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

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

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

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

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

The Absence of TIR-Type Resistance Gene Analogues in the Sugar Beet (Beta vulgaris L.) Genome

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed. These authors (Yanyan Tian, Longjiang Fan) contributed equally to this work.     

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.     

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

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

Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.     

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.     

Association mapping in multiple segregating populations of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

Association mapping in multiple segregating populations (AMMSP) combines high power to detect QTL in genome-wide approaches of linkage mapping with high mapping resolution of association mapping. The main objectives of this study were to (1) examine the applicability of AMMSP in a plant breeding context based on segregating populations of various size of sugar beet (Beta vulgaris L.), (2) compare different biometric approaches for AMMSP, and (3) detect markers with significant main effect across locations for nine traits in sugar beet. We used 768 F _n (n = 2, 3, 4) sugar beet genotypes which were randomly derived from 19 crosses among diploid elite sugar beet clones. For all nine traits, the genotypic and genotype × location interaction variances were highly significant (P < 0.01). Using a one-step AMMSP approach, the total number of significant (P < 0.05) marker-phenotype associations was 44. The identification of genome regions associated with the traits under consideration indicated that not only segregating populations derived from crosses of parental genotypes in a systematic manner could be used for AMMSP but also populations routinely derived in plant breeding programs from multiple, related crosses. Furthermore, our results suggest that data sets, whose size does not permit analysis by the one-step AMMSP approach, might be analyzed using the two-step approach based on adjusted entry means for each location without losing too much power for detection of marker-phenotype associations.     

Analysis of DNA polymorphisms in sugar beet (Beta vulgaris L.) and development of an SNP-based map of expressed genes

A panel of 13 sugar beet lines and one genotype each of the Beta vulgaris cultivars red beet and Swiss chard, and B. vulgaris ssp. maritima were used to identify polymorphisms in alignments of genomic DNA sequences derived from 315 EST- and 43 non-coding RFLP-derived loci. In sugar beet lines, loci of expressed genes showed an average SNP frequency of 1/72 bp, 1 in 58 bp in non-coding sequences, increasing to 1/47 bp upon the addition of the remaining genotypes. Within analysed DNA fragments, alleles at different SNP positions displayed linkage disequilibrium indicative of haplotype structures. On average 2.7 haplotypes were found in sugar beet lines, and haplotype conservation in expressed genes appeared to exceed 500 bp in length. Seven different genotyping techniques including SNP detection by MALDI-TOF mass spectrometry, pyrosequencing and fluorescence scanning of labelled nucleotides were employed to perform 712 segregation analyses for 538 markers in three F₂ populations. Functions were predicted for 492 mapped sequences. Genetic maps comprised 305 loci covering 599.8 cM in population K1, 241 loci distributed over 636.6 cM in population D2, and 166 loci over 507.1 cM in population K2, respectively. Based on 156 markers common to more than one population an integrated map was constructed with 524 loci covering 664.3 cM. For 377 loci the genome positions of the most similar sequences from A. thaliana were identified, but little evidence for previously presented ancestral genome structures was found. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A BAC library of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. for the targeted isolation of centromeric DNA and molecular cytogenetics of <Emphasis Type="Italic">Beta</Emphasis> species

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.     

Phenotypic polymorphism and allele differentiation of isozymes in fodder beet,multigerm sugar beet and monogerm sugar beet

Summary Thirteen enzymes (MDH, SDH, LAP, PGM, PX, IDH, GPI, 6PGD, APH, GOT, GDH, ME and SOD) of 3 cultivated beet (B. vulgaris L.) gene pools, comprising 12 accessions of fodder beet, 11 of old multigerm sugar beet and 10 of modern monogerm sugar beet, were investigated using horizontal starch gel electrophoresis. Eleven accessions of primitive or wild B. vulgaris were also included for the comparison of isozymes. Variation in isozyme phenotypes was investigated to detect diversity in the three cultivated forms of beet. Phenotypic variation was observed in all except ME and SOD, which were monomorphic. A high degree of phenotypic polymorphism (Pj) was found in GDH, PGM, IDH, APH and MDH. Differences in phenotypic polymorphism in MDH, GPI and PX were recognized between fodder beet and both sugar beet groups. Average polymorphism for 13 enzymes in both sugar beets was significantly higher than that in fodder beet. For 13 enzymes, the existence of high isozyme diversity in both sugar beet gene pools was revealed. Allele frequencies in 13 alleles of five enzyme-coding loci, Lap, Px-1, Aph-1, Got-2 and Gdh-2, were investigated. New alleles, Px-1 ¹ and Got-2 ¹, were found in fodder beet accessions. No significant differences of average allele frequencies of five loci between fodder beet and both sugar beets were recognized. Several unique alleles and different isozyme phenotypes were observed in the accessions of B. vulgaris ssp. macrocarpa and ssp. adanensis. Future utilization of cultivated beet gene pools for sugar beet breeding is discussed from the viewpoint of genetic resources.     

