A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

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.

Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris.

The transition from vegetative growth to reproductive development is a complex process that requires an integrated response to multiple environmental cues and endogenous signals. In Arabidopsis thaliana, which has a facultative requirement for vernalization and long days, the genes of the autonomous pathway function as floral promoters by repressing the central repressor and vernalization-regulatory gene FLC. Environmental regulation by seasonal changes in daylength is under control of the photoperiod pathway and its key gene CO. The root and leaf crop species Beta vulgaris in the caryophyllid clade of core eudicots, which is only very distantly related to Arabidopsis, is an obligate long-day plant and includes forms with or without vernalization requirement. FLC and CO homologues with related functions in beet have been identified, but the presence of autonomous pathway genes which function in parallel to the vernalization and photoperiod pathways has not yet been reported. Here, this begins to be addressed by the identification and genetic mapping of full-length homologues of the RNA-regulatory gene FLK and the chromatin-regulatory genes FVE, LD, and LDL1. When overexpressed in A. thaliana, BvFLK accelerates bolting in the Col-0 background and fully complements the late-bolting phenotype of an flk mutant through repression of FLC. In contrast, complementation analysis of BvFVE1 and the presence of a putative paralogue in beet suggest evolutionary divergence of FVE homologues. It is further shown that BvFVE1, unlike FVE in Arabidopsis, is under circadian clock control. Together, the data provide first evidence for evolutionary conservation of components of the autonomous pathway in B. vulgaris, while also suggesting divergence or subfunctionalization of one gene. The results are likely to be of broader relevance because B. vulgaris expands the spectrum of evolutionarily diverse species which are subject to differential developmental and/or environmental regulation of floral transition.

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.

Gibberellin as a factor in floral regulatory networks.

Gibberellins (GAs) function not only to promote the growth of plant organs, but also to induce phase transitions during development. Their involvement in flower initiation in long-day (LD) and biennial plants is well established and there is growing insight into the mechanisms by which floral induction is achieved. The extent to which GAs mediate the photoperiodic stimulus to flowering in LD plants is, with a few exceptions, less clear. Despite evidence for photoperiod-enhanced GA biosynthesis in leaves of many LD plants, through up-regulation of GA 20-oxidase gene expression, a function for GAs as transmitted signals from leaves to apices in response to LD has been demonstrated only in Lolium species. In Arabidopsis thaliana, as one of four quantitative floral pathways, GA signalling has a relatively minor influence on flowering time in LD, while in SD, in the absence of the photoperiod flowering pathway, the GA pathway assumes a major role and becomes obligatory. Gibberellins promote flowering in Arabidopsis through the activation of genes encoding the floral integrators SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), LEAFY (LFY), and FLOWERING LOCUS T (FT) in the inflorescence and floral meristems, and in leaves, respectively. Although GA signalling is not required for floral organ specification, it is essential for the normal growth and development of these organs. The sites of GA production and action within flowers, and the signalling pathways involved are beginning to be revealed.

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.

Modification of gibberellin signalling (metabolism & signal transduction) in sugar beet: analysis of potential targets for crop improvement.

Sugar beet, Beta vulgaris spp. vulgaris is a biennial long day plant with an obligate requirement for vernalization (prolonged exposure to low temperature). As a spring crop in temperate European climates, it is vulnerable to vernalization-induced premature bolting and flowering, resulting in reduced crop yield and quality. Gibberellins (GAs) play important roles in key physiological processes including stem elongation (bolting) and flowering and are, therefore, potential targets for controlling reproductive growth in sugar beet. We show that the BvGA20ox gene, which encodes an enzyme necessary for GA biosynthesis, was transcriptionally activated in apices of sugar beet plants after vernalization and that GA metabolism can be manipulated to delay bolting in vernalized plants. We demonstrate that down-regulation of GA responses by transformation with the Arabidopsis thaliana gai gene (which represses GA signalling), under its own promoter (pgai::gai) or deactivation of GA by over-expression of the Phaseolus coccineus (bean) GA2ox1 gene, which inactivates GA, increased the required post vernalization thermal time (an accurate and stable measure of physiological time), to bolt by approximately 300 degrees Cd. This resulted in agronomically significant bolting time delays of approximately 2 weeks and 3 weeks in the pgai::gai and 35S::PcGA2ox1 plants, respectively. Our data represent the first transgenic sugar beet model to (1) show that GA signalling can be used to improve crops by manipulation of the transition to reproductive growth; and (2) provide evidence that GA is required for seed set in sugar beet.

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.

Transgenic analysis of sugar beet xyloglucan endo-transglucosylase/hydrolase Bv-XTH1 and Bv-XTH2 promoters reveals overlapping tissue-specific and wound-inducible expression profiles.

The identification and analysis of tissue-specific gene regulatory elements will improve our knowledge of the molecular mechanisms that control the growth and development of different plant tissues and offer potentially useful tools for the genetic engineering of plants. A polymerase chain reaction (PCR)-based 5'-genome walk from sequences of an isolated sugar beet xyloglucan endo-transglucosylase hydrolase (XTH) gene led to the isolation of two independent upstream fragments. They were 1332 and 2163 base pairs upstream of the XTH ATG start site, respectively. In vivo transgenic assays in sugar beet hairy roots and Arabidopsis thaliana revealed that both fragments had promoter function and, in A. thaliana, directed expression in vascular tissues within the root, leaves and petals. Promoter activity was also observed in the leaf trichomes and within rapidly expanding stem internodes. Expression driven by both promoters was found to be wound inducible. Overall, the spatial and temporal expression pattern of these promoters suggested that the corresponding Bv-XTH genes (designated Bv-XTH1 and Bv-XTH2) may be involved in secondary cell wall formation. This work provides new insights on molecular mechanisms that could be exploited for the genetic engineering of sugar beet crops.