Positive correlation between recombination rates and levels of genetic variation in natural populations of sea beet (Beta vulgaris subsp. maritima).

The relation between the level of genetic variation and the rate of recombination per physical unit was investigated in sea beet (Beta vulgaris subsp. maritima). The rate of recombination per physical unit was estimated indirectly through marker density in an RFLP linkage map of sugar beet. From this map, we also selected RFLP markers covering two of the nine chromosomes in Beta. The markers were used to estimate the level of genetic variation in three populations of sea beet, two from Italy and one from England. Two estimates of genetic variation were employed, one based on the number of alleles in the sample and the other on heterozygosity. A statistically significant positive correlation was found between recombination rate and genetic variation. Several theoretical explanations for this are discussed, background selection being one. A correlation similar to this has been observed previously in Drosophila, one that was higher than what we obtained for Beta. This is consistent with various biological differences between the two species.     

Influence of sugar beet breeding on populations of Beta vulgaris ssp. maritima in Italy

Abstract. It is highly probable that transgenic cultivars of sugar beet may influence wild beets in the seed-production-area of northern Italy. For this reason a survey of the local wild beet populations and their habitat characteristics was conducted in 1994/1995, i.e. before transgenic beets and their off spring could have become established. Wild beets (Beta vulgaris ssp. maritima) were found at 21 locations between Trieste and Cesenatico, as part of the natural littoral vegetation classified as Atriplicetum tatarici (Cakiletea maritimae) and Crithmetum (Crithmo-Staticetea). The analysis of phenotypic attributes leads to a division into three different sub-populations. Greenhouse studies on the morphology and life-cycle attributes demonstrated actual gene flow between conventional seed beet and the examined wild beet population.     

Low level of gene flow from cultivated beets (Beta vulgaris L. ssp. vulgaris) into Danish populations of sea beet (Beta vulgaris L. ssp. maritima (L.) Arcangeli)

Gene flow from sugar beets to sea beets occurs in the seed propagation areas in southern Europe. Some seed propagation also takes place in Denmark, but here the crop-wild gene flow has not been investigated. Hence, we studied gene flow to sea beet populations from sugar beet lines used in Danish seed propagation areas. A set of 12 Danish, two Swedish, one French, one Italian, one Dutch, and one Irish populations of sea beets, and four lines of sugar beet were analysed. To evaluate the genetic variation and gene flow, eight microsatellite loci were screened. This analysis revealed hybridization with cultivated beet in one of the sea beet populations from the centre of the Danish seed propagation area. Triploid hybrids found in this population were verified with flow cytometry. Possible hybrids or introgressed plants were also found in the French and Italian populations. However, individual assignment test using a Bayesian method provided 100% assignment success of diploid individuals into their correct subspecies of origin, and a Bayesian Markov chain Monte Carlo (MC MC) approach revealed clear distinction of individuals into groups according to their subspecies of origin, with a zero level of genetic admixture among subspecies. This underlines that introgression beyond the first hybridization is not extensive. The overall pattern of genetic distance and structure showed that Danish and Swedish sea beet populations were closely related to each other, and they are both more closely related to the population from Ireland than to the populations from France, the Netherlands, and Italy.     

Genomes of the wild beets Beta patula and Beta vulgaris ssp. maritima

We present draft genome assemblies of Beta patula, a critically endangered wild beet endemic to the Madeira archipelago, and of the closely related Beta vulgaris ssp. maritima (sea beet). Evidence‐based reference gene sets for B. patula and sea beet were generated, consisting of 25 127 and 27 662 genes, respectively. The genomes and gene sets of the two wild beets were compared with their cultivated sister taxon B. vulgaris ssp. vulgaris (sugar beet). Large syntenic regions were identified, and a display tool for automatic genome‐wide synteny image generation was developed. Phylogenetic analysis based on 9861 genes showing 1:1:1 orthology supported the close relationship of B. patula to sea beet and sugar beet. A comparative analysis of the Rz2 locus, responsible for rhizomania resistance, suggested that the sequenced B. patula accession was rhizomania susceptible. Reference karyotypes for the two wild beets were established, and genomic rearrangements were detected. We consider our data as highly valuable and comprehensive resources for wild beet studies, B. patula conservation management, and sugar beet breeding research.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow between sea beet (Beta vulgaris ssp. maritima) populations

Molecular markers can be used to estimate gene flow indirectly by monitoring the relative frequency of alleles in adjacent populations. Sea beet (Beta vulgaris ssp. maritima) is a wild plant species found along the coastlines of many European countries and is closely related to cultivated beets. A set of six simple sequence repeat (SSR) markers that are polymorphic in UK populations have been developed for sea beet to assess the problems of indirect measurement of gene flow in these populations.     

