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.     

Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patellaris and Beta procumbens.

Summary In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n =18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.     

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.     

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     

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.     

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     

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.     

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.).     

Cloning and sequence diversity analysis of <Emphasis Type="Italic">GmHs1</Emphasis><Superscript><Emphasis Type="Italic">pro-1</Emphasis></Superscript> in Chinese domesticated and wild soybeans

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is the most important pathogen in soybean production worldwide and causes substantial yield losses. An apparent narrow genetic base of SCN resistance was observed in current elite soybean cultivars, and searching for novel SCN resistance genes as well as novel resistance sources rather than focusing on the two important genes rhg1 and Rhg4 has become another major objective in soybean research. In the present paper we report a 1,477 bp Hs1 ^pro-1 homolog, named GmHs1 ^pro-1 . This gene was cloned from soybean variety Wenfeng 7 based on information for Hs1 ^pro-1 , a beet cyst nematode resistance gene in sugar beet. It has two domains, Hs1pro-1_N and Hs1pro-1_C, both of which are believed to confer resistance to nematodes. Of the 1,477 bp sequence in GmHs1 ^pro-1 , an open reading frame of 1,314 bp, encoding a protein with 437 amino acids, was flanked by a 5′-untranslated region of 27 bp and a 3′-untranslated region of 135 bp. Fourteen single-nucleotide polymorphisms (SNPs) were observed in 44 soybean accessions including 23 wild soybeans, 8 landraces, and 13 soybean varieties (or lines), among which 5 in wild soybeans and 3 in landrace accessions were unique. Sequence diversity analysis on the 44 soybean accessions showed π = 0.00168 and θ = 0.00218 for GmHs1 ^pro-1 ; landraces had the highest diversity, followed by wild soybeans, with varieties (or lines) having the lowest. Although we did not detect a significant effect of selection on GmHs1 ^pro-1 in the three populations, sequence diversity, unique SNPs, and phylogenetic analysis indicated a slight domestication bottleneck and an intensive selection bottleneck. High sequence diversity, more unique SNPs, and broader representation across the phylogenetic tree in wild soybeans and landraces indicated that wild collections and landrace accessions are invaluable germplasm for broadening the genetic base of elite soybean varieties resistant to SCN. C. Yuan and G. Zhou contributed to this paper equally.     

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.     

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.     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

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.     

Effects of Cycloate on Development of Heterodera schachtii and Growth of Three Beta Species

Greenhouse tests were set up to evaluate the effects of the herbicide, cycloate (S-ethyl cydohexylethylthiocarbamate), oil development of Heterodera schachtii and growth of three Beta species. Cycloate added to infested soil enhanced cyst development/gm root on B. vulgaris and larvae/gm of root in B. patellaris and B. procumbens at 4, 16, and 16 μg(a.i.)/gm of soil, respectively. Total numbers of nematodes/individual root system decreased because of poor root growth of seedlings in cycloate-amended soil. Penetration and larval development through stage three did occur in the wild Beta species in any treatment. Thus, resistance of B. patellaris and B. pocumbens to development of H. schachtii was not altered by cycloate. Cycloate also retarded growth (P = 0.05) of the sugarbeet cultivars and B. patellaris at 4 μg(a.i.)/gm and B. procumbens at 16 μg(a.i.)/gm of soil. Higher concentrations of nematodes/gm root in plants growing in cycloate-amended soil may be attributed to factors such as fewer roots available for penetration, possible effects of cycloate on egg hatch, greater attraction of nematodes to roots, and increased susceptibility of roots to larval penetration. Suppression of seedling growth in cycloate-amended soil may be attributed in part to higher nematode density and in part to direct root damage from cycloate.     

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.     

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.     

Backcrossing of nematode-resistant sugar beet: a second nematode resistance gene at the locus containing Hs1pro-1?

Beet cyst nematode-resistant sugar beet plants, containing the Hs1^pro-1 locus from Beta procumbens, show a female transmission frequency of the resistance of ca. 90%. Such plants often suffer from tumour formation on leaves and root systems, and from the occurrence of a so-called multi-top phenotype. With the aim of obtaining resistant sugar beet material lacking these negative traits, nematode-resistant plants with a reduced size of the chromosome segment of the wild beet that carries the Hs1^pro-1 gene were selected from backcrosses between the resistant stocks B883 or AN1-65-2 and susceptible sugar beet (Beta vulgaris). Analysis of such plants, referred to as Sat-minus plants, showed that the transmission frequency of the resistance to subsequent generations had dropped dramatically to ca. 0.5%. The multi-top phenotype was still present in the newly selected material, indicating that improvement of the resistant sugar beet material by further backcrossing will be hard to achieve. Two of the selected resistant offspring plants were analysed at the molecular level. With the aid of AFLP markers it was found that the size of the alien chromosome segment had decreased to 35% and 17% of the original size, respectively. Surprisingly, both plants had lost the Hs1^pro-1 nematode resistance gene that recently was isolated from the original introgression material. This shows that more than one gene conferring resistance must be present in the locus in B883 and AN1-65-2 carrying the resistance gene Hs1^pro-1.     

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.     

Genetical control and linkage relationships of isozyme markers in sugar beet (B. vulgaris L.)

Summary Five isozyme systems were genetically investigated. The different separation techniques, the developmental expression and the use as marker system in sugar beet genetics and breeding is discussed. Isocitrate dehydrogenase was controlled by two genes. The gene products form inter- as well as intralocus dimers, even with the gene products of the Icd gene in B. procumbens and B. patellaris. Adenylate kinase was controlled by one gene. Three different allelic forms were detected, which were active as monomeric proteins. Glucose phosphate isomerase showed two zones of activity. One zone was polymorphic. Three allelic variants, active as dimers, were found. Phosphoglucomutase also showed two major zones of activity. One zone was polymorphic and coded for monomeric enzymes. Two allelic forms were found in the accessions studied. The cathodal peroxidase system was controlled by two independent genes, of which only one was polymorphic. The gene products are active as monomers. Linkage was found between red hypocotyl color (R) and Icd ₂. Pgm ₁, Gpi ₂, Ak ₁ and the Icd ₂-R linkage group segregated independently.