Populations of Rhizoctonia solani in soil under crops in rotation with sugar beet

Using a soil debris isolation method, populations of Rhizoctonia solani were monitored over a 4 -yr period in four fields which were initially cropped to sugar beet and in which four areas of Rhizoctonia crown rot diseased beets (DA) and four areas of apparently healthy beets (AH) had been selected and precisely located. Soil from these areas was assayed during the subsequent crops, which included sugar beet, tomato, cucumber, maize and soybean. No significant differences in colony counts were found between the soils in DA and AH on any of 30 sampling dates. R. solani population counts were, in general, quite low, except under sugar beet and following tomato harvest. Areas of diseased beet and high R. solani soil populations that developed in subsequent sugar beet crops did not necessarily coincide with the previously selected diseased areas. High R. solani populations developed from parasitic activity on sugar beet or saprophytically on tomato crop residues. Of the other crops, both maize and soybean may have slightly increased the low R. solani residual populations in soil. The monitoring of R. solani populations in the season prior to, and during the early season of sugar beet cropping did not provide a basis for forecasting disease in fields or sites within fields. The initiation of disease patches in these sugar beet fields was therefore governed by factors other than inoculum density.     

Taproot promoters cause tissue specific gene expression within the storage root of sugar beet

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.     

Molecular and chromosomal organization of two repetitive DNA sequences with intercalary locations in sugar beet and other Beta species

 In a search for repetitive DNA sequences in the sugar beet genome, two sequences with repeat unit lengths of 143 and 434 bp were isolated and characterized. The pSV family showed an unusual conservation of restriction sites reflecting homogenization of the analyzed repeats. Members of the family are organized as tandem repeats as revealed by PCR and sequencing of dimeric units. The pSV satellite occurs in large intercalary arrays which are present on all chromosome arms of sugar beet. The pSV sequence family is present in different abundance in the sections Beta, Corollinae and Nanae but is not detectable by Southern hybridization in the section Procumbentes. The pDRV family is characterized by an interspersed genomic organization. The sequence is detectable in all sections of the genus and is amplified in species of the section Beta but was also detected, although at lower abundance, in the remaining three sections. Fluorescent in situ hybridization has shown that the pDRV sequence family is dispersed over all chromosomes of the sugar beet complement with some regions of clustering and centromeric depletion. Received: 18 March 1998 / Accepted: 31 March 1998     

Physical mapping and cloning of a translocation in sugar beet (Beta vulgaris L) carrying a gene for nematode (Heterodera schachtii) resistance from B. procumbens

Two diploid (2n=18) sugar beet (Beta vulgaris L.) lines which carry monogenic traits for nematode (Heterodera schachtii Schm.) resistance located on translocations from the wild beet species Beta procumbens were investigated. Short interspersed repetitive DNA elements exclusively hybridizing with wild beet DNA were found to be dispersed around the translocations. The banding pattern as revealed by genomic Southern hybridization was highly conserved among translocation lines of different origins indicating that the translocations are not affected by recombination events with sugar beet chromosomes. Physical mapping revealed that the entire translocation is represented by a single Sal I fragment 300 kb in size. A representative YAC (yeast artifical chromosome) library consisting of approximately 13,000 recombinant clones (2.2 genome equivalents) with insert sizes ranging between 50 and 450 kb and an average of 130kb has been constructed from the resistant line A906001. Three recombinant YACs were isolated from this library using the wild beet-specific repetitive elements as probes for screening. Colinearity between YAC inserts and donor DNA was confirmed by DNA fingerprinting utilizing these repetitive probes. The YACs were arranged into two contigs with a total size of 215 kb; these represent a minimum of 72% of the translocation.