Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets ( Beta vulgaris ssp. maritima ) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.     

Impact of gene flow from cultivated beet on genetic diversity of wild sea beet populations

Gene flow and introgression from cultivated plants may have important consequences for the conservation of wild plant populations. Cultivated beets (sugar beet, red beet and Swiss chard: Beta vulgaris ssp. vulgaris) are of particular concern because they are cross-compatible with the wild taxon, sea beet (B.vs. ssp. maritima). Cultivated beet seed production areas are sometimes adjacent to sea beet populations; the numbers of flowering individuals in the former typically outnumber those in the populations of the latter. In such situations, gene flow from cultivated beets has the potential to alter the genetic composition of the nearby wild populations. In this study we measured isozyme allele frequencies of 11 polymorphic loci in 26 accessions of cultivated beet, in 20 sea beet accessions growing near a cultivated beet seed production region in northeastern Italy, and 19 wild beet accessions growing far from seed production areas. We found one allele that is specific to sugar beet, relative to other cultivated types, and a second that has a much higher frequency in Swiss chard and red beet than in sugar beet. Both alleles are typically rare in sea beet populations that are distant from seed production areas, but both are common in those that are near the Italian cultivated beet seed production region, supporting the contention that gene flow from the crop to the wild species can be substantial when both grow in proximity. Interestingly, the introgressed populations have higher genetic diversity than those that are isolated from the crop. The crop-to-wild gene flow rates are unknown, as are the fitness consequences of such alleles in the wild. Thus, we are unable to assess the long-term impact of such introgression. However, it is clear that gene flow from a crop to a wild taxon does not necessarily result in a decrease in the genetic diversity of the native plant.     

Evidence for gene flow via seed dispersal from crop to wild relatives in Beta vulgaris (Chenopodiaceae): consequences for the release of genetically modified crop species with weedy lineages

Gene flow and introgression from cultivated to wild plant populations have important evolutionary and ecological consequences and require detailed investigations for risk assessments of transgene escape into natural ecosystems. Sugar beets (Beta vulgaris ssp. vulgaris) are of particular concern because: (i) they are cross-compatible with their wild relatives (the sea beet, B. vulgaris ssp. maritima); (ii) crop-to-wild gene flow is likely to occur via weedy lineages resulting from hybridization events and locally infesting fields. Using a chloroplastic marker and a set of nuclear microsatellite loci, the occurrence of crop-to-wild gene flow was investigated in the French sugar beet production area within a 'contact-zone' in between coastal wild populations and sugar beet fields. The results did not reveal large pollen dispersal from weed to wild beets. However, several pieces of evidence clearly show an escape of weedy lineages from fields via seed flow. Since most studies involving the assessment of transgene escape from crops to wild outcrossing relatives generally focused only on pollen dispersal, this last result was unexpected: it points out the key role of a long-lived seed bank and highlights support for transgene escape via man-mediated long-distance dispersal events.     

Tracing back seed and pollen flow within the crop-wild Beta vulgaris complex: genetic distinctiveness vs. hot spots of hybridization over a regional scale

Hybrids between transgenic crops and wild relatives have been documented successfully in a wide range of cultivated species, having implications on conservation and biosafety management. Nonetheless, the magnitude and frequency of hybridization in the wild is still an open question, in particular when considering several populations at the landscape level. The Beta vulgaris complex provides an excellent biological model to tackle this issue. Weed beets contaminating sugar beet fields are expected to act as a relay between wild populations and crops and from crops-to-crops. In one major European sugar beet production area, nine wild populations and 12 weed populations were genetically characterized using cytoplasmic markers specific to the cultivated lines and nuclear microsatellite loci. A tremendous overall genetic differentiation between neighbouring wild and weed populations was depicted. However, genetic admixture analyses at the individual level revealed clear evidence for gene flow between wild and weed populations. In particular, one wild population displayed a high magnitude of nuclear genetic admixture, reinforced by direct seed flow as evidenced by cytoplasmic markers. Altogether, weed beets were shown to act as relay for gene flow between crops to wild populations and crops to crops by pollen and seeds at a landscape level.     

