Association between resistance to Cercospora and yield in commercial sugarbeet hybrids

This report documents the difficulty breeders have experienced in combining resistance to Cercospora leaf spot (causal agent Cercospora beticola Sacc.) with high yield in sugarbeet (Beta vulgaris L.). Forty commercial hybrids, all recommended for Cercospora-threat areas, were grown in a Cercospora-free and a diseased (inoculated) environment in 1991 and 1992. A 2.9 Mg/ha decrease in root yield associated with each increment increase in susceptibility confirmed that under a severe epiphytotic (1991) Cercospora resistance provided substantial protection. Under less-severe disease conditions (1992) there was no apparent relationship between yield and resistance, suggesting that the benefits of resistance were similar to the yield potential sacrificed to obtain the resistance. In the absence of the disease, root yields increased 2.7 Mg/ha for each increment of increased susceptibility. There was no evidence of association between sucrose concentration and resistance in the Cercospora-free environment. Despite the limited efforts and/or success in developing resistant commercial hybrids, the demonstrated ability of Cercospora to produce fungicide-resistant strains and the possibility that effective fungicides will not be available provide incentives to seek genetic resistance through breeding efforts.     

Mapping of rhizoctonia root rot resistance genes in sugar beet using pathogen response‐related sequences as molecular markers

Rhizoctonia root and crown rot caused by the fungus Rhizoctonia solani is a serious disease of sugar beet. An F_2:3 population from a cross between a resistant and a susceptible parent has been tested for R. solani resistance and a genetic map has been constructed from the corresponding F₂ parents. The map encompasses 38 expressed sequence tags (ESTs) with high similarity to genes which are involved in resistance reactions of plants (R‐ESTs) and 25 bacterial artificial chromosomes (BACs) containing nucleotide binding site (NBS)‐motifs typical for disease resistance genes. Three quantitative trait loci (QTL) for R. solani resistance were found on chromosomes 4, 5 and 7 collectively explaining 71% of the total phenotypic variation. A number of R‐ESTs were mapped in close distance to the R. solani resistance QTL. In contrast, the NBS‐BACs mapped to chromosomes 1, 3, 7 and 9 with two major clusters of NBS‐BACs on chromosome 3. No linkage between NBS‐BACs and R. solani resistance QTL was found. The data are discussed with regard to using R‐ESTs and NBS markers for mapping quantitative disease resistances.     

Identification of a SCAR marker associated with Bm, the beet mosaic virus resistance gene, on chromosome 1 of sugar beet

Beet mosaic virus (BtMV) is an aphid transmitted, viral disease of beet found worldwide. The Bm gene, a resistance gene effective against BtMV, was identified in the sugar beet line 8500 and backcrossed into a C37 background to produce line C719. Three populations were developed from the cross of line C719 with the susceptible line C37 with the intent of developing markers for use in marker‐assisted selection. The F₂ progeny of three crosses were scored for resistance. Two of the three populations conformed to a 3 : 1 ratio, indicating a single gene trait. Sequence characterized amplified region (SCAR) markers were developed by using bulked segregant analysis combined with random amplified polymorphic DNA type markers. The markers showed close association to the Bm resistance gene and were effective in all three populations. The A₁ allele for genetic male sterility also was found to be associated with Bm and the SCAR marker. Development of a single‐nucleotide polymorphism marker from the SCAR sequence was used to validate linkage to chromosome 1 using separate mapping populations. This marker will be useful for the introgression of the Bm gene into germplasm.     

Greenhouse and field techniques for testing sugar beet for resistance to Rhizoctonia root and crown rot