Emergence of gynodioecy in wild beet (Beta vulgaris ssp. maritima L.): a genealogical approach using chloroplastic nucleotide sequences

Gynodioecy is a breeding system where both hermaphroditic and female individuals coexist within plant populations. This dimorphism is the result of a genomic interaction between maternally inherited cytoplasmic male sterility (CMS) genes and bi-parentally inherited nuclear male fertility restorers. As opposed to other gynodioecious species, where every cytoplasm seems to be associated with male sterility, wild beet Beta vulgaris ssp. maritima exhibits a minority of sterilizing cytoplasms among numerous non-sterilizing ones. Many studies on population genetics have explored the molecular diversity of different CMS cytoplasms, but questions remain concerning their evolutionary dynamics. In this paper we report one of the first investigations on phylogenetic relationships between CMS and non-CMS lineages. We investigated the phylogenetic relationships between 35 individuals exhibiting different mitochondrial haplotypes. Relying on the high linkage disequilibrium between chloroplastic and mitochondrial genomes, we chose to analyse the nucleotide sequence diversity of three chloroplastic fragments (trnK intron, trnD-trnT and trnL-trnF intergenic spacers). Nucleotide diversity appeared to be low, suggesting a recent bottleneck during the evolutionary history of B. vulgaris ssp. maritima. Statistical parsimony analyses revealed a star-like genealogy and showed that sterilizing haplotypes all belong to different lineages derived from an ancestral non-sterilizing cytoplasm. These results suggest a rapid evolution of male sterility in this taxon. The emergence of gynodioecy in wild beet is confronted with theoretical expectations, describing either gynodioecy dynamics as the maintenance of CMS factors through balancing selection or as a constant turnover of new CMSs.     

Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet)

The composition of microbial communities on and within seeds may effect their storage and field performance, whether they are indigenous or applied as biocontrol agents. In this study, we have explored the usefulness of profiling small subunit ribosomal (SSR) gene fragments for studying the microflora associated with seeds. DNA was amplified by the polymerase chain reaction (PCR) and the amplicons separated using denaturing gradient gel electrophoresis (DGGE). Primers targeting eukaryotic SSRs were used to investigate fungal communities, and primers targeting bacterial SSRs were employed to study the eubacterial microflora. As a case study, we attempted to profile the fungi and bacteria associated with seeds of Beta vulgaris (sugar beet) to permit an insight into the varying field performance of several well-characterised commercial seed lots. Serious interference with the microbial signals was observed from the plant's own nuclear 18S rRNA genes and chloroplast 16S rRNA genes using standard PCR conditions and DNA extracted from whole seeds as template. Hot-start and touchdown PCR made no appreciable improvement to these signals. Seed imbibition and dissection into operculum and fruit wall and true seed prior to DNA extraction improved signal recovery in the fruit fraction. With primer modification, bacteria and fungi were detected in an excess of plant DNA of 100:1 and 10:1, respectively. With this method, microbial communities on seeds could be profiled, however, it is likely that targeted depletion of plant rDNA targets will be a necessary extra step before this approach can be used to screen seeds routinely.     

Evolutionary conservation of the FLOWERING LOCUS C-mediated vernalization response: evidence from the sugar beet (Beta vulgaris)

In many plant species, exposure to a prolonged period of cold during the winter promotes flowering in the spring, a process termed vernalization. In Arabidopsis thaliana, the vernalization requirement of winter-annual ecotypes is caused by the MADS-box gene FLOWERING LOCUS C (FLC), which is a repressor of flowering. During the vernalization process, FLC is downregulated by alteration of its chromatin structure, thereby permitting flowering to occur. In wheat, a vernalization requirement is imposed by a different repressor of flowering, suggesting that some components of the regulatory network controlling the vernalization response differ between monocots and dicots. The extent to which the molecular mechanisms underlying vernalization have been conserved during the diversification of the angiosperms is not well understood. Using phylogenetic analysis, we identified homologs of FLC in species representing the three major eudicot lineages. FLC homologs have not previously been documented outside the plant family Brassicaceae. We show that the sugar beet FLC homolog BvFL1 functions as a repressor of flowering in transgenic Arabidopsis and is downregulated in response to cold in sugar beet. Cold-induced downregulation of an FLC-like floral repressor may be a central feature of the vernalization response in at least half of eudicot species.     

Disentangling the causes of heterogeneity in male fecundity in gynodioecious Beta vulgaris ssp. maritima

Variation among individuals in reproductive success is advocated as a major process driving evolution of sexual polymorphisms in plants, such as gynodioecy where females and hermaphrodites coexist. In gynodioecious Beta vulgaris ssp. maritima, sex determination involves cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Both restored CMS and non-CMS hermaphrodites co-occur. Genotype-specific differences in male fitness are theoretically expected to explain the maintenance of cytonuclear polymorphism. Using genotypic information on seedlings and flowering plants within two metapopulations, we investigated whether male fecundity was influenced by ecological, phenotypic and genetic factors, while taking into account the shape and scale of pollen dispersal. Along with spatially restricted pollen flow, we showed that male fecundity was affected by flowering synchrony, investment in reproduction, pollen production and cytoplasmic identity of potential fathers. Siring success of non-CMS hermaphrodites was higher than that of restored CMS hermaphrodites. However, the magnitude of the difference in fecundity depended on the likelihood of carrying restorer alleles for non-CMS hermaphrodites. Our results suggest the occurrence of a cost of silent restorers, a condition supported by scarce empirical evidence, but theoretically required to maintain a stable sexual polymorphism in gynodioecious species.     

