DNA markers closely linked to nematode resistance genes in sugar beet (Beta vulgaris L.) mapped using chromosome additions and translocations originating from wild beets of the Procumbentes section

Summary Genes conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) have been transferred to sugar beet (Beta vulgaris L.) from three wild species of the Procumbentes section using monosomic addition and translocation lines, because no meiotic recombination occurs between chromosomes of cultured and wild species. In the course of a project to isolate the nematode resistance genes by strategies of reverse genetics, probes were cloned from DNA of a fragmented B. procumbens chromosome carrying a resistance gene, which had been isolated by pulsed-field gel electrophoresis. One probe (pRK643) hybridized with a short dispersed repetitive DNA element, which was found only in wild beets, and thus may be used as a molecular marker for nematode resistance to progenies of monosomic addition lines segregating resistant and susceptible individuals. Additional probes for the resistance gene region were obtained with a polymerase chain reaction (PCR)-based strategy using repetitive primers to amplify DNA located between repetitive elements. One of these probes established the existence of at least six different chromosomes from wild beet species, each conferring resistance independently of the others. A strict correlation between the length of the wild beet chromatin introduced in fragment addition and translocation lines and the repeat copy number has been used physically to map the region conferring resistance to a chromosome segment of 0.5-3 Mb.     

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

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

A full set of monosomic addition lines in Beta vulgaris from Beta webbiana: morphology and isozyme markers

Summary Nine different monosomic additions in Beta vulgaris from Beta webbiana were characterized through morphological characters and isozyme markers. The effect of the alien chromosome on the morphology of the recipient species is chromosome specific, and nine morphotypes could be distinguished. The added chromosome caused a growth reduction in the recipient plants. Eleven isozyme systems were used as marker systems. A 6PGDH band was found as a marker for chromosome 7, which contains a resistance gene for the beet cyst nematode in monosomic additions from Beta procumbens and Beta webbiana. A difference in the 6PGDH zymogram pattern between the two species with respect to this chromosome has been noted.     

Monosomic additions in beet (Beta vulgaris) carrying extra chromosomes of Beta procumbens

Summary Eleven isozyme systems were used to identify the extra chromosomes, originating from Beta procumbens, in progenies of 33 monosomic additions in beet (B. vulgaris). Nine groups of monosomic additions could be distinguished, representing the nine different chromosome types of B. procumbens.     

High-resolution mapping of YACs and the single-copy gene Hs1pro−1 on Beta vulgaris chromosomes by multi-colour fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful approach for physical mapping of DNA sequences along plant chromosomes. Nematode-resistant sugar beets (Beta vulgaris) carrying aBeta procumbens translocation were investigated by FISH with two differentially labelled YACs originating from the translocation. At mitotic metaphases, the translocation was identified with both YACs in the terminal region on a pair of chromosomes. Meiotic chromosomes, representing a far more extended hybridization target, were used to determine the orientation of YACs with respect to chromosomal domains in combination with chromosomal landmark probes for telomeres and centromeres. The in situ detection of plant single-copy sequences is technically difficult, and the wild beet translocation was used to explore the potential resolution of the FISH approach and to introduce the chromosomal mapping of single-copy genes into genome analysis of Beta species. An internal fragment of the nematode resistance gene Hs1 ^pro–1, 684 bp long, was detected on both chromatids of different Beta chromosomes and represents one of the shortest unique DNA sequences localized on mitotic plant chromosomes so far. Comparative chromosomal mapping of the 684 bp Hs1 ^pro–1 probe in the translocation line, a monosomic addition line and in B. procumbens revealed the origin of the wild beet translocation leading to nematode-resistant sugar beets.     

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 pat-1) on chromosome 1 of B. patellaris

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 ^pat-1. Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11₈₀₀, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11₈₀₀, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11₈₀₀ loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 ^pat-1 locus. Two copies of the middle-repetitive OPX2₁₁₀₀ marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.     

Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 pat−1

A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 ^pat-1 conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 ^pat-1 locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700–800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster.Abbreviations YAC Yeast artificial chromosome - BCN beet cyst nematode - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.     

