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.     

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.     

Phylogenetic relationships between cultivated and wild species of the genusBeta revealed by DNA “fingerprinting”

Forty-one accessions of the genusBeta representing wild and cultivated species of all sections were analyzed by DNA fingerprinting. Four sugar beet minisatellite DNA probes revealed characteristic banding patterns with Southern-hybridizedBeta DNA restricted withHindIII. A total of 111 polymorphic RFLP bands were scored across all accessions. Cluster analysis based on genetic similarity estimates for all 820 combinations of accessions revealed the following results. (1) All accessions could unambiguously be identified by a characteristic RFLP banding pattern. (2) The sugar beet cultivars examined displayed a low level of genetic diversity; they showed high similarity toB. Vulgaris ssp.maritima but low genetic similarity to the other wild species of section I. (3) In most cases, the present taxonomic classification of the genusBeta was confirmed. Species of sections II, III, and IV were clearly distinguishable from those of section I except forB. Macrocarpa, which showed high similarity to wild species of section II. In a second experiment, 108 single-copy RFLP probes from sugar beet were Southern hybridized withB. procumbens DNA. A surprisingly low degree of homology (34%) was found. The results are discussed with regard to the taxonomic classification of the genusBeta.     

Two different satellite DNAs in Beta vulgaris L.: evolution,quantification and distribution in the genus

Summary Two highly repeated EcoRI (0.45 × 10⁶) and BamHI (0.17 × 10⁶) fragments per haploid genome were found in sugar beet genomic DNA. Both fragments were located by 6% acrylamide-gel electrophoresis, purified and cloned in pUC18. Four of the inserts corresponding to each family were chosen for further study. Both fragment families display the main characteristics of the satellite DNA of animals and plants. The EcoRI and BamHI fragment families are arranged in long tandem arrays. Fragments of the EcoRI family (pBVE) were analyzed. They vary both in sequence and in length (158–160 nt) in comparison with the consensus sequence of 159nt. Both families are A-T rich; pBVE is 59% rich while pBVB is 69% rich. The BVESAT family is present in all the members of the section Vulgares. It is conserved in the section Procumbentes with 80% homology and the same length, but is not detectable in the Corollinae. The sequence variation rate and the variation in length (330±5 nt) are of the same order in comparison with those of the BVESAT family. However, the BVBSAT family is present in species of the section Vulgares only. As regards other plant satellite DNAs, the BVESAT family shares homology with Allium cepa satellite DNA, with three of the yeast centromeric sequences, and with three Arabidopsis thaliana sequences. The BVBSAT family is unique to the Vulgares and does not share any homology with other plant or animal satellite DNAs sequences so far.     

Cytoplasmic male sterility inBeta is associated with structural rearrangements of the mitochondrial DNA and is not due to interspecific organelle transfer

Summary Chloroplast (ct) and mitochondrial (mt) DNAs from four cytoplasmic male sterile (cms) and 22 normal fertile sugar beet lines and accessions of wild beets from the genusBeta have been compared with restriction analyses and Southern hybridizations. We have used restriction analyses of ctDNA as a phylogenetic marker to confirm the taxonomic relationships between the different cytoplasms. According to the ctDNA data, all four cms cytoplasms belong to the same taxonomic section,Beta. Restriction patterns of ct and mtDNA from fertile accessions produced analogous trees of similarity and showed a close correlation between the organellar DNA diversity and the accepted taxonomic classification of the species studied. However, the mtDNA restriction profiles of the four cms types differed dramatically from each other and from those of all fertile accessions from the genus. No indication of cytoplasmic introgression was found in any of the four investigated cms types. Southern hybridization to mtDNA revealed variant genomic arrangements in the different fertile and cms cytoplasms, indicating that rearrangement of the mitochondrial genome is a common denominator to the different cms systems inBeta. It may, indeed, be a common property to spontaneously occurring cms in all or most species.     

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.     

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     

Minicircle variation in Beta mitochondrial DNA

Summary Mitochondrial DNAs from nine male fertile and eight cytoplasmic male sterile (cms) accessions of wild and cultivated Beta beets were investigated for the presence of low molecular weight DNA molecules. Five different supercoiled DNA molecules were detected, varying in size from 1.33 to 1.63 kb. Southern hybridizations revealed multimeric forms and sequence homologies between the minicircles. The occurrence of the different minicircles among the 17 accessions was investigated by agarose gel electrophoresis and Southern hybridization using minicircle specific probes. The 1.33 and 1.63 kb minicircles were found in most accessions, the other three minicircles were found in one or two of the wild Beta beet accessions. The presence of a low number of small, more or less homologous, minicircles in all investigated plants makes these molecules a general characteristic of Beta mtDNA. No association is found between the presence or absence of specific minicircles and the expression of male sterility. Neither does the distribution of the different minicircles in Beta beets indicate any essential biological role of these minicircles.     

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.     

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.     

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.     

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.     

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.     

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

Key message

Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.

Abstract

The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R ² > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.     

Construction of a sugar beet BAC library from a hybrid with diverse traits

A bacterial artificial chromosome (BAC) library of the 750-Mbp sugar beet genome represented in hybrid US H20 was constructed fromHind III-digested DNA, with an average insert size of 120 kbp. US H20 is a variety grown in the eastern United States. It exhibits heterosis for emergence and yield, presumably because of its hybridity between eastern and western US germplasm sources. Filter arrays were used to assess the abundance and distribution of particular nucleotide sequences. An rRNA gene probe found that 1.2% of the library carried sequences similar to these highly repetitive and conserved sequences. A simple sequence repeat element (CA)₈ thought to be predominantly distributed throughout centromere regions of all chromosomes was present in 1.7% of clones. For more than half of the 28 randomly chosen expressed sequence tags (ESTs) used as probes, a higher-than-expected number of single-copy hybridization signals was observed. Assuming 6× genome coverage, this suggests that many duplicate genes exist in the beet genome.     

Conservation of a highly repeated DNA family of Aedes albopictus among mosquito genomes (Diptera: Culicidae)

Summary The genomic organization and chromosomal localization of a cloned 0.79-kb highly repeated DNA fragment, H-115, isolated from Aedes albopictus has been examined. The cloned fragment is a part of a larger unit of 1.86 kb that is tandemly repeated in the Ae. albopictus genome. The H-115 family of sequences are located at the intercalary position on chromosome 1 in Ae. albopictus. Similar patterns of in situ and Southern blot hybridization results are obtained in Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. Alcasidi, and Ae. katherinensis. The H-115 sequences are widely conserved in Culicidae and are found in Haemagogus equinus, Tripteroides bambusa, and Anopheles quadrimaculatus by hybridization under high stringency conditions. The H-115 sequences are also tandemly repeated in Hg. equinus with a monomer unit of 1.86 kb and in Tp. bambusa with a slightly diverged monomer unit of 1.90kb. In Anopheles quadrimaculatus, the H-115 sequences are dispersed throughout the genome. Partial sequence analysis shows that the H-115 insert is 62% AT and contains two perfect inverted repeats and numerous perfect direct repeats. The occurrence of inverted repeats with potential to form intrastrand palindromic structure suggests that the H-115 family of sequences may be involved in chromatin condensation.     

The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation

Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next‐generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome‐wide fluorescent in situ hybridization complemented with immunostaining and super‐resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44–52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.