Rhizoctonia‐resistant sugar beet varieties are the key to an integrated control strategy for Rhizoctonia root rot. Because of the unpredictable occurrence of Rhizoctonia solani in the field testing of sugar beet for resistance to Rhizoctonia root rot is difficult. The aim of the study was to develop advanced greenhouse and field techniques which allow a reliable assessment of sugar beet for resistance to R. solani. A highly infectious liquid inoculum was used for the first time in this study. It can be produced in large quantities of a standardized quality, sterile, and exactly quantified according to its carbon content. In a greenhouse trial, sugar beet grown in the same way as field grown beet was inoculated by applying a suspension of Rhizoctonia mycelium (equivalent to 10 mg carbon per plant) to the beet crown. After 3 weeks, inoculation had led to uniform and severe root rot. Disease symptoms were similar to those found under natural conditions in the field. No ‘escapes’, i.e. susceptible plants apparently expressing resistance were observed in the test. A new nine‐class disease scale was established and a Rhizoctonia index (RI) was calculated. Reliability of disease assessment was demonstrated on progeny of plants, selected from segregating populations, showing Rhizoctonia resistance more closely related to the resistant parent lines than to the susceptible ones. Sugar beet varieties could be assessed in the greenhouse within only 11 weeks. All varieties were affected by the pathogen but partially resistant varieties could be clearly recognized by a significantly lower Rhizoctonia index. Significant differences in susceptibility were also found within the group of new resistant genotypes. Infection studies performed in the field showed the superiority of the new liquid inoculum compared with a solid form and revealed the influence of inoculation date and inoculum level on the development of Rhizoctonia root rot. In field tests performed at different sites under different environmental conditions, susceptible and partially resistant sugar beet varieties could be reproducibly rated according to their susceptibility to R. solani. On average, susceptible varieties showed a Rhizoctonia index of 8 while resistant genotypes ranged from 5 to 6. The newly developed techniques allow fast and reliable evaluation of sugar beet for resistance to R. solani.     

Changes in sugar beet mitochondrial DNA induced during callus stage

The organization of mitochondrial DNA was studied in seed‐derived plants of sugar beet, Beta vulgaris L., and compared with the structure of DNA isolated from calli propagated in vitro. Tissues representing three genotypes (diploid male sterile, diploid maintainer and diploid fertile) were maintained under in vitro conditions for different periods before the DNA was analysed. Restriction fragment length polymorphism with BamHI and XhoI enzymes, and Southern hybridization with atpA and atp6 homologous probes were used. Hybridization experiments showed conspicuous differences in the organization of both genes in long‐term callus cultures (2 years or older). The novel organization of mitochondrial DNA in the calli of the male sterile genotype showed an additional 3.9 kb fragment after hybridization with the atp6, and 7.9 kb and 3.5 kb bands after hybridization with atpA. In the fertile genotype, changes in mitochondrial DNA structure were manifested in the disappearance of a 2.1 kb fragment after probing with atpA. Alterations induced in the mitochondrial genome of the male sterile line showed unique features in mtDNA rearrangements.     

A genetic map of sugar beet (Beta vulgaris) based on RAPD markers

Linkage analysis of sugar beet (Beta vulgaris L.) was performed with random amplified polymorphic DNA (RAPD)-markers. From three segregating populations, a combined genetic map was constructed which comprises 85 RAPD, five isozyme, one RFLP marker and the genes for resistance against the nematode Heterodera schachtii Schm., one restorer locus for male sterility and the genes for annuality and hypocotyl colour. For mapping of the two unlinked restorer genes a statistical model was developed based on the maximum-likelihood function.     

QTL analysis of Cercospora leaf spot resistance in sugar beet

The inheritance of Cercospora leaf spot resistance in sugar beet was investigated by means of quantitative trait loci (QTL) analysis of a segregating population of 193 individuals, using 110 AFLP and 35 restriction fragment length polymorphism (RFLP) markers. Five QTL were found through composite interval mapping on linkage groups 1, 2, 3, and 9, respectively, two of which were linked on linkage group 3. The significance of these QTL was tested by permutation analysis The QTL had mostly additive, but also certain negative dominance effects; all the resistance alleles came from the Cercospora-resistant parent. Each quantitative trait locus accounted for 7-18% of the phenotypic variation, leaving 37% of the variation unexplained. The results are discussed in relation to the potential use of marker-assisted breeding for Cercospora leaf spot resistance in sugar beet.     

An EMS mutagenesis protocol for sugar beet and isolation of non-bolting mutants

An annual sugar beet line homozygous for the dominant gene for early bolting (B) has been mutagenized with different doses of ethylmeth‐anesulfonate (EMS). Approximately 15 000 M1 seeds were treated with EMS doses between 0.5 and 1% for 4, 6, 8, 12 and 14 h. Among 10 066 M1s, plants with chlorophyll defects and other abnormalities were found. Germination rates ranged between 30 and 100%, whereas the fertility of M1s dropped to 36%. A dose of 1% EMS applied for 8 h was found to yield an acceptable rate of M2 sterility (16%). Exactly 0.5% of the M2 families contained plants with altered bolting behaviour. After selfing these M2 plants, five non‐bolting M3 lines were selected. These plants do not exhibit shoot elongation even after cultivation under long‐day conditions. Thus, they are homozygous for new mutagenized, recessive non‐bolting alleles. Moreover, four M3 lines showed delayed bolting which was clearly different from the early bolting parent. This demonstrates varying activities of the bolting gene due to different mutational events.     

