Polygalacturonase (PG) is the major cell wall degrading enzyme of tomato fruit. It is developmentally regulated and is synthesised de novo in ripening fruit. Genomic clones encoding a PG gene of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) have been isolated, mapped and sequenced. The sequence of the protein-coding region is identical to that of a PG cDNA [20]. Comparison of the cloned restriction fragments with genomic Southern data suggests that there may only be one gene for PG per haploid genome. The PG gene, which covers approximately 7 kb, is interrupted by 8 intervening sequences ranging in size from 99 bp to 953 bp. The transcription start point was identified by S1 mapping and primer extension analysis. About 1.4 kb of 5' flanking DNA has been sequenced. This contains putative TATA and CAAT boxes and also direct repeat sequences. A transcriptional fusion has been constructed between the putative 1.4 kb promoter fragment and the chloramphenicol acetyl transferase (CAT) gene. Constructs containing this gene have been transferred to tomato using binary vectors. Regenerated transgenic plants express CAT in ripe tomato fruit, but not in unripe tomatoes, leaves, or roots.

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Background Brachypodium distachyon is emerging as the model plant for temperate grass research and the genome of the community line Bd21 has been sequenced. Additionally, techniques have been developed for Agrobacterium-mediated transformation for the generation of T-DNA insertional lines. Recently, it was reported that expression of the polyubiquitin genes, Ubi4 and Ubi10 are stable in different tissues and growth hormone-treated plant samples, leading to the conclusion that both Ubi4 and Ubi10 are good reference genes for normalization of gene expression data using real-time, quantitative PCR (qPCR).Principal FindingsMining of the Joint Genome Institute (JGI) 8X Brachypodium distachyon genome assembly showed that Ubi4 and Ubi10 share a high level of sequence identity (89%), and in silico analyses of the sequences of Ubi4 (Bradi3g04730) and Ubi10 (Bradi1g32860) showed that the primers used previously exhibit multiple binding sites within the coding sequences arising from the presence of tandem repeats of the coding regions. This can potentially result in over-estimation of steady-state levels of Ubi4 and Ubi10. Additionally, due to the high level of sequence identity between both genes, primers used previously for amplification of Ubi4 can bind to Ubi10 and vice versa, resulting in the formation of non-specific amplification products.ConclusionsThe results from this study indicate that the primers used previously were not sufficiently robust and specific. Additionally, their use would result in over-estimation of the steady-state expression levels of Ubi4. Our results question the validity of using the previously proposed primer sets for qPCR amplification of Ubi4 and Ubi10. We demonstrate that primers designed to target the 3′-UTRs of Ubi4 and Ubi10 are better suited for real-time normalization of steady-state expression levels in Brachypodium distachyon.

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