Fast and Sensitive LC-MS/MS Method for the Quantitation of Saponins in Various Sugar Beet Materials.

An LC-MS/MS method was developed for the simultaneous quantitative analysis of the following 11 triterpene saponins within different sugar beet materials and plant compartments: betavulgaroside I (1), betavulgaroside II (2), betavulgaroside III (3), betavulgaroside IV (4), betavulgaroside VIII (5), boussingoside A2 (6), 3-O-[ß-d-glucopyranosyl-(1 → 2)-(ß-d-xylopyranosyl-(1 → 3))-ß-d-glucuronopyranosyl]-28-O-ß-d-glucopyranosyl-3ß-hydroxyolean-12-en-28-oic acid (7), betavulgaroside V (8), chikusetsusaponin IVa (9), calenduloside E (10), and ginsenoside R0 (11). Our results showed highly varying amounts of saponins within different varieties, roots, and leaves as well as different plant compartments. The amounts for sugar beet roots were in the range of 862 mg/kg to 2 452 mg/kg. They were mostly higher for leaves compared to roots of the same variety with amounts ranging from 907 mg/kg to 5 398 mg/kg. Furthermore, the occurrence of sugar beet saponins within different side streams was examined; in this context, sugar beet fiber contained the highest amounts of saponins for all investigated plant constituents and byproduct streams with a total amount of 12.7 g/kg. Finally, this is the first publication about the occurrence of individual saponins in sugar beets.

Insights into the genetic relationships among plants of Beta section Beta using SNP markers.

KEY MESSAGE: Using a much higher number of SNP markers and larger sample sizes than all the previous studies, we characterized the genetic relationships among wild and cultivated plants of section Beta. We analyzed the genetic variation of Beta section Beta, which includes wild taxa (Beta macrocarpa, B. patula, B. vulgaris subsp. adanensis and B. vulgaris subsp. maritima) and cultivars (fodder beet, sugar beet, garden beet, leaf beet, and swiss chards), using 9724 single nucleotide polymorphism markers. The analyses conducted at the individual level without a priori groups confirmed the strong differentiation of B. macrocarpa and B. vulgaris subsp. adanensis from the other taxa. B. vulgaris subsp. maritima showed a complex genetic structure partly following a geographical pattern, which confounded the differences between this taxon and the cultivated varieties. Cultivated varieties were structured into three main groups: garden beets, fodder and sugar beets, and leaf beets and swiss chards. The genetic structure described here will be helpful to correctly estimate linkage disequilibrium and to test for statistical associations between genetic markers and environmental variables.

Potencial do óleo essencial de erva-luísa (Aloysia citriodora Palau) no controle de Fusarium sp. in vitro / Essential oil potential herb-luisa (Aloysia citriodora Palau) in the control of Fusarium sp. in vitro

RESUMO O presente trabalho teve como objetivo principal avaliar o potencial do uso do óleo essencial de Aloysia citriodora no controle in vitro de Fusarium sp., isolado de plântulas de beterraba infectadas com o mesmo. O trabalho foi realizado por meio de dois experimentos: um sobre o efeito do óleo essencial no crescimento micelial, e outro sobre o efeito na germinação de conídios do fungo. No primeiro trabalho, avaliou-se em delineamento inteiramente ao acaso, o efeito das concentrações 0,0155%; 0,0315%; 0,0625%; 0,125%; 0,250% e 0,500% do óleo essencial de A. citriodora em placas de Petri® com meio de cultivo BDA, mais a testemunha, com meio BDA puro. Cada placa foi considerada uma repetição, as quais foram incubadas a 24ºC±1ºC e submetidas a fotoperíodo de doze horas. Avaliou-se o crescimento radial do patógeno em função do crescimento micelial do patógeno quando este atingia as bordas da primeira placa. No segundo experimento, as mesmas concentrações foram testadas, nas mesmas condições de incubação, no entanto, em lâminas de microscopia com meio BD. Utilizou-se delineamento inteiramente ao acaso, sendo considerada como unidade experimental cada lâmina utilizada. Avaliou-se 24 horas após a incubação, em microscópio óptico, a germinação dos primeiros vinte conídios visualizados a partir do canto esquerdo superior para o direito. Os resultados do segundo experimento foram expressos em porcentagem de germinação de conídios. Os resultados obtidos permitiram concluir que este óleo essencial possui efeito fungistático e fungicida sobre o crescimento micelial e na germinação de conídios de Fusarium sp.. Além disso este efeito é maior em função do aumento da concentração do óleo essencial.

