The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation

Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next‐generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome‐wide fluorescent in situ hybridization complemented with immunostaining and super‐resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44–52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome‐arm identification,integration of genetic linkage groups and analysis of major repeat family distribution

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North–South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome‐specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S‐5.8S‐25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber‐FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

Plant regeneration from protoplasts of sugar beet (Beta vulgaris)

Mesophyll protoplasts from two of five sugar beet lines tested were regenerated into plants. Mesophyll protoplasts of all lines showed high plating efficiencies up to 4.0% developed hard compact callus, and two of the lines also developed white, soft and friable callus consisting of starch grain-containing cells. Whereas the compact callus never regenerated into plants, the white friable ones frequently developed globular structures, which became green in the light and formed adventitious shoots after cytokinin (BAP or thidiazuron) treatment. Genetic analysis by PCR-fingerprinting and flow cytometry showed uniformity and unchanged ploidy levels in 15 independently regenerated plantlets in line NF. but altered ploidy level (from diploid to triploid) in a regenerated plantlet of clone VRB.     

Effects of zinc toxicity on sugar beet (Beta vulgaris L.) plants grown in hydroponics

The effects of high Zn concentration were investigated in sugar beet ( Beta vulgaris L.) plants grown in a controlled environment in hydroponics. High concentrations of Zn sulphate in the nutrient solution (50, 100 and 300 μ m ) decreased root and shoot fresh and dry mass, and increased root/shoot ratios, when compared to control conditions (1.2 μ m Zn). Plants grown with excess Zn had inward-rolled leaf edges and a damaged and brownish root system, with short lateral roots. High Zn decreased N, Mg, K and Mn concentrations in all plant parts, whereas P and Ca concentrations increased, but only in shoots. Leaves of plants treated with 50 and 100 μ m Zn developed symptoms of Fe deficiency, including decreases in Fe, chlorophyll and carotenoid concentrations, increases in carotenoid/chlorophyll and chlorophyll a / b ratios and de-epoxidation of violaxanthin cycle pigments. Plants grown with 300 μ m Zn had decreased photosystem II efficiency and further growth decreases but did not have leaf Fe deficiency symptoms. Leaf Zn concentrations of plants grown with excess Zn were high but fairly constant (230–260 μg·g⁻¹ dry weight), whereas total Zn uptake per plant decreased markedly with high Zn supply. These data indicate that sugar beet could be a good model to investigate Zn homeostasis mechanisms in plants, but is not an efficient species for Zn phytoremediation.     

Cadmium effects on leaf transpiration of sugar beet (Beta vulgaris)

Seedlings of sugar beet ( Beta vulgaris L. cv Monohill) were cultivated for 4 weeks in nutrient solution containing different concentrations of CdCl₂ (0 to 10 μ M ). The effects of Cd on appearance and function of stomata and leaf cuticle were investigated by water loss measurements and microscopy. The leaf transpiration rate increased with increasing Cd concentrations while the sum total of stomatal aperture area per unit leaf area decreased. Already at low Cd levels. an increase of defective and undeveloped stomata was found in Cd treated plants. These stomata are closed or have small apertures and probably lack a functional closing mechanism. The number of intact stomata per unit leaf area was lower in leaves of Cd treated plants than in controls, and Cd induced closure of intact stomata. The total number of stomata per leaf area slightly increases with increasing Cd concentration. as does the percentage of small stomata. Furthermore. specific leaf area increased, while the density of leaf structure was decreased by Cd. From this observation we conclude that the increase in transpiration rate caused by Cd is primarily due to effects on the permeability of the leaf cuticle to water.     