SUGAR BEET YELLOWS: A PRELIMINARY STUDY OF THE DISTRIBUTION AND INTERRELATIONSHIPS OF VIRUSES AND VIRUS STRAINS FOUND IN EAST ANGLIA, 1955-57

Aphid transmissions to sugar beet seedlings from yellowed sugar beet leaves collected from commercial fields in East Anglia during the summers of 1955, 1956 and 1957, showed the occurrence of two yellowing viruses. One was sugar beet yellows virus (SBYV) and produced vein-etch and yellowing symptoms on beet seedlings in the glasshouse; the other produced yellowing but no etch. These two viruses were apparently unrelated, so that sugar beet tolerant to one of them would not necessarily be tolerant to the other. The second virus, called 'sugar beet mild yellowing virus' (SBMYV), decreased the root yield of sugar beet plants grown under glass, by as much as did the milder SBYV strains, but less than did the severe SBYV strains. The proportions of the two viruses in the samples differed from year to year and from place to place.     

利用mRNA差异显示技术分离甜菜M14品系特异表达基因的cDNA片段

二倍体栽培甜菜与白花甜菜杂交、进一步回交而获得的单体附加系M14,其染色体组成中除了含有18条栽培甜菜染色体外,还附加有一条野生白花甜菜第9号染色体,该附加染色体通过母本的传递率为96.5%;单体附加系传递率如此高的原因是因为M14中有无融合生殖基因的存在。本实验采用mRNA差异展示技术对甜菜无融合生殖品系M14和正常有性生殖的二倍体栽培甜菜A2Y花蕾减数分裂时期的基因表达进行了差异分析。采用GT15A,GT15G,GT15C3种锚定引物,共筛选了20个随机引物,通过RT-PCR检测,获得了6个阳性差异表达的cDNA片段,应用NCBI的BLASTx软件对测序结果进行同源序列、相似序列检索,为进一步克隆无融合生殖基因提供侯选cDNA片段。     

The control of Alternaria species on leaves of sugar beet infected with yellowing viruses

Leaves of virus-free sugar-beet plants rarely became infected with Alternaria spp. in two field experiments at Cambridge in 1965. Infection with beet yellows virus (BYV) increased susceptibility of plants to Alternaria only slightly but infection with beet mild yellowing virus (BMYV) increased it greatly. There was a close association between the severity of Alternaria symptoms, shown by different breeding lines and varieties of sugar beet, and the losses of sugar yield which they sustained after infection with BYV and BMYV. Many lines and varieties were resistant to Alternaria even when infected with BMYV and their resistance seemed to be inherited as a dominant character. Individual plants of any one line or variety differed greatly in resistance to Alternaria, suggesting that selection should improve the present level of resistance. Spraying the foliage of Alternaria-susceptible varieties with fungicides had little effect on the severity of Alternaria symptoms or on sugar yield. This was probably because the wet summer of 1965 was ideal for the spread of Alternaria and because rain washed the fungicide deposits from the sprayed leaves.     

Fine-scale geographical structure of genetic diversity in inland wild beet populations

Introgression arising from crop-to-wild gene flow provides novel sources of genetic variation in plant species complexes. Hybridization within the Beta vulgaris species complex is of immediate concern; crop lineages ( B .  vulgaris ssp. vulgaris ) hybridize easily with their wild relatives ( B .  vulgaris ssp. maritima ) thereby threatening wild beet gene diversity with genetic swamping. Hybridization 'hotspots' occur in European seed production areas because inland ruderal wild beets occur and reproduce in sympatry with cultivated beets. We studied gene flow occurring between seed-producing cultivars and ruderal wild B .  vulgaris in southwestern France to determine whether feral beets, arising from unharvested cultivated seed, represent an opportunity for crop-to-wild gene flow. We surveyed 42 inland ruderal beet populations located near seed production fields for nucleo-cytoplasmic variation and used a cytoplasmic marker diagnostic of cultivated lines. Occurrence of cultivated-type cytoplasm within ruderal populations clearly reflected events of crop seed escape. However, we found no genetic signatures of nuclear cultivated gene introgression, which suggests past introgression of cultivated cytoplasm into a wild nuclear background through seed escape rather than recent direct pollen flow. Overall, patterns of genetic structure suggested that inland ruderal wild beet populations act as a metapopulation, with founding events involving a few sib groups, followed by low rates of seed or pollen gene flow after populations are established. Altogether, our results indicate that a long-lived seed bank plays a key role in maintaining cultivated-type cytoplasm in the wild and highlight the need for careful management of seed production areas where wild and cultivated relatives co-occur.     