ABSTRACT This study aimed to evaluate the potential use of Aloysia citriodora essential oilisolated from beet seedlings infected with it. The study was performed by conducting two experiments evaluating the effect of the essential oil on mycelial growth and fungus conidia germination. In the first study, in a completely randomized design, the effect concentrations (0.0155%; 0.0315%; 0.0625%; 0.125%; 0.250% and 0.500%) of essential oil of A. citriodorawas evaluated on Petri® dish with a PDA cultivation medium, plus the control, with half pure PDA. Each plate was taken as a repeat, and incubated at 24°C±1°C and a photoperiod of twelve hours. The radial growth of the pathogen, when the first plate was hit by the mycelial growth of the pathogen on its edges was evaluated. In the second experiment, the same concentrations were tested under the same incubationconditions, however, on microscope slides with half PD medium. The design was used completely randomized, each microscope slide used was considered as one experimental unit. Germination of the first 2 conidia strains, viewed from the upper left to the right was evaluated24 hours after incubation, using an optical microscope. The results of the second experiment were expressed as a percentage of conidia germination. The results obtained showed that this essential oil has fungistatic and fungicidal effect on the mycelial growth and at the conidia germination of Fusarium sp., which has a greater effect with increasing concentrations.

HPLC-MS(n) Identification of Betalain Profile of Different Beetroot (Beta vulgaris L. ssp. vulgaris) Parts and Cultivars.

The distribution of betalains in peel, flesh, and petioles of yellow and red beetroot cultivars has been investigated using an High-performance liquid chromatography (HPLC) system with electrospray mass spectrometry. Differences in the levels of betacyanins and betaxanthins between different colored cultivars were individually determined for 3 plant parts. The content of almost all analyzed compounds decreased in the following order: peel > flesh > petiole. Betanin/isobetanin pigments comprised a major portion of the relative peak area measured in red beetroot peel. Isobetanin relative peak areas were also high in leaf petioles (68.94% to 74.16%) of red colored cultivars. However, betacyanins were completely absent from the extracts of all analyzed parts of yellow beet. Glutamine-bx represented a very high relative peak area (59.54% to 64.18%) in flesh of red-colored cultivars analyzed in the study. Our results indicate that red beet cultivars can be utilized as a potential source of red and yellow natural colorants. However, differences in pigment composition among different beetroot parts must be considered and in order to maximize the pigment yields petioles can also be used as a source rich in specific betalain compounds.

Breeding patterns and cultivated beets origins by genetic diversity and linkage disequilibrium analyses.

KEY MESSAGE: Genetic diversity in worldwide population of beets is strongly affected by the domestication history, and the comparison of linkage disequilibrium in worldwide and elite populations highlights strong selection pressure. Genetic relationships and linkage disequilibrium (LD) were evaluated in a set of 2035 worldwide beet accessions and in another of 1338 elite sugar beet lines, using 320 and 769 single nucleotide polymorphisms, respectively. The structures of the populations were analyzed using four different approaches. Within the worldwide population, three of the methods gave a very coherent picture of the population structure. Fodder beet and sugar beet accessions were grouped together, separated from garden beets and sea beets, reflecting well the origins of beet domestication. The structure of the elite panel, however, was less stable between clustering methods, which was probably because of the high level of genetic mixing in breeding programs. For the linkage disequilibrium analysis, the usual measure (r (2)) was used, and compared with others that correct for population structure and relatedness (r S (2) , r V (2) , r VS (2)). The LD as measured by r (2) persisted beyond 10 cM within the elite panel and fell below 0.1 after less than 2 cM in the worldwide population, for almost all chromosomes. With correction for relatedness, LD decreased under 0.1 by 1 cM for almost all chromosomes in both populations, except for chromosomes 3 and 9 within the elite panel. In these regions, the larger extent of LD could be explained by strong selection pressure.

Geosmin (2ß,6α-dimethylbicyclo[4.4.0]decan-1ß-ol) production associated with Beta vulgaris ssp. vulgaris is cultivar specific.

The characteristic earthy flavor and aroma of table beet [Beta vulgaris ssp. vulgaris (garden beet group)] is due to the presence of geosmin, C12H22O, a volatile terpenoid compound commonly produced by many soil microorganisms. This study screened beet and related subspecies cultivars grown in three different environments (field, greenhouse in nonautoclaved soil, greenhouse in autoclaved soil) to evaluate the effect of cultivar and environment on geosmin level in table beet. There was no significant difference between years or between cultivars grown in autoclaved and nonautoclaved soil, indicating geosmin content may not be primarily attributable to microbial associations. A significant interaction between cultivar and environment was found, but generalizations could be made for high- or low-producing cultivars, demonstrating that geosmin levels were cultivar specific. 'Bull's Blood', 'Chioggia', and sugar beet exhibited the highest geosmin levels. Cultivars grown in the field had the smallest range of geosmin production, from 4.84 to 20.82 µg geosmin (kg root tissue)⁻¹. The high degree of consistency in cultivar performance across years and in ranking for geosmin levels across environments as well as the lack of a significant difference between plants grown in autoclaved and nonautoclaved soil suggests characteristic levels of geosmin may be present in and produced endogenously by cultivars of table beet. It may be possible to establish breeding populations with defined geosmin levels and to identify variety-specific aroma and flavor intensities that would be durable across environments.