Transgenic sugar beet tolerant to glyphosate

Phenology (i.e. the influence of environment on ontogeny) is the most important single factor influencing crop adaptation. The timing of flowering is particularly important since it largely determines when annual cereal, pulse and oilseed crops will subsequently be ripe for harvest. Two environmental factors are of overriding importance in the induction of flowering – photoperiod (daylength) and temperature. In seeking to predict times from sowing to flowering, f, it has proved profitable to analyse photothermal responses in terms of the rate of progress from sowing to flowering, 1/f. This paper summarises the advantages of a model based on rates rather than the traditional approach based on f. Over a wide range of photothermal regimes, the model involves just six coefficients, all of which (and their derivatives) have clearly defined biological meaning. Of paramount importance too is that the coefficients are not affected by the environment; they are genetic characters which determine phenotypic responses to the environment in a quantitative and predictable way.     

Marker analysis for quantitative traits in sugar beet

Many economically important traits are inherited quantitatively and are analysed by breeders in replicated field trials. If dense maps are available, chromosomal regions containing quantitative trait loci (QTL) can be identified and this opens up the possibility of preselecting for quantitative traits in the laboratory. In this study, QTL analysis for yield and yield components in sugar beet is used in two different populations tested in several environments in both populations, QTL were detected for all traits investigated, and their predictive value in breeding schemes was analysed by correlating predicted with observed values. Tolerance to Rhizomania, caused by a gene on chromosome 3, was the main source of genotype‐environment interaction in one population, allowing selection on a QTL basis within macro‐environments with or without Rhizomania infestation, respectively. No clear results were found for the second population tested in environments with and with‐out Cercospora infestation. Consequences for breeding strategies are discussed.     

Ecological aspects of transgenic sugar beet: transfer and expression of herbicide resistance in hybrids with wild beets

Summary An increasing number of genetically engineered cultivars of several crops is being experimentally released into the environment. In future, crops with new transgenic traits will probably play an important role in agricultural practice. The long-term effect of transgenes on community ecology will depend on the distribution and establishment of transgenic plants in the wild, on the sexual transfer of their new genes to the environment and on the potential ecological impact of the transgenic trait. The starting point was the use of transgenic sugar beet lines, Beta vulgaris subspec. vulgaris var. altissima DÖLL (Helm 1957), with transgenes coding for rhizomania and herbicide (BASTA^®) resistance. The first two questions to answer were: Can the transgenes be transferred via pollen to wild beets, Beta vulgaris subspec. maritima (L.) ARCANG. or cultivated relatives such as red beet or spinach beet and are they expressed in the hybrids? Can transgenes be monitored in young Beta vulgaris-hybrids? The experimental transfer of transgenes was conducted in 1993 at a field location in northern Germany. The beets were hand-pollinated with transgenic pollen. In a non destructive biotest, the hybrid seedlings were tested for herbicide resistance. Transgenic plants showed no noxious phenotypic effects whereas control plants developed leaf necroses. All herbicide resistant hybrids within the biotest were assumed to be transgenic.     

A marker-assisted analysis of bolting tendency in sugar beet (Beta vulgaris L.)

