Abstract
BackgroundApomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris.FindingsAmong tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction.ConclusionOur gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.
Highlights
Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant
All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation
Six reference gene sequences (EF1alpha, EIF4A, UBCE, ACT2, GAPDH and TUBA) from other plant species were used in BlastN analysis against the C. ciliaris pistil EST library at NCBI where C. ciliaris EST sequences with high similarity to all six selected reference genes (E-value = 0.0) were retrieved
Summary
Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. Sexual and asexual modes of reproduction occur in flowering plants. Apomixis is a natural form of asexual reproduction through seeds. The global regulatory effects of polyploidy and hybridity have been proposed to be the possible triggers for conversion of sexual to apomictic forms of reproduction [1,7]. Genetic data from several species indicate that at least two genes, one for apomeiosis and one for parthenogenesis [2], are required for apomixis, lack of recombination in some species manifests as monogenic inheritance of the trait
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