Empirical evaluation of DArT, SNP, and SSR marker-systems for genotyping, clustering, and assigning sugar beet hybrid varieties into populations.

Dominant and co-dominant molecular markers are routinely used in plant genetic research. In the present study we assessed the success-rate of three marker-systems for estimating genotypic diversity, clustering varieties into populations, and assigning a single variety into the expected population. A set of 54 diploid sugar beet (Beta vulgaris L. ssp. vulgaris) hybrid varieties from five seed companies was genotyped with 702 Diversity Array-Technology (DArT), 34 Single Nucleotide Polymorphisms (SNP), and 30 Simple Sequence Repeats (SSR) markers. Analysis of the population structure revealed three well-defined populations and clustering of varieties that generally correlates with their seed company origin. Two populations each contained varieties from two different seed companies indicating genetic similarity of this material. The third population was comprised only of varieties from a single seed company. Analysis of the SSR and SNP datasets indicates that some of the hybrid varieties likely have a common (or very closely related) parent. Comparison of the three marker-systems revealed substantial differences in the number of loci needed for analyses. Varietal clustering required approximately 1.8-2×more SSR, 3-4.5×more SNP, and 4.8×more DArT markers than were required for detection of genotypic diversity. When marker-systems were compared across different types of analyses per locus success-rate was the highest for the SSR and the lowest for the DArT markers. Generally, about 1.4-3×more SNPs, and 4.9-13.3×more DArTs then SSRs were needed to achieve the 100% success-rate. However, using only DArT markers with a high level of polymorphism decreased the number of DArT loci needed for analyses by 38-61%. Results from the present work provide a premise to selecting the type(s) and number of markers that are needed for genetic diversity analysis of sugar beet hybrid varieties.

Structural and content diversity of mitochondrial genome in beet: a comparative genomic analysis.

Despite their monophyletic origin, mitochondrial (mt) genomes of plants and animals have developed contrasted evolutionary paths over time. Animal mt genomes are generally small, compact, and exhibit high mutation rates, whereas plant mt genomes exhibit low mutation rates, little compactness, larger sizes, and highly rearranged structures. We present the (nearly) whole sequences of five new mt genomes in the Beta genus: four from Beta vulgaris and one from B. macrocarpa, a sister species belonging to the same Beta section. We pooled our results with two previously sequenced genomes of B. vulgaris and studied genome diversity at the species level with an emphasis on cytoplasmic male-sterilizing (CMS) genomes. We showed that, contrary to what was previously assumed, all three CMS genomes belong to a single sterile lineage. In addition, the CMSs seem to have undergone an acceleration of the rates of substitution and rearrangement. This study suggests that male sterility emergence might have been favored by faster rates of evolution, unless CMS itself caused faster evolution.

Sugar beet contains a large CONSTANS-LIKE gene family including a CO homologue that is independent of the early-bolting (B) gene locus.

Floral transition in the obligate long-day (LD) plant sugar beet (Beta vulgaris ssp. vulgaris) is tightly linked to the B gene, a dominant early-bolting quantitative trait locus, the expression of which is positively regulated by LD photoperiod. Thus, photoperiod regulators like CONSTANS (CO) and CONSTANS-LIKE (COL) genes identified in many LD and short-day (SD)-responsive plants have long been considered constituents and/or candidates for the B gene. Until now, the photoperiod response pathway of sugar beet (a Caryophyllid), diverged from the Rosids and Asterids has not been identified. Here, evidence supporting the existence of a COL gene family is provided and the presence of Group I, II, and III COL genes in sugar beet, as characterized by different zinc-finger (B-box) and CCT (CO, CO-like, TOC) domains is demonstrated. BvCOL1 is identified as a close-homologue of Group 1a (AtCO, AtCOL1, AtCOL2) COL genes, hence a good candidate for flowering time control and it is shown that it maps to chromosome II but distant from the B gene locus. The late-flowering phenotype of A. thaliana co-2 mutants was rescued by over-expression of BvCOL1 thereby suggesting functional equivalence with AtCO, and it is shown that BvCOL1 interacts appropriately with the endogenous downstream genes, AtFT and AtSOC1 in the transgenic plants. Curiously, BvCOL1 has a dawn-phased diurnal pattern of transcription, mimicking that of AtCOL1 and AtCOL2 while contrasting with AtCO. Taken together, these data suggest that BvCOL1 plays an important role in the photoperiod response of sugar beet.