Bolting tendency in sugar beet varies among breeding lines and cultivars. Four crosses were made between breeding lines susceptible and resistant to bolting in order to study the genetic basis of bolting tendency. Bolting percentage in F₂, after 8 weeks of low temperature treatment, varied among the crosses, suggesting a complicated genetic control of bolting tendency. Different segregation ratios were observed, in particular, between families derived from the bolting F₁ plants and those from their non-bolting siblings, the former families showing a significantly higher bolting percentage than the latter. A marker-assisted analysis with seven isozyme loci, Ak1, Gdh2, Idh1, Lap, Mdh1, Pgi2 and Pgm1, revealed that a locus with marked effect on bolting tendency was located near Idh1. Because of a close linkage of Idh1 with B for annuality, the gene tagged by Idh1 appeared to be equivalent or similar to B′ for easy bolting allelic to B as reported by Owen et al. (1940). The results obtained suggest that the B locus may not only have an important role in determining growth habits but also control various degrees of bolting tendency in individual sugar beet plants. A linked pair, Ak1-Lap, and Pgi2 also were found to affect bolting tendency, although their effects varied depending on the crosses and families tested. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selection for sugar yield in sugar beet, Beta vulgaris, using different selection indices

In sugar beet breeding, sugar yield is mainly influenced by root yield and sugar content. In this short communication several indices to select for both traits simultaneously are compared in order to find the best one. The indices are correlated with the base index of Williams (1962) from independent experiments. The indices differ in the amount of information necessary for the calculation of their weights. Three different series of each eight sites gave similar results. The optimum index using all information from phenotypic and genotypic variances and covariances, did not perform best. Sugar content with its higher herit ability must have a larger weight than root yield. Heritabilities as index weights performed best, but two other indices using heritabilities and phenotypic but no genetic covariances also performed well.     

Overwintering of genetically modified sugar beet, Beta vulgaris L. subsp. vulgaris, as a source for dispersal of transgenic pollen

The potential impact of transgenic crops on community ecology will depend on the distribution and establishment of the new transgenic traits, on the sexual transfer of their new genes to the environment (Bartsch &; Pohl-Orf, 1996) and on the potential ecological impact of the transgenic trait. Flowering and pollen dispersal is important for outcrossing of the genetically engineered trait. For a biennial plant, like the cultivars of Beta vulgaris L., overwintering is normally necessary to become generative and to produce pollen and seeds (Abe et al., 1997), which usually does not happen with sugar beet as a field crop harvested in autumn (Longden 1989). The starting point for the project was a transgenic sugar beet, Beta vulgaris L. subsp. vulgaris (Lange et al., 1998), with rhizomania and herbicide ( Basta®, Liberty®) resistance. Cold tolerance is one of the most important factors for survival of sugar beet in Central- and North-Europe. Among other ways, spreading of transgenic traits into weed beet (Boudry et al., 1993) or wild beet can occur if genetically engineered – biennial – plants survive the winter, flower in spring and spread their pollen. Field experiments were performed with transgenic breeding lines and their hybrids, transgenic and non-transgenic hybrids with Swiss chard and three conventional beet cultivars to evaluate winter survival rates at seven different field sites. We could show that survival of sugar beet – transgenic as well as conventional ones – in Germany and at the Dutch border is possible. Survival rates were well correlated with temperature data and were unexpectedly high. Differences between sugar beet hybrids and breeding lines could be detected but not within different breeding lines or hybrids. There were no differences detectable between transgenic and non-transgenic plants. The data are crucial for the risk assessment of the release of transgenic sugar beet and are the basis for further experiments towards outcrossing and establishment.     

A gene complex for annual habit in sugar beet (Beta vulgaris L.)

Annual habit in sugar beet has been shown to be controlled by a dominant gene, B, which induces bolting under long days without the cold requirement usually essential for biennial cultivars. The induction of bolting by B, however, is often influenced by both environmental and genetic factors. We studied the genetic basis for bolting suppression, caused by delayed planting, in lines derived from a cross between annual and biennial lines. The F₂ progeny of a late-bolting F₁ plant yielded an unexpected segregation ratio of annuality from monogenic inheritance when planted in late May, there being an excess of non-bolting biennials, although the expected segregation was observed in the late April sowing. Bolting suppression was caused by restricted daylengths due to delayed planting because the segregation was normal under artificially induced long days. The analysis with Idh1, an isozyme marker proximal to B, demonstrated that heterozygous B plants were more susceptible to bolting suppression due to delayed planting than homozygous B plants. The results suggest that bolting suppression was controlled by a number of genes responsible for long daylength requirement, one of which was closely linked to B and formed a gene complex for annuality. The annual habit was controlled singly by B under long days but modified by the genes for long daylength requirement under restricted daylengths. This revised version was published online in July 2006 with corrections to the Cover Date.     