The influence of size and duration of aphid infestation on host plant quality, and its effect on sugar beet yellowing virus epidemiology

This paper studies the influence of previous infestation on the host quality of sugar beet (Beta vulgaris L.) for aphids and the influence of previous infestation on sugar beet yellowing virus epidemiology. Sugar beet previously infested with Myzus persicae (Sulzer) or Aphis fabae Scopoli (Homoptera: Aphididae) had an improved host quality for subsequently infesting aphids of the same species. There was a significant negative relationship between the number of M. persicae infesting a plant and the proportion of those that died with a dark deposit in their stomachs, and a significant positive relationship between the number that settled on a plant and the number that infested it previously. Nymphs feeding on previously infested plants grew more rapidly than those on control plants. The beneficial effect of previous infestation persisted for at least 2 weeks and prolongation of the infestation beyond 2 weeks was of no further benefit to the aphids. Field grown sugar beet, previously colonised by M. persicae, was more susceptible to natural infestation by M. persicae up to 5 days after exposure. Previously infested plants were also more susceptible to infection with beet mild yellowing virus (BMYV) but not beet yellows virus (BYV), suggesting that the aphids on the previously infested sugar beet settled more readily and were more inclined to feed (and thus transmit BMYV) than aphids on the previously uninfested plants. The consequences for the control of sugar beet yellowing virus vectors are discussed.     

Estimating genetic variation in sugar beets and wild beets using pools of individuals.

The study describes the genetic structure in sugar beets and in wild beets (Beta vulgaris) using 30 RFLP markers. Samples consisting of pooled plant material of 100 individuals from each line and population were used to analyse 120 sugar beet breeding lines and 91 wild beet populations. Greater variation was found among the wild populations than among the breeding lines. Although the two major groups of breeding lines, monogerm and multigerm, had approximately equal amounts of genetic variation, in the monogerm group more of this variation was partitioned among the lines than within the lines. Furthermore, despite most of the variation being shared by the two groups, the two groups were found to be separated along the first two components in a principal component analysis. Computer simulations were carried out to evaluate the usefulness of the pooled-sample strategy employed in the investigation. These simulations showed the use of pooled samples to be a better alternative than that of analysing a few plants individually.     

Nuclear and cytoplasmic genetic diversity in weed beet and sugar beet accessions compared to wild relatives: new insights into the genetic relationships within the <Emphasis Type="Italic">Beta vulgaris</Emphasis> 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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization and chromosomal distribution of species-specific repetitive DNA sequences from Beta corolliflora, a wild relative of sugar beet.

Repetitive DNA sequences have been isolated from a Sau3AI plasmid library of tetraploid Beta corolliflora (2n = 4x = 36), a wild relative of sugar beet (B. vulgaris). The library was screened by differential hybridization with genomic DNA of B. corolliflora and B. vulgaris. When used as probes for Southern hybridization of genomic DNA, six clones were determined to represent highly repetitive DNA families present only in the B. corolliflora genome. Five other sequences were highly repetitive in B. corolliflora and low or single copy in B. vulgaris. The insert size varied between 43 bp and 448 bp. Two sequences pBC1279 and pBC1944 displayed strong homology to a previously cloned satellite DNA from B. nana. With one exception, sequences are tandemly arranged as revealed by a typical ladder pattern after genomic Southern hybridization. The chromosomal distribution of five probes was determined by fluorescence in situ hybridization (FISH) of mitotic metaphases from B. corolliflora and a triploid hybrid between B. vulgaris and B. corolliflora. Three sequences were spread along all chromosome arms of B. corolliflora while one sequence was present on only six chromosomes. The chromosome-specific sequence pBC216 was found in close vicinity to the 5S rDNA located on B. corolliflora chromosome IV. This set of species-specific sequences has the potential to be used as probes for the identification of monosomic alien addition lines and for marker-assisted gene transfer from wild beet to cultivated beet.     

