Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha
© Silveira et al; licensee BioMed Central Ltd. 2009
Received: 25 November 2008
Accepted: 02 July 2009
Published: 02 July 2009
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.
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.
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.
Brachiaria is an important Poaceae genus introduced in Brazil from Africa. This genus consists of around 100 species, and the two most important cultivars in Brazil are B. brizantha cv. Marandu and B. decumbens cv. Basilisk . They show qualities of forage grass, good adaptability to cerrado areas (dry-tropical savanna, Brazil), and are cultivated in more than 40 million hectares in Brazil . Both cultivars reproduce asexually through seeds by apomixis , which is classified as a pseudogamous aposporic type [4–9]. Apomixis is present in more than 300 angiosperm species  and is being investigated by many groups due to the biotechnological interest of controlling the process of cloning through seeds.
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. The identification of genes involved in apomictic development will open the possibility of controlling the expression of this trait and engineering crops with higher productivity and a reduced risk of gene transfer. One way of comparing these different molecular pathways is by comparing the transcript expression profiles of genes related to ovary development in sexual plants, which have a Polygonum-type embryo sac, to an apomictic plant, which has a Panicum-type embryo sac . Analysis of a Brachiaria germplasm collection assembled at CIAT-Colombia pointed to a majority of polyploids apomicts, whereas the diploids are sexual [3, 11]. In B. brizantha among 275 accessions identified to date only one is diploid, BRA 002747 . Sexual tetraploids were obtained with colchicine treatment of the diploid plants [12, 13]. These plants are under analysis at the breeding program aiming to produce intraspecific hybrids and to identify molecular markers associated with the apomixis trait. Currently, comparative studies of the molecular biology of Brachiaria reproductive processes are being performed with BRA 002747 and BRA 00591 [13, 14]. Both accessions are very important for these comparative studies since the sexual diploid BRA 002747 is the only sexual accession among all the accessions, while BRA 00591 is the most apomictic accession, with 98% of aposporous embryo sacs .
Quantitative real-time PCR (qRT-PCR) has emerged as an important technique to compare the expression profiles of target genes in different species, tissues or treatments and also to validate high-throughput gene expression profiles [15, 16]. One of the methodologies to determine gene expression levels in qRT-PCR is by comparing the expression of the gene of interest in different conditions with reference genes whose expressions do not change under the various experimental conditions. Based on these requirements, statistical analysis methods have been developed in order to identify the best reference genes to a certain organism or experimental condition [17–19]. The use of reference genes without prior verification of their expression stability can lead to inaccurate data interpretation and thus generate incorrect results.
According to previous work concerning the best reference genes for transcription normalization in plants, the most reliable ones are those constitutively expressed and involved in basic cellular processes, such as protein and sugar metabolism and cell structure [18, 20–22]. A large-scale comparative analysis of the most stable genes of Arabidopsis has shown that the best reference genes are those related to the ubiquitin degradation process, such as polyubiquitin, ubiquitin-conjugating enzymes and ubiquitin ligases . In the qRT-PCR expression profile analysis of suitable reference genes for poplar (Populus trichocarpa × P. deltoides, cottonwood hybrid) and vitis (Vitis vinifera), tubulin and actin were stably expressed and considered the most reliable ones [18, 22]. In a similar approach, Jain et al. (2006) showed that the best genes among the different tested tissue samples in Oryza sativa were ubiquitin 5 and elongation factor-1 alpha. For species with both sexual and apomictic reproductive mode, the best reference genes for qRT-PCR experiments have not been reported yet. Real time PCR has been done to validate other differential expression experiments using absolute qRT-PCR or using internal control genes tested by other differential expression techniques [24, 25].
In this work, we evaluated eight potential reference genes isolated from EST ovary libraries for Brachiaria brizantha qRT-PCR experiments. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested. The relative transcription levels of the genes were determined in ovaries and anthers at different developmental stages, sporogenesis and gametogenesis, in spikelets, leaves and roots all together. Also, it was determined the most stable genes only for spikelets and ovaries, where differences in the expression profile between sexual and apomictic plants must occur, from both sexual and apomictic accessions.