Combined linkage maps and QTLs in sugar beet (Beta vulgaris L.) from different populations

The construction of genetic maps is an expensive and time-consuming process. The breeder is therefore interested in using maps developed from other mapping populations but this is only possible if the genetic structure is similar for the chromosomal regions of interest. In this paper, maps of three populations of sugar beet (Beta vulgaris L.) with common polymorphic marker loci are compared. Maps were constructed with MAPMAKER 3.0 and JOINMAP 2.0. Both mapping programs gave, in general, the same order for common markers. However, the number of common markers was too low to construct a combined map for all chromosomes. For one population, in contrast to the other two, the map constructed with MAPMAKER 3.0 was much longer than that constructed with JOINMAP 2.0. For two of these populations yield traits were also available from different environments. For quantitative trait loci (QTL) analysis of the yield data, the packages MAPMAKER/QTL 1.1 and PLABQTL were used. No QTL common for the two populations could be detected. The program and the version used strongly influenced the estimated positions of QTLs. There was also a strong interaction with environments.     

Integration of AFLP markers into a linkage map of sugar beet (Beta vulgaris L.)

The AFLP (amplified fragment length polymorphism) technique has been applied in establishing an extended linkage map of sugar beet. A total of 120 AFLPs were integrated into an existing linkage map based on RFLP markers. Four primer combinations yielded between 19 and 40 polymorphic bands in an F₂ population consisting of 94 plants. The AFLP loci were evenly distributed over the nine linkage groups, with the exception of linkage group V where the number of AFLPs was significantly low. The AFLPs were found to be reproducible even against the background of different combinations of Taq DNA polymerases and buffers. However, the quantity of higher molecular weight fragments (>400 bp) was reduced when using plant DNA of poor quality as a template. The results of these experiments are discussed, together with possible applications of AFLPs in sugar beet breeding.     

Combining different linkage maps in sugar beet (Beta vulgaris L.) to make one map

Several genetic maps for sugar beet (Beta vulgaris L.), from different German research groups, have been published and it is now possible to consider combining them with the aid of the common markers. The computer program JOINMAP (versions 1.3 and 2.0) was used for pair-wise combination of three populations. Several problems arose: the genetic background of the populations, different population structures (F₂ versus F₁× F₁), different number of polymorphic loci for common probes in the populations to be combined, different estimates of the recombination rates between the same markers and differences between the Join Map versions. The maps from two F₂ populations could be integrated into a single map, but it was more appropriate to construct separate maps for the F₂ populations and the F₁× F₁ population using common markers as reference points only.     

Monosomic addition lines of Beta corolliflora in sugar beet: plant morphology and leaf spot resistance

Monosomic addition lines in Beta vulgaris from Beta corolliflora were described morphologically and characterized for disease resistance. Monosomic addition plants (2n= 19) were selected among segregating offspring by a squash dot technique in combination with B. corolliflora‐specific probes. Plants carrying an added chromosome were characterized by leaf shape, plant size and plant vigour. In this way, most addition lines could be distinguished from diploid beets, however, to identify those plants unequivocally, molecular marker analysis was also necessary. Transmission frequencies of each addition line were determined to be in the range 13.9% (Cor‐4) to 60% (Cor‐9). High transmission rate of addition line Cor‐9 was assumed to be due to apomictic propagation because transmission rate after selfing cannot exceed 50%. Cercospora leaf spot resistance tests were performed on 167 monosomic plants from seven different addition lines, two fragment addition lines and 89 diploid controls. No line exhibited complete resistance, but the monosomic additions Cor‐3 and Cor‐4 showed significantly lower infection rates than their diploid sibling plants. The identification of monosomic addition lines with apomictic and disease resistance characters offers the possibility of transferring those genes to sugar beet.     

The efficiency of between and within full-sib family selection in a recurrent selection programme in sugar beet (Beta vulgaris L.)

The efficiency of between and within family selection in a full-sib family recurrent selection programme was investigated using data from three cycles of recurrent selection. Correlations between mid-parent values and offspring were high for sugar content and juice purity characters, but low for root weight and sugar yield. This suggests that single root selection within full-sib families is effective in the improvement of sugar content and juice purity, but ineffective for root weight and sugar yield. Root weight and sugar yield will respond better to between full-sib family selection. This revised version was published online in July 2006 with corrections to the Cover Date.