Protocol: precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli.

Plant genome sequence data now provide opportunities to conduct molecular genetic studies at the level of the whole gene locus and above. Such studies will be greatly facilitated by adopting and developing further the new generation of genetic engineering tools, based on homologous recombination cloning in Escherichia coli, which are free from the constraints imposed by the availability of suitably positioned restriction sites. Here we describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

A new RNASeq-based reference transcriptome for sugar beet and its application in transcriptome-scale analysis of vernalization and gibberellin responses.

BACKGROUND: Sugar beet (Beta vulgaris sp. vulgaris) crops account for about 30% of world sugar. Sugar yield is compromised by reproductive growth hence crops must remain vegetative until harvest. Prolonged exposure to cold temperature (vernalization) in the range 6 °C to 12 °C induces reproductive growth, leading to bolting (rapid elongation of the main stem) and flowering. Spring cultivation of crops in cool temperate climates makes them vulnerable to vernalization and hence bolting, which is initiated in the apical shoot meristem in processes involving interaction between gibberellin (GA) hormones and vernalization. The underlying mechanisms are unknown and genome scale next generation sequencing approaches now offer comprehensive strategies to investigate them; enabling the identification of novel targets for bolting control in sugar beet crops. In this study, we demonstrate the application of an mRNA-Seq based strategy for this purpose. RESULTS: There is no sugar beet reference genome, or public expression array platforms. We therefore used RNA-Seq to generate the first reference transcriptome. We next performed digital gene expression profiling using shoot apex mRNA from two sugar beet cultivars with and without applied GA, and also a vernalized cultivar with and without applied GA. Subsequent bioinformatics analyses identified transcriptional changes associated with genotypic difference and experimental treatments. Analysis of expression profiles in response to vernalization and GA treatment suggested previously unsuspected roles for a RAV1-like AP2/B3 domain protein in vernalization and efflux transporters in the GA response. CONCLUSIONS: Next generation RNA-Seq enabled the generation of the first reference transcriptome for sugar beet and the study of global transcriptional responses in the shoot apex to vernalization and GA treatment, without the need for a reference genome or established array platforms. Comprehensive bioinformatic analysis identified transcriptional programmes associated with different sugar beet genotypes as well as biological treatments; thus providing important new opportunities for basic scientists and sugar beet breeders. Transcriptome-scale identification of agronomically important traits as used in this study should be widely applicable to all crop plants where genomic resources are limiting.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Bolting and flowering control in sugar beet: relationships and effects of gibberellin, the bolting gene B and vernalization

Using a co-dominant genotypic PCR marker we show for the first time that, in sugar beet, the GA and B-gene pathways are independent for bolt initiation. We show that vernalization permits GA-dependant stem elongation and that the B-allele influences subsequent flowering

Protocol: precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli.

Plant genome sequence data now provide opportunities to conduct molecular genetic studies at the level of the whole gene locus and above. Such studies will be greatly facilitated by adopting and developing further the new generation of genetic engineering tools, based on homologous recombination cloning in Escherichia coli, which are free from the constraints imposed by the availability of suitably positioned restriction sites. Here we describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Screening for genetic elements involved in the non-host response of sugar beet to the plasmodiophorid cereal root parasite Polymyxa graminis by representational difference analysis

Representational difference analysis (RDA) was used to select and clone cDNA fragments of genes whose steady state transcription was upregulated in sugar beet challenged with the nonhost parasite Polymyxa graminis. In silico analysis revealed that sequences with similarities to plant defence genes as well as genes of unknown function were represented amongst the cloned cDNAs. The utility of RDA was verified when, in material from the nonhost interaction, semiquantitative RT-PCR confirmed transcriptional upregulation of at least 10 of 17 genes randomly selected from the RDA library. Time-course transcriptional analysis of two plant defence gene-like sequences demonstrated that, in sugar beet, both were upregulated within 1 h in response to P. graminis but not to P. betae. This work comprises the first report of an active response by sugar beet to P. graminisPeer reviewe

Barley elicits a similar early basal defence response during host and non-host interactions with Polymyxa root parasites

Plant viruses transmitted by the obligate root-infecting plasmodiophorid parasites Polymyxa graminis and Polymyxa betae cause devastating yield losses to cereal and sugar beet crops worldwide. Barley is a non-host for P. betae but is a host for P. graminis. Using the Barley1 GeneChip® microarray we have investigated the transcriptional re-programming of barley roots during the earliest non-host and host interactions with zoospores of these protist species. At high confidence levels we detected 20 and 13 genes with increased transcriptional activity in response to P. betae and P. graminis, respectively, compared to unchallenged barley roots. Functional classification of the induced genes showed that a majority of the genes from both responses were associated with a classic defence response. Validation by quantitative RT-PCR analysis indicated that all of the genes examined were induced to comparable levels in both non-host and host responses. Our results also demonstrated that the barley defence-associated genes, RAR1, ROR1 or ROR2 were not essential for limiting the establishment of P. betae infection in barley. These data suggest that in barley roots the Polymyxa species induce a similar basal defence response whether the interaction is with a non-host or host. Thus, the early response to protist plant parasites appears to be part of the general 'frontline' defence against invading microbes.Peer reviewe

The Role of a Pseudo-Response Regulator Gene in Life Cycle Adaptation and Domestication of Beet

Highlights: Map-based cloning of B in beet led to isolation of the PRR gene BvBTC1 BvBTC1 controls life cycle through differential regulation of the BvFT1/BvFT2 module BvBTC1 mediates floral transition in response to both long days and vernalization Beet domestication involved selection of a rare Bvbtc1 allele conferring bienniality Summary: Life cycle adaptation to latitudinal and seasonal variation in photoperiod and temperature is a major determinant of evolutionary success in flowering plants. Whereas the life cycle of the dicotyledonous model species Arabidopsis thaliana is controlled by two epistatic genes, FLOWERING LOCUS C and FRIGIDA [1,2,3], three unrelated loci (VERNALIZATION 1–3) determine the spring and winter habits of monocotyledonous plants such as temperate cereals [4,5,6]. In the core eudicot species Beta vulgaris, whose lineage diverged from that leading to Arabidopsis shortly after the monocot-dicot split 140 million years ago [7,8], the bolting locus B [9] is a master switch distinguishing annuals from biennials. Here, we isolated B and show that the pseudo-response regulator gene BOLTING TIME CONTROL 1 (BvBTC1), through regulation of the FLOWERING LOCUS T genes [10], is absolutely necessary for flowering and mediates the response to both long days and vernalization. Our results suggest that domestication of beets involved the selection of a rare partial loss-of-function BvBTC1 allele that imparts reduced sensitivity to photoperiod that is restored by vernalization, thus conferring bienniality, and illustrate how evolutionary plasticity at a key regulatory point can enable new life cycle strategies.Peer reviewe