Adenylate patterns of autumn-sown sugar beet differ from spring-sown sugar beet. Implications for root quality

Sugar beet ( Beta vulgaris L) is generally cultivated using two different planting and harvest patterns. In northern zones, spring sugar beet is sown in spring and harvested in autumn, whereas in subtropical latitudes, autumn sugar beet is sown in autumn and harvested in summer. The industrial quality of the root is frequently higher in spring-sown sugar beet crops. In order to explore physiological changes associated with this fact, this study has been focused on the seasonal changes of adenosine 5'-triphosphate and adenosine 5'-diphosphate levels in the storage roots of sugar beet plants, as an index of its metabolic status. The results obtained correspond to a different metabolic status of spring and autumn sugar beet at the moment of harvest. The adenylate patterns of autumn beets suggested a functional and active respiratory system. On the contrary, the patterns shown by spring beets corresponded to those we would expect to see in plants becoming dormant. The proline and glucose contents, which decrease the industrial quality of the root, and the respiratory rate measured in autumn-sown sugar beets, were nearly twice those of spring-sown sugar beets. The combination of an active respiratory system, which allows the carbohydrate catabolism and the synthesis of stress molecules, with the environmental factors at the time of the harvest, could be the underlying physiological mechanism causing some of the differences between spring- and autumn-sown sugar beet crops.     

Restricted host range of the weevil Aubeonymus mariaefranciscae, a new sugar-beet pest

The extent of feeding, fecundity and fertility of a new sugar beet pest, Aubeonymus mariaefranciscae Roudier (Coleoptera: Curculionidae), on a range of plants from different families was determined. We have found that the adult weevils were only able to feed and oviposit on two closely related cultivated plants, beet and sugar beet, both subspecies of Beta vulgaris L., suggesting a restricted host range for this curculionid. None of 16 secondary metabolites (widely distributed among plants, and representative of different chemical groups), nor any of 19 non-host extracts from 13 different families, deterred the adults of this curculionid from feeding on sugar beet. We found a phagostimulatory effect when beet and sugar beet homogenates from various plant tissues were supplied to adult weevils on weeds that they do not feed on. Conversely, no effect was observed when sucrose was provided. These findings suggest that host selection by A. mariaefranciscae adults is due to the presence of an undetermined phagostimulant compound(s) in beet and sugar beet, instead of the recognition of deterrent substances among the non-host plants.     

The effects of Beet mild yellowing virus and Beet chlorosis virus on the yield of UK field-grown sugar beet in 1997,1999 and 2000

The separate effects of the aphid‐transmitted poleroviruses; Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV), on the yield of field‐grown sugar beet were studied following different inoculation dates from May to July in 1997,1999 and 2000. Each sugar beet plant within the appropriate plots was infected with virus using at least 10 wingless viruliferous Myzus persicae per plant. In all 3 years, overall yield losses caused by BMYV were negatively correlated with time of infection with early season (May) inoculations causing 18–27% losses in sugar yield but late season losses only 4–15%. BChV decreased the sugar yield and sugar content of beet following early season inoculations, although the effects on sugar yield were more variable (range 8–24%) and the virus appeared to be less damaging compared to BMYV. However, inoculations with BChV in July of each year caused greater root and sugar losses than inoculations with BMYV at that time. Both poleroviruses increased the sodium content of the roots early in the season, although neither virus had an effect on potassium levels at any stage.     

A revision of Beta section Vulgares (Chenopodiaceae), with new light on the origin of cultivated beets

Taxonomic investigations of the variation within Beta vulgaris sensu lato, including the use of numerical techniques, have led to the following conclusions: 1. The variation in morphology of the modern cultivated types of beet exhibit noda in a continuous variation pattern. 2. Microspeciation within wild forms of B. vulgaris is considered to have occurred in the centre of diversity, while in some areas hybridization within Section Vulgares has produced a complex taxonomic situation. 3. Because of the existence of this continuous variation, the classification within the section has been simplified. A new theory for the origin of cultivated beets is proposed.     

