DNA fingerprinting in sugar beet (Beta vulgaris) — identification of double-haploid breeding lines

Summary The distribution and abundance of simple repetitive sequences complementary to the synthetic oligonucleotides (GACA)₄, (GATA)₄, (GTG)₅ and (CA)₈ in the genomes of several cultivars of Beta vulgaris and in the wild beet B. vulgaris ssp. maritima were investigated. Hybridization experiments revealed that all four motifs were present, though at different abundances, in the genomes of all of the investigated beet cultivars. Considerable intraspecific variation of the resulting DNA fingerprints was observed. The extent of polymorphism depends on the oligonucleotide probe. The most informative banding patterns were obtained with the (GATA)₄ probe hybridized to HinfI-, HaeIII-, or RsaI-restricted DNA, respectively. DNA fingerprinting with (GATA)₄ allowed a clear differentiation of double-haploid breeding lines (DH lines). We demonstrated that the application of oligonucleotide probes for DNA fingerprinting is a sensitive tool for genome diagnosis in cultivated beet.     

DNA fingerprinting in rice using oligonucleotide probes specific for simple repetitive DNA sequences

In this report we describe the use of five oligonucleotide probes, namely (GATA)₄, (GACA)₄, (GGAT)₄, (GAA)₆ and (CAC)₅, to reveal highly polymorphic DNA regions in rice. With each of the oligonucleotide probes, the level of polymorphism was high enough to distinguish several rice genotypes. Moreover, individual plants of one cultivar showed the same cultivar-specific DNA fingerprint. The multilocus fingerprint patterns were somatically stable. Our study demonstrates that microsatellite-derived DNA fingerprints are ideally suited for the identification of rice genotypes. As the majority of the probes detected a high level of polymorphism, they can be very useful in monitoring and aiding gene introgression from wild rice into cultivars.     

The organization of the evolutionarily conserved GATA/GACA repeats in the mouse genome

Simple repeated GATA and GACA sequences which were originally isolated from sex-specific snake satellite DNA have been found subsequently in all eukaryotes studied. The organization of these sequences within the mouse genome was investigated here by using synthetic oligonucleotide probes as a novel tool in comparison with conventional hybridization probes. Southern blot hybridization showed sex-specific patterns with both the (GATA)₄ and (GACA)₄ oligonucleotide probes, as previously described with conventional probes. The quantitative analysis of two mouse DNA phage libraries and of 25 isolated GATA-positive phage clones revealed intensive interspersion of GATA sequences with GACA, and with other repetitive and single-copy sequences. Ubiquitous interspersion and homogeneous genomic distribution of GATA and GACA sequences were confirmed by hybridization in situ of the oligonucleotide probes to metaphase chromosomes. The lengths of the GATA and GACA stretches were found to vary considerably in the individual phage clones. DNA inserts from 20 phages were assigned to autosomes and sex chromosomes and three genomic fragments were found to be confined to the Y chromosome. The organization of GATA and GACA sequences is discussed in the context of their evolutionary potential and possible conservation mechanisms.     

Microsatellite fingerprinting in the genus Phaseolus.

The genetic variability of the genus Phaseolus was investigated by nonradioactive DNA fingerprinting. The simple repetitive sequences (GATA)4, (GACA)4, (CAC)5, and (CA)8 were used as probes to differentiate 18 species comprised of 90 genotypes. (GATA)4, (CAC)5, and (CA)8 could be detected in the genome of nearly all species, while the (GACA)4 motif occurred only in 13 species. Almost all fragments that hybridized with (GACA)4 also hybridized with (GATA)4. All but two cultivars of Phaseolus vulgaris, P. lunatus, P. acutifolius, and P. polyanthus showed specific banding patterns with (GATA)4. The other repetitive motifs revealed only limited or no intraspecific variation. In P. vulgaris, two group-specific patterns were found with (GATA)4, giving further evidence for a Middle American and an Andean origin of the P. vulgaris genotypes. The high intraspecific pattern variation that was revealed with (GATA)4 in the predominantly self-pollinating species P. vulgaris and P. lunatus can probably be explained by there being at least two primary centres of domestication and, hence, genetic diversification. In cross-pollinating species (e.g., P. coccineus), the observed intraspecific variation was, surprisingly, rather low. The present study shows that DNA fingerprinting with microsatellites successfully distinguishes among gene pools, cultivars, and, in some cases, among individuals.     