Nuclear and cytoplasmic genetic diversity in weed beet and sugar beet accessions compared to wild relatives: new insights into the genetic relationships within the Beta vulgaris complex species.

Hybridization between cultivated species and their wild relatives is now widely considered to be common. In the Beta vulgaris complex, the sugar beet seed multiplication areas have been the scene of inadvertent pollination of sugar beet seed bearers by wild ruderal pollen donors, generating a weedy form of beet which infests sugar beet fields in European countries. Up to now, investigations of evolutionary dynamics of genetic diversity within the B. vulgaris complex were addressed using few genetical markers and few accessions. In this study, we tackled this issue using a panel of complementary markers: five nuclear microsatellite loci, four mitochondrial minisatellite loci and one chloroplastic PCR-RFLP marker. We sampled 1,640 individuals that illustrate the actual distribution of inland ruderal beets of South Western France, weed beets and wild sea beets of northern France as well as the diversity of 35 contemporary European diploid cultivars. Nuclear genetic diversity in weed beets appeared to be as high as those of ruderal beets and sea beets, whereas the narrowness of cultivar accessions was confirmed. This genetic bottleneck in cultivars is even more important in the cytoplasmic genome as only one haplotype was found among all sugar beet cultivars. The large majority of weed beet populations also presented this unique cytoplasmic haplotype, as expected owing to their maternal cultivated origin. Nonetheless, various cytoplasmic haplotypes were found within three populations of weed beets, implying wild-to-weed seed flows. Finally, our findings gave new insights into the genetical relationships between the components of the B. vulgaris complex: (1) we found a very strong genetic divergence between wild sea beet and other relatives, which was unexpected given the recent evolutionary history and the full cross-compatibility of all taxa and (2) we definitely confirmed that the classification into cultivated, wild, ruderal and weed forms according to their geographical location, phenotype or their domesticated status is clearly in accordance with genetic clustering despite the very recent domestication process of sugar beet.

Mitochondrial DNA phylogeny of cultivated and wild beets: relationships among cytoplasmic male-sterility-inducing and nonsterilizing cytoplasms.

Cytoplasmic male sterility (CMS), the maternally inherited failure to produce functional pollen, has been used in the breeding of sugar beet (Beta vulgaris ssp. vulgaris). At least three different sources of CMS can be distinguished from one another as well as from normal fertile cytoplasm by polymorphisms in their mitochondrial genomes. Here we analyzed 50 accessions of cultivated and wild beets to investigate the phylogenetic relationships among male-sterility-inducing and normal cytoplasms. The haplotypes were characterized by the nucleotide sequence of the mitochondrial cox2-cox1 spacer region and mitochondrial minisatellite loci. The results indicated that (1) a normal cytoplasm line, cv. TK81-O, was situated at the major core node of the haplotype network, and (2) the three sterilizing cytoplasms in question derived independently from the core haplotype. The evolutionary pathway was investigated by physical mapping study of the mitochondrial genome of a wild beet (B. vulgaris ssp. orientalis) accession BGRC56777 which shared the same mitochondrial haplotype with TK81-O, but was not identical to TK81-O for the RFLP profiles of mitochondrial DNA. Interestingly, three sets of inverted repeated sequences appeared to have been involved in a series of recombination events during the course of evolution between the BGRC56777 and the TK81-O mitochondrial genomes.

Resistance gene analogues are clustered on chromosome 3 of sugar beet and cosegregate with QTL for rhizomania resistance.

Worldwide, rhizomania is the most important disease of sugar beet. The only way to control this disease is to use resistant varieties. Four full-length resistance gene analogues (RGAs) from sugar beet (cZR-1, cZR-3, cZR-7, and cZR-9) were used in this study. Their predicted polypeptides carry typical nucleotide-binding sites (NBSs) and leucin-rich repeat (LRR) regions, and share high homology to various plant virus resistance genes. Their corresponding alleles were cloned and sequenced from a rhizomania resistant genotype. The 4 RGAs were mapped as molecular markers, using sequence-specific primers to determine their linkage to the rhizomania resistance locus Rz1 in a population segregating for rhizomania resistance. One cZR-3 allele, named Rz-C, together with 5 other molecular markers, mapped to the Rz1 locus on chromosome 3 and cosegregated with quantitative trait loci for rhizomania resistance. After screening a bacterial artificial chromosome (BAC) library, 25 cZR-3-positive BACs were identified. Of these, 15 mapped within an interval of approximately 14 cM on chromosome 3, in clusters close to the Rz1 locus. Rz-C differentiates between susceptible and resistant beet varieties, and its transcripts could be detected in all rhizomania resistant varieties investigated. The potential of this RGA marker for cloning of rhizomania resistance genes is discussed.