Results and discussion
Candidate reference genes
Gene description, primer sequences and efficiency of the selected ESTs.
Gene identification/Gene description
E value/ID (%)
Primer sequence/Amplicon size
Amplification efficiency ± SD*
GeneBank Accession Number
Elongation factor-1 alpha
0.87 ± 0.012
Eukaryotic initiation factor 4A
0.94 ± 0.011
0.97 ± 0.009
1.01 ± 0.009
succinyl-CoA ligase (GDP-forming) beta-chain
1.00 ± 0.008
putative tubulin alpha-5 chain
1.01 ± 0.019
0.92 ± 0.013
0.95 ± 0.015
0.94 ± 0.020
Primer efficiency and Ct variation
In order to find the best reference genes for relative quantification, a high quality starting material is needed. For that, total RNA was extracted from all tissue samples using the same extraction protocol  for the different Brachiaria organs. All samples were treated with DNAse to avoid misinterpretation of qRT-PCR results by genomic DNA contamination in cDNA samples. RNA quality analysis and quantitation were performed by agarose gel analysis and a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies) measurement, respectively. This procedure was crucial to guaranteeing the same amount of starting material and equivalent efficiency of cDNA synthesis from total RNA samples.
Based on DNA analysis by agarose gel electrophoresis and the dissociation curves (additional file 1), one single PCR product with the expected size was amplified for each of the nine sets of primers selected for this analysis (not shown). After the PCR reaction, the entire raw fluorescence data generated in Opticon3 was used for the primer amplification efficiency calculation and Ct determination with the miner algorithm . This algorithm accounts for each PCR exponential curve, making it is possible to have accurate values for the quantification of qRT-PCR. The amplification efficiency using this program can vary between 50% and 150%, and for the nine tested primer pairs it varied from 0.87 ± 0.012 (87%) to 1.01 ± 0.009 (101%), which are expected amplification efficiencies between compared genes .
Gene expression stability of candidate reference genes
From the eight housekeeping genes tested in this study, the ones encoding for the ubiquitin-conjugating enzyme (BbrizUBCE) and elongation factor-1 (BbrizEF1) were considered most stable based on the transcriptional profile and geNORM analysis when considering both vegetative and reproductive tissues.
These two genes have been suggested as reference genes in other plants for qRT-PCR analysis, but also for other experimental techniques such as RT-PCR and northern blot analysis [21, 31, 32]. Even though the two genes exhibited the desired stability values, the best experimental designs use reference genes that act independently and are involved in different cellular processes. Therefore, BbrizUBCE and BbrizEF1 will be used as the reference genes in further experiments of B. brizantha vegetative and reproductive developmental tissues. This is the first report to clone, sequence and test reference genes for the transcriptional analysis of plants of the Brachiaria genus. 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.
Plant material and tissue samples
Two accessions of Brachiaria brizantha from Embrapa were used in this work: BRA 002747 (B105), a sexual diploid (2n = 2x = 18), and BRA 00591 (B030), a facultative apomictic tetraploid (2n = 4x = 36) named B. brizantha (A. Rich) Stapf cv. Marandu, which were both cultivated in the field at the Embrapa Genetic Resources and Biotechnology.
For analysis of the most stable genes during male and female gametophyte development, ovaries and anthers of both accessions were dissected from flowers at four different stages of development before anthesis, as previously described by Rodrigues et al. (2003). For each RNA extraction experiment, around 1000 ovaries and 50 anthers of each of the four stages were isolated. Stages I and II are related to sporogenesis, and stages III and IV are related to gametogenesis [9, 6]. In addition, whole spikelet, leaf and root tissues were isolated from both B. brizantha accessions for RNA extraction.
Total RNA was extracted from each pool sample with TRIZOL (1/10 w/v) (Invitrogen™) with a modified method from the manufacturer's instructions. Samples were ground with a drill (AD-18 S Bionic Drill set) holding an RNAse-free polystyrene pistil. After extraction, the RNA sample was dissolved in 15–20 μl of 0.1% diethyl pyrocarbonate (DEPC)-treated water. RNA was treated with DNAse using on-column Qiagen DNAse Treatment (RNeasy MicroKit, Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The RNA concentration and A260/A280 ratios were determined before and after DNAse I treatment using a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies), and 1.1% agarose gel electrophoresis was conducted to visualize the integrity of the RNA. Only the RNA samples with A260/A280 ratios between 1.9 and 2.1 and A260/A230 ratios greater than 2.0 were used for the analysis.
