A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops

Analysis of Gene Expression in Resynthesized Brassica napus Allopolyploids Using Arabidopsis 70mer Oligo Microarrays

Background Studies in resynthesized Brassica napus allopolyploids indicate that homoeologous chromosome exchanges in advanced generations (S5:6) alter gene expression through the loss and doubling of homoeologous genes within the rearrangements. Rearrangements may also indirectly affect global gene expression if homoeologous copies of gene regulators within rearrangements have differential affects on the transcription of genes in networks. Methodology/Principal Findings We utilized Arabidopsis 70mer oligonucleotide microarrays for exploring gene expression in three resynthesized B. napus lineages at the S0:1 and S5:6 generations as well as their diploid progenitors B. rapa and B. oleracea. Differential gene expression between the progenitors and additive (midparent) expression in the allopolyploids were tested. The S5:6 lines differed in the number of genetic rearrangements, allowing us to test if the number of genes displaying nonadditive expression was related to the number of rearrangements. Estimates using per-gene and common variance ANOVA models indicated that 6–15% of 26,107 genes were differentially expressed between the progenitors. Individual allopolyploids showed nonadditive expression for 1.6–32% of all genes. Less than 0.3% of genes displayed nonadditive expression in all S0:1lines and 0.1–0.2% were nonadditive among all S5:6 lines. Differentially expressed genes in the polyploids were over-represented by genes differential between the progenitors. The total number of differentially expressed genes was correlated with the number of genetic changes in S5:6 lines under the common variance model; however, there was no relationship using a per-gene variance model, and many genes showed nonadditive expression in S0:1 lines. Conclusions/Significance Few genes reproducibly demonstrated nonadditive expression among lineages, suggesting few changes resulted from a general response to polyploidization. Furthermore, our microarray analysis did not provide strong evidence that homoeologous rearrangements were a determinant of genome-wide nonadditive gene expression. In light of the inherent limitations of the Arabidopsis microarray to measure gene expression in polyploid Brassicas, further studies are warranted

Induced genetic variation, TILLING and NGS-based cloning.

Mutagenesis is one of the most important tools available to barley geneticists and breeders in order to investigate trait inheritance and to provide useful genetic variation to breeding programmes