Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.     

A Family of Differentially Amplified Repetitive DNA Sequences in the Genus Beta Reveals Genetic Variation in Beta vulgaris Subspecies and Cultivars

Members of a highly abundant restriction satellite family have been isolated from the wild beet species Beta nana. The satellite DNA sequence is characterized by a conserved RsaI restriction site and is present in three of four sections of the genus Beta, namely Nanae, Corollinae, and Beta. It was not detected in species of the evolutionary old section Procumbentes, suggesting its amplification after separation of this section. Sequences of eight monomers were aligned revealing a size variation from 209 to 233 bp and an AT content ranging from 56.5% to 60.5%. The similarity between monomers in B. nana varied from 77.7% to 92.2%. Diverged subfamilies were identified by sequence analysis and Southern hybridization. A comparative study of this repetitive DNA element by fluorescent in situ hybridization and Southern analyses in three representative species was performed showing a variable genomic organization and heterogeneous localizations along metaphase chromosomes both within and between species. In B. nana the copy number of this satellite, with some 30,000 per haploid genome, is more than tenfold higher than in Beta lomatogona and up to 200 times higher than in Beta vulgaris, indicating different levels of sequence amplification during evolution in the genus Beta. In sugar beet (B. vulgaris), the large-scale organization of this tandem repeat was examined by pulsed-field gel electrophoresis. Southern hybridization to genomic DNA digested with DraI demonstrated that satellite arrays are located in AT-rich regions and the tandem repeat is a useful probe for the detection of genetic variation in closely related B. vulgaris cultivars, accessions, and subspecies. Received: 24 May 1996 / Accepted: 13 September 1996     

High-resolution mapping of repetitive DNA by in situ hybridization: molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens

Members of three prominent DNA families of Beta procumbens have been isolated as Sau3A repeats. Two families consisting of repeats of about 158 bp and 312 bp are organized as satellite DNAs (Sau3A satellites I and II), whereas the third family with a repeat length of 202 bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differentially labelled repeats. The physical order of satellite subfamily arrays along a chromosome was visualized and provided evidence that large arrays of plant satellite repeats are not contiguous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau3A satellite family I. It is known that arrays of Sau3A satellite I repeats are tightly linked to a nematode (Heterodera schachtii) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau3A satellite II were found in centromeric regions of 16 chromosomes and, in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202 bp repeats is uniformly distributed over all chromosomes and largely excluded from the rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes 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.     

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.     

Genetic evidence for the origin of Californian wild beets (genus Beta)

The genus Beta L. is a morphologically and genetically variable group composed of wild, weedy, and domesticated forms that are used for sugar production or as vegetables. In this study, we have evaluated genetic variation in 64 germplasm accessions of wild and domesticated beets and examined the origin of wild beet accessions in California using allozyme analysis. UPGMA analysis showed overall that domesticated and wild beets form genetically coherent groups. Wild beets in California have two different origins, from European Beta vulgaris or from Beta macrocarpa. Population-level patterns of allozyme variation for wild California beets related to B. vulgaris suggest that those populations evolved from naturalized populations of the cultivated B. vulgaris ssp. vulgaris which had hybridized to varying degrees with the sea beets B. vulgaris ssp. maritima. Wild California beets related to B. macrocarpa are essentially genetically identical to European accessions. In addition, we found substantial evidence for hybridization and introgression of B. vulgaris alleles in one B. macrocarpa accession in California. The obligate outcrosser B. vulgaris exhibits more allelic diversity than the self-compatible B. macrocarpa. Beta vulgaris ssp. maritima exhibits more genetic diversity than domesticated B. vulgaris ssp. vulgaris. Received: 2 November 1998 / Accepted: 29 April 1999     

High frequency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and plant regeneration via direct shoot formation from leaf explants in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and <Emphasis Type="Italic">Beta maritima</Emphasis>

We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.     

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.     

