Effect of transgenes on global gene expression in soybean is within the natural range of variation of conventional cultivars.

Current safety assessment for novel crops, including transgenic crops, uses a targeted approach, which relies on compositional analysis. The possibility that transgene expression could lead to unintended effects remains a debated issue. This study used transcriptome profiling as a nontargeted approach to evaluate overall molecular changes in transgenic soybean cultivars. Global gene expression was measured in the first trifoliate leaves of two transgenic and three conventional soybean cultivars using the soybean Affymetrix GeneChip. It was found that gene expression differs more between the two conventional cultivars than between the transgenics and their closest conventional cultivar investigated and that the magnitudes of differences measured in gene expression and genotype (determined by SSR analysis) do not necessarily correlate. A MySQL database coupled with a CGI Web interface was developed to store and present the results ( http://soyxpress.agrenv.mcgill.ca/). By integrating the microarray data with gene annotations and other soybean data, a comprehensive view of differences in gene expression is explored between cultivars.

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.

Bacillus thuringiensis cry3Ca1 protein is toxic to the Colorado potato beetle, Leptinotarsa decemlineata (Say).

We expressed the wild-type cry3Aa3 and cry3Ca1 Bacillus thuringiensis genes, which code for insecticidal proteins, in an Escherichia coli expression system. Highly purified preparations of the soluble delta-endotoxins were used to perform comparative bioassays with third-instar larvae of the Colorado potato beetle (CPB). Acute mortality data showed that Cry3Ca1 (LD(50) = 320.1 ng) was 2-fold more toxic than Cry3Aa3 (LD(50) = 672.9 ng). We also compared the chronic effects of sublethal doses of these toxins by measuring the consumption of untreated foliage and monitoring survival and development for 6 days after intoxication. No significant additional mortality was recorded, but we found that surviving larvae fed Cry3Ca1 consumed foliage at a slower rate than the larvae fed Cry3Aa3, suggesting more damage to their digestive epithelium. This study, the first assessment of the toxicity of cry3Ca1 in third-instar CPB, suggests cry3Ca1 will prove useful for the control of this important insect pest.

Premature polyadenylation contributes to the poor expression of the Bacillus thuringiensis cry3Ca1 gene in transgenic potato plants.

The cry genes that code for the insecticidal crystal proteins of Bacillus thuringiensis (B.t.) have been widely used to develop insect-resistant transgenic plants. The cry3Ca1 gene has been reported to code for a crystal protein which is particularly potent against the Colorado potato beetle (CPB). To explore the biotechnological potential of cry3Ca1, we introduced this gene into transgenic potato plants under the control of the CaMV 35S promoter. In the resulting transformants, the cry3-Ca1 gene was very poorly expressed. In fact, no full-length transcript (2300 nt) could be detected. Instead, only short transcripts of approximately 1100 nt were observed. Analysis of these short transcripts by Northern hybridization, RT-PCR as well as by cloning and sequencing showed that they resulted from premature polyadenylation. These processing events occurred at four sites within the cry3Ca1 coding region (at positions 652, 669, 914 and 981 relative to the translation start site). The sites at which premature polyadenylation took place were not those that showed the highest degree of identity to the canonical AAUAAA motif. Together with other recent data, our findings suggest that premature polyadenylation is an important mechanism which can contribute to the poor expression of transgenes in a foreign host.

Hairpin elements, the first family of foldback transposons (FTs) in Arabidopsis thaliana.

We report here on the identification in Arabidopsis thaliana of a new family of transposable elements named Hairpin. These elements are related to foldback transposons (FTs), a large and heterogeneous group of transposable elements first described in Drosophila and recently in Solanaceae. Hairpin elements are the first family of FTs reported in Arabidopsis thaliana and the first family of FTs of type 3 to be described in the plant kingdom. In contrast to previous FTs described, Hairpin appears to be a homogeneous family in size (238 +/- 7 bp) as well as in structure. Hairpin elements are dispersed in the Arabidopsis genome and Southern hybridization revealed that they are present in relatively low copy numbers. Finally, we discuss the potential usefulness of these elements in studying the phylogenetic relationship between Arabidopsis ecotypes.

Regional insertional mutagenesis on chromosome III of Arabidopsis thaliana using the maize Ac element.

The authors describe the production and characterization of a collection of Arabidopsis lines each carrying a transposed Ac (trAc) element. A total of 507 lines were obtained following germinal transpositions of a single Ac element located on the upper portion of chromosome III. Southern analysis revealed that up to 90% of the lines in this collection harbour distinct insertions of Ac in the Arabidopsis genome. As previous studies on the behaviour of Ac in Arabidopsis have indicated that approximately two out of three transposition events occur to linked loci, the authors hypothesized that this collection could be of great use in isolating insertional mutants for genes located in the vicinity of the donor locus. PCR and phenotypic screens were performed to identify mutations in five loci located within a 40 cm region of chromosome III centered on the donor locus. Molecular analyses confirmed the presence of germinal insertions of Ac in three of the loci (NPTII, ABI3 and EST #210A22). At a fourth locus (AtDMC1), despite the absence of a germinal insertion, one line in which somatic insertions occurred regularly was identified and may be of use in isolating a germinal insertion. This collection of trAc lines constitutes a useful complement to the existing collection of T-DNA insertion lines and will soon be made available through the Arabidopsis Biological Resource Center.