Genetic localization of four genes for nematode (Heterodera schachtii Schm.) resistance in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is highly susceptible to the beet cyst nematode (Heterodera schachtii Schm.). Three resistance genes originating from the wild beets B. procumbens (Hs1 ^pro-1) and B. webbiana (Hs1 ^web-1, Hs2 ^web-7) have been transferred to sugar beet via species hybridization. We describe the genetic localization of the nematode resistance genes in four different sugar beet lines using segregating F₂ populations and RFLP markers from our current sugar beet linkage map. The mapping studies yielded a surprising result. Although the four parental lines carrying the wild beet translocations were not related to each other, the four genes mapped to the same locus in sugar beet independent of the original translocation event. Close linkage (0–4.6 cM) was found with marker loci at one end of linkage group IV. In two populations, RFLP loci showed segregation distortion due to gametic selection. For the first time, the non-randomness of the translocation process promoting gene transfer from the wild beet to the sugar beet is demonstrated. The data suggest that the resistance genes were incorporated into the sugar beet chromosomes by non-allelic homologous recombination. The finding that the different resistance genes are allelic will have major implications on future attempts to breed sugar beet combining the different resistance genes.     

Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host

Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT‐PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.     

Evaluation of nematode-resistant sugar beet (Beta vulgaris L.) lines by molecular analysis

 Thirty sugar beet (Beta vulgaris) lines conferring complete resistance to the beet cyst nematode (BCN, Heterodera schachtii) originating from interspecific crosses with wild beets of the section Procumbentes (B. procumbens, B. webbiana and B. patellaris) were investigated by morphology and wild beet-specific molecular markers. The beet lines carrying chromosome mutations consisted of monosomic additions (2n=18+1), fragment additions (2n=18+fragment) and translocations (2n=18) from the wild beets. Genome-specific single-copy, satellite and repetitive probes were applied to study the origin, chromosomal assignment and presence of nematode resistance genes. Within the wild beet species at least three different resistance genes located on different chromosomes were distinguished: Hs1 on the homoelogous chromosomes I of each species, Hs2 on the homoelogous chromosomes VII of B. procumbens and B. webbiana and Hs3 on chromosome VIII of B. webbiana. A clear distinction between the three chromosomes was possible by morphological and molecular means. The translocation lines were separated into two different groups: one containing the resistance gene Hs1 from chromosome I and the other carrying a different nematode resistance gene. The molecular data combined with sequence analyses of Hs1 of the three wild beet species revealed a clear distinction between B. procumbens and B. webbiana. The evolutionary and taxonomical relationship of these species supporting the idea of three different species originating from a common ancestor is discussed. Received: 6 April 1998 / Accepted: 22 April 1998     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

An analysis of genetic variation in sugar beet (Beta vulgaris L. using an augmented biparental (ABIP) mating design

An augmented biparental (ABIP) mating design was used to investigate the quantitative variation, particularly the dominance variation, for morphological and chemical characters in sugar beet. Diploid O-type plants were both crossed and selfed and the progeny were grown in a single-plant randomised field trial. A comparison of the two kinds of family provided tests for both dominance variation and directional dominance effects. Estimates of the narrow heritability were also obtained for each character. Germination problems reduced the size of the final analysis but evidence was obtained of dominance variation and positive directional dominance effects for leaf length, root weight and potassium concentration and, to a lesser extent, sugar concentration. Genetic control of sodium and alpha-amino nitrogen concentrations appeared to be mostly additive. Hybrid varieties of sugar beet should exploit these directional dominance effects and the more closely varieties approach true F, hybrids the more they will capitalise on these advantages. It is possible that other factors such as epistasis, contamination, competition and seed effects may cause us to under- or overestimate the importance of dominance.     

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.     

Survey of sugar beet (Beta vulgaris L.) hAT transposons and MITE-like hATpin derivatives

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.     

Myxosporea (Myxozoa,Cnidaria) Lack DNA Cytosine Methylation

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.     

