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Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera.

ABSTRACT: BACKGROUND: Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. RESULTS: Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEf?1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEf?1 or BoechUBQ and BoechEf?1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEf?1), in anthers from both reproductive systems (BoechACT2 and BoechEf?1), in apomictic vegetative tissues (BoechEf?1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEf?1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEf?1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively) CONCLUSIONS: From a total of six tested genes, BoechRPS18, BoechEf?1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.

SUBMITTER: Pellino M

PROVIDER: S-EPMC3171723 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera.

Pellino Marco M Sharbel Timothy F TF Mau Martin M Amiteye Samuel S Corral José María JM

BMC research notes 20110819

<h4>Background</h4>Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genom ...[more]

PMID: 21851639

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Project description:BackgroundApomixis or asexual seed formation represents a potentially important agronomic trait whose introduction into crop plants could be an effective way to fix and perpetuate a desirable genotype through successive seed generations. However, the gene regulatory pathways underlying apomixis remain unknown. In particular, the potential function of microRNAs, which are known to play crucial roles in many aspects of plant growth and development, remains to be determined with regards to the switch from sexual to apomictic reproduction.ResultsUsing bioinformatics and microarray validation procedures, 51 miRNA families conserved among angiosperms were identified in Boechera. Microarray assay confirmed 15 of the miRNA families that were identified by bioinformatics techniques. 30 cDNA sequences representing 26 miRNAs could fold back into stable pre-miRNAs. 19 of these pre-miRNAs had miRNAs with Boechera-specific nucleotide substitutions (NSs). Analysis of the Gibbs free energy (ΔG) of these pre-miRNA stem-loops with NSs showed that the Boechera-specific miRNA NSs significantly (p ≤ 0.05) enhance the stability of stem-loops. Furthermore, six transcription factors, the Squamosa promoter binding protein like SPL6, SPL11 and SPL15, Myb domain protein 120 (MYB120), RELATED TO AP2.7 DNA binding (RAP2.7, TOE1 RAP2.7) and TCP family transcription factor 10 (TCP10) were found to be expressed in sexual or apomictic ovules. However, only SPL11 showed differential expression with significant (p ≤ 0.05) up-regulation at the megaspore mother cell (MMC) stage of ovule development in apomictic genotypes.ConclusionsThis study constitutes the first extensive insight into the conservation and expression of microRNAs in Boechera sexual and apomictic species. The miR156/157 target squamosa promoter binding protein-like 11 (SPL11) was found differentially expressed with significant (p ≤ 0.05) up-regulation at the MMC stage of ovule development in apomictic genotypes. The results also demonstrate that nucleotide changes in mature miRNAs significantly (p ≤ 0.05) enhance the thermodynamic stability of pre-miRNA stem-loops.

Project description:BackgroundBrachiaria 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.ResultsEight 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.ConclusionIn 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.

Project description:Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO gene encoding aspartate glutamate aspartate aspartate histidine exonuclease is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Furthermore, we characterize the structure of CENH3 by bioinformatic tools, study expression levels of APOLLO and CENH3 transcripts by Real-Time Polymerase Chain Reaction RT-PCR in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of meiosis and before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until the 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes. The results obtained may indicate to a role of the CENH3 and APOLLO genes in the development of apomixis in species of the genus Boechera.

Dataset Information

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera.

Publications

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera.

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