Molecular cloning and analysis of one member of a polymorphic family of GACA-hybridising DNA repeats in tomato

Simple sequence repeat oligonucleotides were used to probe the tomato genome for elements displaying variability amongst commercial cultivars. The oligonucleotide (GACA)₄ was found to be particularly informative on genotype screening blots, hybridising to a highly polymorphic family of elements, and was used to clone one such member from a lambda library. The GACA-hybridisation was localised to a 1.3-kbHinfI fragment within the original 15-kb lambda insert. This 1,349-bp subclone (pT-GACA-2:1.3) was used to probe 27 Californian processing varieties and found to be capable of distinguishing all from each other, thus demonstrating its utility as a genetic fingerprinting probe for cultivar identification. Hybridisation occurred to approximately 10 major high molecular weight (> 4-kb) bands, most of which segregated independently in F₂ populations, as well as a large number of less clearly resolvable smaller fragments. Sequence analysis of the cloned element reveals that it is almost entirely composed of GACA or GATA repeats. These tetranucleotides are organised into distinct repetitive domains, consisting either of tandem arrays of each tetranucleotide or interspersions of GACA and GATA to form dodecanucleotides that are then further repeated. The boundaries between domains contain sufficient departures from the concensus repeat to allow construction of unique polymerase chain reaction (PCR) primers. Amplification from two such contiguous regions identifies length variation in both, thus yielding a genotype screen appropriate for high-throughput applications, such as assessment of purity in F₁ hybrid seed lots.     

The contribution of short repeats of low sequence complexity to large conifer genomes

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC)₅ and (GACA)₄ are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus. The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG)_n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine (Pinus elliottii Engelm. var. elliottii), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA)₈, (GA)₈, (GGAT)₄ and (GATA)₄, and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated. Received: 1 October 1999 / Accepted: 3 November 1999     

Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences

Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically demonstrable sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)_n/(gaca)_m are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)₄ probe. The (GATA)₄ oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In contrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)₄. Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)₄ probe.by E.R. SchmidtDedicated to Professor Karl Sperling on the occasion of his 50th birthday     

Simple GA C T A repeats characterize the X chromosomal heterochromatin of Microtus agrestis,European field vole (Rodentia,Cricetidae)

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GA _C ^T A probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)_n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.     

Increased detectability of somatic changes in the DNA from human tumours after probing with “synthetic” and “genome-derived” hypervariable multilocus probes

Summary DNA fingerprinting with two minisatellite (33.15, M13) and two simple repeat probes [(GACA)₄, (CAC)₄/ (GTG)₅] was performed to screen for somatic changes in the DNA from various solid human tumours in comparison with constitutional DNA from the same patient. Loss of bands or changes in band intensitities were observed. Together the probes 33.15 and (CAC)₅/(GTG)₅ detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)₄ and (CAC)₅/(GTG)₅.     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori , and their relative discriminatory abilities were assessed. Four probes, (GACA)₄, (GT)₈, (GTG)₅ and (GGAT)₄, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)₄-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori .     