Foliar uptake of Cd by pea (Pisum sativum) and sugar beet (Beta vulgaris)

Pea ( Pisum sativum L. cv. Fenomen) and sugar beet ( Beta vulgaris L. cv. Monohill) were cultivated in nutrient media without or with 10 μM CdCl₂. Leaves of the same size and stage of development, detached or still attached to the intact plants, were submerged into redistilled water containing 1 to 250 μM CdCl₂. The uptake experiments were run for 1 to 8 h at pH 3.6 and 5.1. Cuticular transpiration rate, density of leaf and density of stomata were also measured. Percentage of open stomata was studied at different pH.
Foliar uptake of Cd into the leaf is evident since Cd is transported from the exposed part of the pea leaves, through the petioles and into the stipules, and since the Cd concentration of the leaves increases with time and external Cd concentration. The foliar uptake depends on the permeability of the cuticular membrane, which is increased by a high intrinsic Cd level, which in turn enhances the foliar uptake of Cd in sugar beet. Higher cuticular permeability in pea than in sugar beet is shown by a 2.5 times higher cuticular transpiration rate and a 4 times lower density of leaf for pea, which causes a 7 times higher foliar uptake in pea than in sugar beet. Low pH decreases the net uptake of Cd, probably by an exchange reaction in the cutin and pectin of the cuticular membrane. Stomata are not directly involved in the Cd uptake, and the differences in the sum total of stomatal aperture area per unit leaf area is not related to differences in foliar uptake of Cd. Percentage of open stomata, calculated as average of both sides of the leaves, was not affected by changes in pH: but especially at high pH. proportionally more stomata were open on the adaxial than on the abaxial side.     

Emergence of seedlings of sugar beet (Beta vulgaris L.) as affected by the size,roughness and position of aggregates in the seedbed

Laboratory and field experiments were carried out to quantify the effects of the size and roughness of aggregates placed in the seedling path of sugar beet, in order to help in decision making for soil tillage and sowing operations. Graded aggregates (10, 15, 20, 30, 40, 50, 60 and 70 mm longest axis) were either laid on the soil surface or included in the soil over the seeds. The percent emergence decreased exponentially with aggregate size over 10 mm when the aggregates were included in the seedbed. The result was the same with aggregates laid on the soil surface, but for aggregates over 30 mm (mass>10 g). Aggregates on the soil surface could be lifted by the seedlings until their weight exceeded the seedling emergence force. Larger aggregates or aggregates in the soil layer could not be moved. Seedlings which did not emerge remained blocked in small cavities in the aggregate surface. No seedlings were blocked under smooth aggregates or glass beads. The experimental results fits with a model giving the probability to meet a hole according the distance covered and the diameter and density of holes. The results obtained under controlled conditions were similar to those obtained in field experiments for a wide range of aggregate sizes. These results will be incorporated into a computerised seedbed generator to simulate the effects of seedbed structure on seedling emergence.     

Responses of sugar beet (Beta vulgaris L.) to drought and nutrient deficiency stress

The responses of two sugar beet genotypes, 24367 (putative droughttolerant) and N6 (putative drought intolerant), to drought and nutrientdeficiency stress were investigated in an attempt to identify reliable andsensitive indicators of stress tolerance. In glasshouse-grown plants of bothgenotypes, relative water content (RWC) of the leaves decreased and leaftemperature increased in response to drought stress. Genotype differences inresponse to drought included leaf RWC, glycine betaine accumulation, alterationof shoot/root ratio and production of fibrous roots. Thus, in comparison to N6,genotype 24367 lost less water from leaves, produced more fibrous roots,produced more glycine betaine in shoots and tap roots and had a much reducedshoot/root ratio in response to withholding water for up to 215 h.The hydraulic conductance and sap flow of sugar beet seedlings grown innutrientculture decreased when subjected to nitrogen deficiency stress. Under nitrogensufficient conditions sap flow was greater in 24367 than in N6. The resultsindicate that genotype 24367 is more tolerant to stresses induced by water andnitrogen deficiency and that increased fibrous root development may be a majorfactor in increasing sap flow via a concomitant enhancement of aquaporinactivity.