The role of a pseudo-response regulator gene in life cycle adaptation and domestication of beet.

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, three unrelated loci (VERNALIZATION) determine the spring and winter habits of monocotyledonous plants such as temperate cereals. 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, the bolting locus B 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, 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.

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.

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

BACKGROUND AND AIMS: Bolting, the first visible sign of reproductive transition in beets (Beta vulgaris), is controlled by the dominant bolting gene B (B allele), which allows for flowering under long days (LDs, >14 h light) without prior vernalization. The B-locus carries recessive alleles (bb) in sugar beet (Beta vulgaris L. spp. vulgaris), so that vernalization and LDs are required for bolting and flowering. Gibberellin growth hormones (GAs) control stem elongation and reproductive development, but their role during these processes in sugar beet is not defined. We aimed to investigate the involvement of GAs in bolting and flowering in sugar beet, and also its relationship with the vernalization requirement as defined by the B-gene. METHODOLOGY: Plants segregating for the B allele were treated with exogenous GA(4) under inductive (16 h light) and non-inductive (8 h light) photoperiods, with and without prior vernalization treatment. A co-dominant polymerase chain reaction (PCR) marker was used to genotype the B-gene locus. Bolting and flowering dates were scored, and bolt heights were measured as appropriate. Analysis of variance was used to determine the effects and interactions of GAs, the B allele and vernalization on bolting and flowering. The effects of the B allele on bolting were also verified in the field. PRINCIPAL RESULTS: Application of GAs or the B allele could initiate bolting independently. When the B allele was absent, the applied GAs promoted stem growth, but did so only in vernalized plants, irrespective of photoperiod. Under LDs, bolt height before flowering in plants carrying the B allele (BB; Bb) was not significantly influenced by GAs. The timing and frequency of flowering were influenced by the B allele without interactive effects from GAs. CONCLUSIONS: In sugar beet, GA acts independently of the B allele and photoperiod to induce bolting. Vernalization enables GA action independently of the B allele; hence, the dominant B allele may not directly participate in vernalization-induced bolting.

Progress towards the understanding and control of sugar beet rhizomania disease.

Rhizomania is a soil-borne disease that occurs throughout the major sugar beet growing regions of the world, causing severe yield losses in the absence of effective control measures. It is caused by Beet necrotic yellow vein virus (BNYVV), which is transmitted by the obligate root-infecting parasite Polymyxa betae. BNYVV has a multipartite RNA genome with all natural isolates containing four RNA species, although some isolates have a fifth RNA. The larger RNA1 and RNA2 contain the housekeeping genes of the virus and are always required for infection, whereas the smaller RNAs are involved in pathogenicity and vector transmission. RNA5-containing isolates are restricted to Asia and some parts of Europe, and these isolates tend to be more aggressive. With no acceptable pesticides available to restrict the vector, the control of rhizomania is now achieved almost exclusively through the use of resistant cultivars. A single dominant resistance gene, Rz1, has been used to manage the disease worldwide in recent years, although this gene confers only partial resistance. More recently, new variants of BNYVV have evolved (both with and without RNA5) that are able to cause significant yield penalties on resistant cultivars. These isolates are not yet widespread, but their appearance has resulted in accelerated searches for new sources of resistance to both the virus and the vector. Combined virus and vector resistance, achieved either by conventional or transgenic breeding, offers the sugar beet industry a new approach in its continuing struggle against rhizomania.

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.