First strand cDNAs were synthesized from 1.5 μg of total RNA with OligodT and Superscript II enzyme (Invitrogen™). The first strand synthesis system was used according to the manufacturer's instructions.
PCR primer design
Several described plant housekeeping genes were selected for the analysis. Genes already described as good reference genes for other plant species were used to BLAST search against B. brizantha EST (expressed sequence tag) ovaries libraries, and the list of selected sequences is shown in Table 1. Primers were designed within 800 bp of the polyadenylation site, since the primers came from an EST library constructed using the OligodT priming strategy. Primer 3.0 software was used for primer design. Amplicon lengths varied from 100 to 200 bp, with melting temperatures (Tm) varying between 59 – 60°C and primer lengths between 20–23 bp. The primers were screened for hairpins, dimmer formation, and target specificity by BLASTN http://www.ncbi.nlm.nih.gov/BLAST against the nr databank. Primer pairs were tested for specificity by RT-PCR and also by qRT-PCR, followed by a dissociation curve and agarose gel electrophoresis.
Real-time PCR conditions and analysis
PCR reactions were performed in 96-well plates with the Chromo4 Real-Time PCR Detector System (BioRad®) using SYBR® Green to detect dsDNA synthesis. Reactions were done in 20 μL volumes containing PCR Buffer (Invitrogen™), 1.5 mM MgCl2, 0.1 mM dNTPs, 0.25 U Taq Platinum (Invitrogen™), 0.1× SYBR Green (Amersham™), 200 nM of each primer and 10 μl sscDNA (corresponding to 5 ng of total RNA). Aliquots from the same sscDNA sample were used with all primer sets in two separate experiments. Two biological replicates for each of the 20 samples were used for real-time PCR analysis, and three technical replicates were analyzed for each biological replicate.
Reactions were run in a BioRad qRT-PCR machine using the following cycling parameters: 94°C for 5 min, 40 cycles of 94°C for 15 s, 60°C for 10 s, 72°C for 15 s and 60°C for 35 s. No-template controls (NTC) were included for each primer pair, and each PCR reaction was performed in triplicate. Dissociation curves for each amplicon and agarose gel were then analyzed to verify the specificity of each amplification reaction; the dissociation curve was obtained by heating the amplicon from 40°C to 100°C and reading at each °C.
Primer efficiency calculation and Ct determination
The calculation of primer amplification efficiency and Ct determination were done using the miner algorithm . Raw fluorescence data generated with the Opticon 3 software (BioRad) was used for these calculations. After running the miner algorithm, Ct values were transferred as a Microsoft Excel file (Microsoft, Redmond, WA) for further gene expression stability analysis.
Analysis of gene expression stability
For analysis of gene expression stability and rank, geNorm v. 3.4 software was used. The Microsoft Excel file (Microsoft, Redmond, WA) with the raw expression Ct values for each tested gene in the 22 different samples generated with the miner algorithm was first analyzed with the qBase software version 1.3.4 http://medgen.ugent.be/qbase/ and then transferred into the expression stability program geNorm, version 3.4 http://medgen.ugent.be/~jvdesomp/genorm/, as described by Vandesompele et al. (2002). This application defines the most stable genes by calculating the mean pairwise variation between a particular gene and all the others used in one experiment and determines an M value. The highest M value corresponds to the least stable expression in a set of samples. As a result, the normalization factor (NF) is defined, by considering the M value of the most stable genes. This information allows for the establishment of the minimum number of reference genes required for an accurate calculation of the relative expression of a target gene. This ideal number is given by the inclusion of a certain number of genes in the NF calculation until there is no significant contribution to an additional gene. The raw data from the two biological replicas was used for gene stability analysis and both showed similar results.
This work was funded by grants from CNPq and CBAB and a Ph.D. fellowship from CAPES, Brazil.
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