A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes

We have used a standard protocol established for human chromosomes to create a chromosome-specific plasmid library from a Beta patellaris chromosome conferring nematode resistance. A monosomic addition line was chosen carrying 18 sugar-beet (Beta vulgaris L.) and one wild-beet (B. patellaris) chromosome. The wild-beet chromosome can readily be identified as a univalent during metaphase I of meiosis. Highly synchronized meiotic material was used to excise the univalents from four pollen mother cells. The chromatin was lysed in a 1 nl collection drop, the DNA purified and restricted with Rsa I, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. The amplified DNA was inserted into a standard plasmid vector and cloned. Approximately 23 000 recombinant plasmids were obtained of which 15 800 could be utilized. Their insert sizes ranged from 80 to 700 bp with an average of 130 bp. 61 clones were tested in more detail by genomic Southern hybridization with sugar-beet and wild-beet DNA. Of these 32 plasmids (52%) contained single-copy inserts, 11 (18%) were specific for wild-beet DNA indicating that the DNA cloned originates in the univalent chromosome. The application of this technique for establishing high-density RFLP maps for discrete regions of plant genomes is discussed.     

Monosomic additions in beet (Beta vulgaris) carrying extra chromosomes of B. procumbens

Summary Alien monosomic additions in beet (Beta vulgaris), each carrying one of the nine chromosomes of B. procumbens, were grown in vivo and in vitro to study the effect of the alien chromosomes on plant development. All additional chromosomes caused a reduction of the growth rate in vivo, which, in one case was so strong that some of the plants died as seedlings. In general, the morphological plant characteristics were not very useful to distinguish the addition types; this could have been the results of the wide variation in the recipient parent. However, some developmental characteristics proved to be highly chromosome-specific; for plants in vivo this was annuality, in combination with early or late flowering. If grown in vitro, chromosome specificity was observed for growth type (rosette or elongated stem), occurrence and rate of vitrification, occurrence and morphology of wound callus, formation of additional meristems on the midribs of leaves, formation of roots and a specific reaction to benzylaminopurine (BAP) the medium. Two chromosome types of B. procumbens caused resistance to the beet cyst nematode.     

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.     

Construction of a binary BAC library for an apomictic monosomic addition line of<Emphasis Type="Italic"> Beta corolliflora</Emphasis> in sugar beet and identification of the clones derived from the alien chromosome

A plant-transformation-competent binary BAC library was constructed from the genomic DNA of the chromosome 9 monosomic addition line of Beta corolliflora Zoss. in sugar beet (B. vulgaris. L). This monosomic addition line (designated M14) is characterized by diplosporic reproduction caused by the alien chromosome carrying the gene(s) responsible for diplospory. The library consists of 49,920 clones with an average insert size of 127 kb, representing approximately 7.5 haploid genome equivalents and providing a greater than 99% probability of isolating a single-copy DNA sequence from the library. To develop the scaffold of a physical map for the alien chromosome, B. corolliflora genome-specific dispersed repetitive DNA sequences were used as probes to isolate BAC clones derived from the alien chromosome in the library. A total of 2,365 positive clones were obtained and arrayed into a sublibrary specific for B. corolliflora chromosome 9 (designated bcBAC-IX). The bcBAC-IX sublibrary was further screened with a subtractive cDNA pool generated from the ovules of M14 and the floral buds of B. vulgaris by the suppression subtractive hybridization method. One hundred and three positive binary BACs were obtained, which potentially contain the genes of the alien chromosome specifically expressed during the ovule and embryo development of M14, and may be associated with apomictic reproduction. Thus, these binary BAC clones will be useful for identification of the genes for apomixis by genetic transformation.Communicated by H. C. Becker     

Monosomic addition lines of Beta corolliflora Zoss in sugar beet: cytological and molecular-marker analysis

Beta corolliflora is a wild relative of sugar beet (Beta vulgaris) with 2n=4x=36 chromosomes. Monosomic addition lines (2n=19) of B. corolliflora in B. vulgaris were identified from backcross progenies between triploid hybrids (genome constitution VVC) and sugar beet. They were characterized by DNA-fingerprinting using nine different B. corolliflora-specific repetitive sequences as probes and by fluorescence in situ hybridization (FISH) using two B. corollifora specific sequences and two rDNA probes. Unique banding patterns obtained after genomic Southern hybridization enabled the classification of monosomic addition lines into 11 clusters, three of which proved to have a wild beet chromosome fragment in addition to the sugar beet chromosomes as revealed by FISH. Repetitive sequences pBC216 and pBC1416 were found to be present only on wild beet chromosomes IV and V. Chromosomes I and IV were found to carry genes for 18S and 5S rRNA, respectively. An idiogram of B. corolliflora was established in the triploid VVC hybrid on the basis of chromosome size and FISH. Eight B. corolliflora addition lines could be unequivocally identified by Southern hybridization and FISH, one addition line carrying the missing wild beet chromosome is probably not viable under greenhouse conditions. The monosomic addition lines will serve as a bridge for transferring genes from wild species to sugar beet and will help to uncover genetic relationships between species of the genus Beta.     

