Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR

<p>Abstract</p> <p>Background</p> <p>The wild grass species <it>Brachypodium distachyon </it>(Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.</p> <p>Results</p> <p>A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST) databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR) using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (<it>UBC18</it>) was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (<it>Ubi4 </it>and <it>Ubi10</it>) was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (<it>SamDC</it>) ranked was most stable in plants grown under various environmental stresses.</p> <p>Conclusion</p> <p>This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.</p

The chromosome content and genotype of two wheat cell lines and of their somatic fusion product with oat

Somatic hybridization seeks to genetically combine phylogenetically distant parents. An effective system has been established in bread wheat (Triticum aestivum L.) involving protoplasts from a non-totipotent cell line adapted to in vitro culture (T1) in combination with totipotent protoplasts harvested from embryogenic calli (T2). Here, we report the karyotype and genotype of T1 and T2. Line T1 carries nine A (A-genome of wheat), seven B (B-genome of wheat) and eight D (D-genome of wheat) genome chromosomes, while T2 cells have 12 A, 10 B and 12 D genome chromosomes. Rates of chromosome aberration in the B- and D-genomes were more than 25%, higher than in the A-genome. DNA deletion rates were 55.6% in T1 and 19.4% in T2, and DNA variation rates were 8.3% in T1 and 13.9% in T2. Rate of DNA elimination was B- > D- > A-genome in both T1 and T2. The same set of cytological and genetic assays was applied to a derivative of the somatic fusion between protoplasts of T1, T2 and oat (Avena sativa L.). The regenerant plants were near euploid with respect to their wheat complement. Six wheat chromosome arms—4AL, 3BS, 4BL, 3DS, 6DL and 7DL—carried small introgressed segments of oat chromatin. A genotypic analysis of the hybrid largely confirmed this cytologically-based diagnosis

The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members) using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress