Characterization of the horseweed (Conyza canadensis) transcriptome using GS-FLX 454 pyrosequencing and its application for expression analysis of candidate non-target herbicide resistance genes.

BACKGROUND: The de novo transcriptome sequencing of a weedy plant using GS-FLX 454 technologies is reported. Horseweed (Conyza canadensis L.) was the first broadleaf weed to evolve glyphosate resistance in agriculture, and also is the most widely distributed glyphosate-resistant weed in the United States and the world. However, available sequence data for this species are scant. The transcriptomic sequence should be useful for gene discovery, and to help elucidate the non-target-based glyphosate resistance mechanism and the genomic basis of weediness. RESULTS: Sequencing experiments yielded 411 962 raw reads, an average read length of 233 bp and a total dataset of 95.8 Mb (NCBI accession number SRA010952). After trimming and quality control, 379 152 high-quality sequences were retained and assembled into contigs. The assembly resulted in 31 783 unique transcripts, including 16 102 contigs and 15 681 singletons. The average coverage depth for each contig and each nucleotide position was 22-fold and 12-fold respectively. A total of 16 306 unique sequences were annotated by searching a custom plant protein database. The utility of the transcriptome data was demonstrated by further exploration of ABC transporters, which were previously hypothesized to play a role in non-target glyphosate resistance. Real-time RT-PCR primers were designed from the transcriptome data, which made it possible to assess expression patterns of 17 ABC transporters from resistant and susceptible horseweed accessions from Tennessee, with and without glyphosate treatment. CONCLUSION: These results show that GS-FLX 454 sequencing is a powerful and cost-effective platform for the development of functional genomic tools for a weed species.

Species identification of plant tissues from the gut of An. sergentii by DNA analysis.

There are three commonly used assays to identify plant material in insect guts: the cold anthrone test for fructose, the cellulose staining test for visualizing plant tissue and gas chromatography for seeking unique sugar content profiles. Though sugar and cellulose tests can distinguish between the general sources of sugar meal (nectar versus tissue), they cannot identify the species of plant sources. Even gas chromatography profiles can be problematic; there are reported instances of intra-specific variation as well as inter-specific and intergeneric variation that can mar results. Here, we explore the potential for DNA analysis to help resolve this issue. First, Anopheles sergentii were exposed to branches of two species of highly attractive flowering bushes in the laboratory and the great majority ( approximately 90-98%) were positive for sugar from nectar while very few were positive for cellulose ( approximately 0.5-8%) and DNA (6-19%). Moreover, laboratory An. sergentii showed opposing preferences, tending to obtain sugar from nectar of one plant (Tamarix nilotica) but to feed more on tissue from the other (Ochradenus baccatus). An. sergentii are exposed to a wide variety of plants in their natural desert habitats and in the absence of flowers in the dry season, they resort to feeding specifically on tissues of a few plants. According to DNA analysis the favorite plants were Suaeda asphaltica, Malva nicaeensis and Conyza dioscoridis, which are succulents that account for less than 1% of vegetation in the area.

Ferritin2 gene in paraquat-susceptible and resistant biotypes of horseweed Conyza canadensis (L.) Cronq.

Ferritins, the multimeric iron storage proteins, are the main regulators of the cellular level of uncomplexed iron. Ferritins are encoded by small gene families and expressed differentially under various developmental conditions depending on iron availability, effect of hormones or oxygen radical generating agents. In the present work the primary structure of the ferritin2 gene from resistant and susceptible biotypes of horseweed Conyza canadensis was determined. This gene was found to exhibit great similarity and possess all the structural characteristics of known plant ferritin2 genes. The C. canadensis ferritin2 genes had identical primary structure in the two biotypes and were upregulated by paraquat (Pq) in both susceptible and resistant plants. The enhanced expression level was probably connected with defence reactions in the plants after Pq treatment.