Genetic study of a new color of the beet Beta vulgaris L.

A new genetic character of the beet Beta vulgaris L., named stem color, was described and studied genetically. This character was shown to be dominant and monogenically inherited. The first-year beet plants with the genotype Stc/_ have red leafstalks, weakly colored central rib, and colored storage root; however, the root itself is not colored. The second-year plants have a red-colored low third of the floral shoot. The plants with the genotype stc/stc are uncolored. The Stc gene was localized to the first linkage group at a distance of 17.5 ± 2.1% crossing over units from the gene B (Bolting), which controls the annual-perennial habit of beet.     

Gynodioecy in structured populations: understanding fine-scale sex ratio variation in Beta vulgaris ssp. maritima

Natural selection, random processes and gene flow are known to generate sex ratio variations among sexually polymorphic plant populations. In gynodioecious species, in which hermaphrodites and females coexist, the relative effect of these processes on the maintenance of sex polymorphism is still up for debate. The aim of this study was to document sex ratio and cytonuclear genetic variation at a very local scale in wind-pollinated gynodioecious Beta vulgaris ssp. maritima and attempt to elucidate which processes explained the observed variation. The study sites were characterized by geographically distinct patches of individuals and appeared to be dynamic entities, with recurrent establishment of distinct haplotypes through independent founder events. Along with substantial variation in sex ratio and unexpectedly low gene flow within study sites, our results showed a high genetic differentiation among a mosaic of genetically distinct demes, with isolation by distance or abrupt genetic discontinuities taking place within a few tens of metres. Overall, random founder events with restricted gene flow could be primary determinants of sex structure, by promoting the clumping of sex-determining genes. Such high levels of sex structure provide a landscape for differential selection acting on sex-determining genes, which could modify the conditions of maintenance of gynodioecy in structured populations.     

Chemical Composition and Biological Studies of the Essential Oil from Aerial Parts of Beta vulgaris subsp. maritima (L.) Arcang. Growing in Tunisia

The chemical composition, antioxidant, cytotoxic, anticholinesterase and anti‐tyrosinase activities of the hydrodistilled essential oil of the aerial parts of Beta vulgaris subsp. maritime (L.) Arcang . from Tunisia have been evaluated. The chemical composition of the oil (yield 0.037% [w/w]), determined by GC‐FID and GC/MS is reported for the first time. Twenty five components, accounting for 98.1% of the total oil have been identified. The oil was characterized by a high proportion of oxygenated sesquiterpenes (39.2%), followed by sesquiterpene hydrocarbons (30.3%) and one apocarotenoids (26.3%). The main compounds were γ‐irone (26.3%), α‐cadinol (12.1%), T‐cadinol (10.6%), bicyclogermacrene (10.4%) and δ‐cadinene (6.0%). The isolated oil was tested for its antioxidant activity using the DPPH^·, ABTS^+·, catalase, and paraoxonase assays and also for its cytotoxic, anticholinesterase, and anti‐tyrosinase activities. The essential oil exhibited high antioxidant activity (IC₅₀ = 0.055 ± 0.006 mg/ml) and important result oncatalase (524.447 ± 2.58 Units/mg protein). Furthermore, it exerted a significant cytotoxic effect against A549 cell line, with IC₅₀ = 42.44 ± 1.40 μg/ml. The results indicate that the essential oil of B. vulgaris subsp. maritima (L.) Arcang . aerial parts may be used in future as an alternative to synthetic antioxidant agents, with potential application in the food and pharmaceutical industries.     

beta-Glucosidase of leaves and roots of the common beet Beta vulgaris

beta-Glucosidases detected in the leaves and roots of common beet, Beta vulgaris, have been demonstrated to catalyze hydrolysis of native betacyanins. A method is described for the isolation and purification of beta-glucosidase from the roots, which involves ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex gel filtration. Maximum activity of the enzyme is detected at 50 degrees C and pH 8.0; it retains stability within the pH range from 5.1 to 9.2. In the leaves, beta-glucosidase is chloroplast membrane-associated; solubilization of the membranes results in the enzyme inactivation.     

Monosomic additions in beet (Beta vulgaris) carrying extra chromosomes of Beta procumbens

Summary Eleven isozyme systems were used to identify the extra chromosomes, originating from Beta procumbens, in progenies of 33 monosomic additions in beet (B. vulgaris). Nine groups of monosomic additions could be distinguished, representing the nine different chromosome types of B. procumbens.     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.