Studies on beet western yellows virus in oilseed rape (Brassica napus ssp. oleifera) and sugar beet (Beta vulgaris)

Oilseed rape (Brassica napus L. ssp. oleifera) was studied as a potential overwintering host for the sugar-beet yellowing viruses, beet yellows virus (BYV) and beet mild yellowing virus (BMYV), and their principal vector, Myzus persicae. In spring 1982, plants infected with a virus which reacted positively in enzyme-linked immunosorbent assay (ELISA) with BMYV antibody globulin were found in oilseed-rape crops; none of the plants contained virus which reacted with BYV antibody globulin. This virus was subsequently identified as beet western yellows virus (BWYV). No leaf symptoms could be consistently associated with infection of oilseed rape, but the virus was reliably detected by sampling any leaf on an infected oilseed-rape plant. Some isolates from oilseed rape did infect sugar beet in glasshouse tests, but the proportions of inoculated plants which became infected were low. Apparently there is therefore little danger of much direct transmission of BWYV by M. persicae from oilseed rape to sugar beet in spring. BWYV was introduced to and spread within oilseed-rape crops in autumn by M. persicae, and autumn-sown oilseed rape proved to be a potentially important overwintering host for M. persicae. In a survey of 80 autumn-sown crops of oilseed rape in East Anglia, northern England and Scotland in spring 1983, 78 were shown to be extensively infected with BWYV. Experimental plots of oilseed rape with 100% BWYV-infection yielded approximately 13.4% less oil than plots with 18% virus infection, the result of a decrease in both seed yield and oil content.     

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.     

SUGAR-BEET YELLOWS: FURTHER STUDIES ON VIRUSES AND VIRUS STRAINS AND THEIR DISTRIBUTION IN EAST ANGLIA, 1958-59

Experiments have shown that, as in the years 1955-57, two yellowing viruses, beet yellows virus (SBYV) and sugar-beet mild yellowing virus (SBMYV), were present in commercial sugar-beet crops in East Anglia in 1958 and 1959. The evidence that they are not closely related viruses has been confirmed. In both years the prevalence of the two viruses was estimated by aphid transmissions from yellowed sugar-beet leaves to healthy sugar beet and Chenopodium capitatum seedlings in the glasshouse, and in 1959 additionally by examination of symptoms on field plants. SBMYV was more common than SBYV over the whole region in 1958, but in 1959 SBYV was slightly more prevalent than SBMYV. In both years SBYV was found more often in the southern than in the northern parts of the region. The results described in this paper suggest that breeding for tolerance to SBMYV may be at least as important economically in East Anglia as breeding for tolerance to SBYV. A wide range of SBYV strains was present in East Anglia in 1959, most of the strains being those which caused severe symptoms in sugar beet and C. capitatum.     

STUDIES ON THE IMPORTANCE OF WILD BEET AS A SOURCE OF PATHOGENS FOR THE SUGAR-BEET CROP

Beet yellows virus, beet mosaic virus, rust ( Uromyces betae (Pers) Lév.), and downy mildew ( Peronospora schachtii Fuckel.) were found to be common in wild beet ( Beta vulgaris s.-sp. maritima L.) growing on the foreshores of south Wales and southern England. The virus diseases were more prevalent in southeast England than in the west, rust more in the west than in the east, and downy mildew is equally prevalent in all regions.
Beet yellows is the most commercially important disease and is more common in sugar-beet crops in East Anglia than elsewhere in Great Britain. There was no evidence that beet yellows spread in East Anglia from wild beet to nearby sugar-beet crops during the springs of 1958 or 1959, and Myzus persicae Sulz., the principal vector of yellows, was rarely found on wild beet growing on the foreshore.
In glasshouse experiments aphids colonized sugar-beet plants watered with tap water in preference to those watered with sea water. Daily watering with sea water made plants unpalatable to aphids within 14 days. Aphids also preferred leaves sprayed with distilled water to those that had been sprayed with sea water. Salt solutions gave results similar to those obtained with sea water.