The Owen mitochondrial genome in sugar beet (Beta vulgaris L.): possible mechanisms of extensive rearrangements and the origin of the mitotype-unique regions.

The mitochondrial genomes of normal fertile and male-sterile (Owen CMS) cytoplasms of sugar beet are highly rearranged relative to each other and dozens of inversional recombinations and other reshuffling events must be postulated to interconvert the two genomes. In this paper, a comparative analysis of the entire nucleotide sequences of the two genomes revealed that most of the inversional recombinations involved short repeats present at their endpoints. Attention was also focused on the origin of the Owen CMS-unique mtDNA regions, which occupy 13.6% of the Owen genome and are absent from the normal mtDNA. BLAST search was performed to assign the sequences, and as a result, 7.6% of the unique regions showed significant homology to previously determined mitochondrial sequences, 17.9% to nuclear DNA, 4.6% to mitochondrial episomes, and 0.1% to plastid DNA. Southern blot analysis revealed that additional sequences of nuclear origin may be included within the unique regions. We also found that the copies of many short repeat families are scattered throughout the unique regions. This suggests that, in addition to the incorporation of foreign DNAs, extensive duplication of short repetitive sequences and continued scrambling of mtDNA sequences may be implicated in the generation of the Owen CMS-unique regions.

Nutrient digestibility in sheep fed diets containing Roundup Ready or conventional fodder beet, sugar beet, and beet pulp.

The objective of this digestibility assessment was to determine whether there are significant differences in the digestibility of Roundup Ready (glyphosate-tolerant) and conventional sugar beet, fodder beet, and beet pulp produced from sugar beet varieties when fed to sheep (seven wethers per treatment group). Three experiments were conducted in this assessment. Experiment 1 (35 wethers) compared one glyphosate-tolerant fodder beet variety with four conventional varieties, Exp. 2 (42 wethers) compared one glyphosate-tolerant sugar beet variety with five conventional varieties, and Exp. 3 (42 wethers) compared beet pulp derived from glyphosate-tolerant sugar beet with beet pulp from five European locations. The experimental phase consisted of a 2-wk preliminary period followed by a 1-wk collection period for Exp. 1 and 2, and a 1-wk preliminary period followed by a 1-wk digestibility collection period for Exp. 3. Diets were comprised of grass hay at 30, 30, and 20% of DM for Exp. 1, 2, and 3, respectively, with the balance being beet components. Urea and sodium sulfate were supplemented (8 and 2.9 g, respectively, for Exp. 1 and 2; and 6 g and 2.16 g, respectively, for Exp. 3) to supply sufficient dietary N and S. Each diet was fed to sheep (96 +/- 0.9 kg) in the three experiments to at or near maintenance energy levels. Treatment differences were considered significant at P < 0.05. Apparent digestibilities of DM, OM, CP, NDF, ADF, and DE for glyphosate-tolerant fodder and sugar beets did not differ from those for commercial fodder and sugar beets in Exp. 1 and 2. There were differences (P < 0.05) in DM, OM, CP, NDF, ADF, and DE digestibilities influenced by the different varieties of beet pulp in Exp. 3, but these were not unique to just the Roundup Ready sugar beet variety. Digestibilities and feeding values of Roundup Ready fodder beet, sugar beet, and beet pulp produced from Roundup Ready sugar beet varieties were not influenced by the introduction of the Roundup Ready trait compared with conventional varieties.

Variability of cathodic peroxidases in sugar beet (Beta vulgaris L.) cultivars.

Eighteen varieties of sugar beet (Beta vulgaris L.), originating from various European countries, were compared in respect of peroxidase variability level. They were cultivated in the same experimental plot. The cultivars differed in ploidy level: one variety was tetraploid, three were diploid and 14 varieties were triploid. The cathodic peroxidase system is controlled by four independent genes, of which only one is polymorphic. Consequently, the varieties were characterised by frequencies of 3 allozymes belonging to one locus. Only one variety proved to be fully monomorphic. Genetic similarities between the cultivars were illustrated by a dendrogram (UPGMA) and show different groups of varieties not related to their ploidy level.