside the anthers and ovaries. There is therefore much interest in identifying genes expressed in these organs and their corresponding upstream regulatory sequences. In this work we characterized three cDNA from ovaries of B. brizantha plants (CL 09, CL10, and CL21) which show similarity in databases with genes encoding ribosomal proteins S8, S15a, and L41 and were named BbrizRPS8, BbrizRPS15a, and BbrizRPL41, respectively. These clones show higher expression in ovaries, anthers and roots, mitotically active tissues, when compared to leaves of B. brizantha. Localization of transcripts of BbrizRPS8, BbrizRPS15a, and BbrizRPL41 was investigated in the reproductive organs, ovaries, and anthers, from the beginning of development up to maturity. Their activity was higher in early stages of anther development, while expression was detected in all developmental stages in the ovaries, except for BbrizS15a, which was detected only in synergids of apomictic plants.