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     

Chloroplastic and nuclear diversity of wild beets at a large geographical scale: Insights into the evolutionary history of the Beta section

Historical demographic processes and mating systems are believed to be major factors in the shaping of the intraspecies genetic diversity of plants. Among Caryophyllales, the Beta section of the genus Beta, within the Amaranthaceae/Chenopodiaceae alliance, is an interesting study model with species and subspecies (Beta macrocarpa, Beta patula, Beta vulgaris maritima and B.v. adanensis) differing in geographical distribution and mating system. In addition, one of the species, B. macrocarpa, mainly diploid, varies in its level of ploidy with a tetraploid cytotype described in the Canary Islands and in Portugal. In this study, we analyzed the nucleotide diversity of chloroplastic and nuclear sequences on a representative sampling of species and subspecies of the Beta section (except B. patula). Our objectives were (1) to assess their genetic relationships through phylogenetic and multivariate analyses, (2) relate their genetic diversity to their mating system, and (3) reconsider the ploidy status and the origin of the Canarian Beta macrocarpa.     

High yields of cytoplasts from protoplasts of Lolium perenne and Beta vulgaris using gradient centrifugation

Methods for the isolation of cytoplasts from suspension culture-derived protoplasts of the monocot Lolium perenne (perennial ryegrass) and the dicot Beta vulgaris (sugarbeet) have been determined. After comparing a range of gradients it was found that a discontinuous sucrose/mannitol gradient gave the highest cytoplast yields for both species tested: of the protoplasts loaded onto the gradient, for Beta >30% and for Lolium up to 45% could be recovered as cytoplasts. Sufficient protoplasts could be loaded onto the gradient to produce suitable numbers of cytoplasts for use in asymmetric somatic hybridisation experiments. Cytoplasts could be isolated from several suspension cultures of different ages. The cytoplast fraction was recovered from the upper part of the gradient in all cases and was only slightly contaminated (2–8%) with protoplasts. Lolium cytoplasts were small, evacuolate cells with granular cytoplasm. In contrast, Beta cytoplasts were larger and predominantly vacuolate. Both contained mitochondria as determined using fluorescence staining.Abbreviations 2,4-d 2,4 dichlorophenoxyacetic acid - M mannitol - S sucrose - P Percoll - S/M sucrose/mannitol gradient     

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.     

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.     

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.     

Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima

Summary The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349–352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168–169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.     

Structure Analysis of Two <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="BoldItalic">Neospora caninum</Emphasis> Satellite DNA Families and Evolution of Their Common Monomeric Sequence

A family of repetitive DNA elements of approximately 350 bp—Sat350—that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1) an arrangement of AB1CB2 350-bp repeats and (2) an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database ( ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.     

Restriction enzyme analysis of a highly diverged satellite DNA from Drosophila nasutoides

The satellite II DNA of Drosophila nasutoides is a highly diverged repetitive DNA, showing about 17% base changes between repeat units (Cordeiro-Stone and Lee, 1976). This DNA is cleaved by four different restriction enzymes to produce multimeric fragmentation patterns, indicating that their restriction sites are regularly arranged. Moreover, all four enzymes produce identical fragment lengths, the size of a monomer being 96 base pairs. Such multimeric patterns are expected for a diverged repetitive DNA, since many restriction sequences could have undergone changes during sequence divergence. Further restriction analyses of this DNA by double digestions and cloning reveal that there are three different sequences in satellite II DNA with respect to the presence and the arrangement of various restriction sites (Fig. 7). As an example, one sequence contains many EcoRI sites and fewer Hinfl sites (20% of EcoRI sites), which are arranged regularly. These observations suggest that satellite II DNA of D. nasutoides might have evolved through different modes of sequence divergence.     

Structure and variability of nuclear ribosomal genes in the genus Helianthus

Summary The restriction map of the rDNA unit of Helianthus annuus was constructed using EcoRI, BamHI, HindIII, KpnI and SacI restriction enzymes. Variations in this map among 61 ecotypes representing 39 species of the genus Helianthus were analyzed. The sizes of the rDNA unit ranged from 9.8 to 11.0 kbp, due to a length-repeat heterogeneity of the external non-transcribed spacer by increments of 200 base pair segments. Lengthrepeat heterogeneity and restriction polymorphism were found to be characteristic of populations or species of Helianthus. Restriction patterns and thermal melting with probes of a cloned H. annuus ENTS segment allowed us to differentiate species from each other. However, most lines of the cultivated sunflower were found to be identical on the basis of the physical properties of their ribosomal DNA.     

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species

Summary Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1–3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A — H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B — H. leporinum; Map C — H. vulgare, H. spontaneum, H. agriocrithon; Map D — H. chilense, H. bogdanii; and Map E — remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.Contribution No. 1169, Plant Research Centre