Long-range organization of a satellite DNA family flanking the beet cyst nematode resistance locus (Hs1) on chromosome-1 of B. patellaris and B. procumbens

New members of a satellite DNA family (Sat 121), specific for wild beets of the section Procumbentes of the genus Beta, were isolated. Sequence analysis showed that the members of Sat-121 fall into two distinct classes. The organization of Sat-121 in the vicinity of a beet cyst nematode (Heterodera schachtii Schm.) resistance locus (Hs1) in B. patellaris and B. procumbens was investigated by pulsed-field gel electrophoresis (PFGE) using DNA from a series of resistant monosomic fragment additions, each containing an extra chromosome fragment of B. patellaris chromosome-1 (pat-1) in B. vulgaris. In this way several clusters of Sat-121 flanking the Hs1 ^pat-1 locus were identified. In nematode resistant diploid introgressions (2n=18), which contain small segments of B. procumbens chromosome-1 (pro-1) in B. vulgaris, only two major Sat-121 clusters were detected near the Hs1 ^pro-1 locus.     

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.     

Construction of Beta procumbens-specific DNA probes and their application for the screening of B. vulgaris x B. procumbens (2n = 19) addition lines

Summary Beta procumbens-specific DNA probes have been constructed by cloning digested total DNA in E. coli and screening the resulting recombinant plasmids in dot blot hybridizations with labelled B. procumbens and B. vulgaris DNA. Four clones (pTS1-4) have been analyzed in detail determining their degree of specificity and DNA sequence. Two clones (pTS1 and pTS2) with the highest degree of B. procumbens specificity were adapted for the squash dot hybridization with the aim of screening large numbers of individual hybrid plants (B. vulgaris x B. procumbens) carrying an alien B. procumbens chromosome (2n = 19). These addition lines carry in some cases B. procumbens resistance genes to the beet cyst nematode (Heterodea schachtii Schm.).     

Characterization of nematode resistance genes in the section Procumbentes genus Beta: response to two populations of Heterodera schachtii

Three species of the section Procumbentes genus Beta, nine monosomic additions, and five translocation lines were tested for resistance to two Heterodera schachtii populations. Nematode population 129-v (129-virulent) was selected for virulence to resistance gene(s) transferred from chromosome 1 of Beta procumbens to the diploid resistant sugar beet KWS-NR1. This population is considered to be a pathotype. The unselected sib population 129-av (129-avirulent) was reared continuously on fodder rape, Brassica napus cv Velox. Monosomic additions with chromosome 1 from the three species of the section Procumbentes were susceptible to population 129-v, regardless of the origin of the alien chromosome. Translocations with a gene(s) for resistance from chromosome 7 of B. procumbens and B. webbiana were also susceptible to the pathotype. However, a monosomic addition with chromosome 7 of B. webbiana was resistant to population 129-v. The three wild beets of the section Procumbentes, Beta procumbens, Beta webbiana and Beta patellaris, also were highly resistant to the two populations. The results indicate the existence of just two different major genes for resistance to H. schachtii in the entire Procumbentes section.     

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.     

Comparison of chloroplast and mitochondrial DNA from five morphologically distinct Beta vulgaris cultivars: sugar beet,fodder beet,beet root,foliage beet,and Swiss chard

Summary Two cytoplasms, N and S, are used in the breeding of sugar beet, Beta vulgaris var. altissima. These cytoplasms can be distinguished by their mitochondrial DNA. In an attempt to detect new cytoplasms, we compared the restriction profiles of chloroplast and mitochondrial DNA from five different cultivars of Beta vulgaris. All restriction patterns of chloroplast DNA were identical. With the exception of sugar beet with S-cytoplasm, all cultivars studied showed the same restriction profile of mitochondrial DNA, indicating that these cultivars all contain the N-cytoplasm. These results are discussed with regard to the large morphological differences of the cultivars and the cytoplasmic variability found in natural populations of the wild beet, Beta maritima.     

Distribution and evolution of two satellite DNAs in the genus Beta

Summary EcoRI monomers of a highly repetitive DNA family of Beta vulgaris have been cloned. Sequence analysis revealed that the repeat length varies between 157–160 bp. The percentage of AT-residues is 62% on average. The basic repeat does not show significant homology to the BamHI sequence family of B. vulgaris that was analyzed by us earlier. Both the EcoRI and BamHI sequences are investigated and compared to each other with respect to their genomic organization in the genus Beta. Both repeats were found to be tandemly arranged in the genome of B. vulgaris in a satellite-like manner. The EcoRI satellite DNA is present in three sections (Beta, Corollinae and Nanae) of the genus, whereas the BamHI satellite DNA exists only in the section Beta. The distribution of the EcoRI and BamHI satellite families in the genus is discussed with respect to their evolution.