Differential abundance of simple repetitive sequences in species ofBrassica and relatedBrassicaceae

SixBrassica species, known as the triangle of U, and four species from related genera were characterized by DNA fingerprinting with simple repetitive oligonucleotide probes. Our results show that CT-, TCC-, and GTG-repeat motifs are equally abundant in the genomes of the sixBrassica species. In contrast, GATA-, GGAT-, and GACA-multimers are unevenly distributed among different species. As judged from the number and strength of hybridization signals, the highest copy number of all three motifs occurs inBrassica nigra, while the lowest is observed inB. oleracea. The abundance of GATA-and GACA-repeats varies in a coordinate way. The amphidiploid genomes ofB. juncea, B. carinata, andB. napus each harbour intermediate amounts of (GATA)₄ and (GACA)₄-detected repeats as compared to their diploid progenitors, thus supporting the concept of the U triangle. GATA-, GACA-, and GGAT-repeats were also abundant inEruca sativa andSinapis arvensis, but not inRaphanus sativus andSinapis alba. These results support the idea thatBrassica nigra is more closely related toSinapis arvensis than to otherBrassica species such asB. rapa andB. oleracea.     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

Random amplification of polymorphic DNA with conserved sequences reveals genome-specific monomorphic amplicons: Implications in clad identification

The enzymatic amplification of genomic DNA with an arbitrary primer generates informative band profile useful for genome analysis. We used a set of synthetic oligodeoxyribonucleotide primers OAT15.2 (GACA)_3.75, OAT18. 2 (GACA)_4.5, OAT24.2 (GACA)₆, OAT36 (GACA)₉, comprising 4–9 consecutive units of GACA repeat, O33.15 (CACCTCTCCACCTGCC) and 033.6 (CCTCCAGCCCTCCTCCAGCCCT) for RAPD reactions of genomic DNA from different sources. The GACA based oligos of 15 and 18 base residues generated discernible genome specific amplicons whereas primers larger than 18 bases revealed smeary signals. The other oligos O33.15 and O33.6 also generated genome specific amplicons with more bands compared with those obtained from OAT15.2 or OAT18.2. The presence of OAT15.1 (GATA)_3.75 and OAT15.2 (GACA)_3.75 sequences in different genomes were ascertained by independent dot-blot hybridization prior to using them for RAPD reactions. The RAPD amplicons generated by evolutionarily conserved primer(s) or sequences shared by many species may be useful for clad identification in controversial systematics, comparative genome analysis, and for establishing the phylogenetic status of an organism.     

Chromosomal Location of Ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n Telomeric Repeats in the Neogastropod Fasciolaria Lignaria (Mollusca: Prosobranchia)

This paper reports on a successful application of fluorescent in situhybridization (FISH) with three repetitive DNA probes (ribosomal DNA (rDNA), (GATA)_nand (TTAGGG)_n) in the chromosomes of Fasciolaria lignaria(Mollusca: Prosobranchia: Neogastropoda). rDNA FISH consistently identified four chromosome pairs per spread in the three examined specimens. The telomeric sequence (TTAGGG)_nhybridized with termini of all chromosomes. GATA FISH revealed abundant, dispersed minisatellite regions which were not associated to the XY sex-determining mechanism as indicated by the absence of a Y specific pattern of labelling. This revised version was published online in July 2006 with corrections to the Cover Date.     

Simple repetitive sequences are associated with differentiation of the sex chromosomes in the guppy fish

Summary Hybridization of restriction enzymedigested genomic guppy (Poecilia reticulata, Poeciliidae) DNA with the oligonucleotide probe (GACA)₄ revealed a male-specific simple tandem repeat locus, which defines the Y chromosome in outbred populations. The related (GATA)₄ probe identifies certain males with the red color phenotype. In contrast only in two out of eight laboratory guppy strains was the typical (GACA)₄ band observed. By specific staining of the constitutive heterochromatin one pair of chromosomes could also be identified as the sex chromosomes, confirming the XX/XY mechanism of sex determination. All males exhibit Y chromosomes with a large region of telomeric heterochromatin. Hybridization in situ with nonradioactively labeled oligonucleotide probes localized the (GACA)_n repeats to this heterochromatic portion. Together these results may be regarded as a recent paradigm for the differentiation of heteromorphic sex chromosomes from a pair of autosomes during the course of evolution. According to the fish model system, this may have happened in several independent consecutive steps.     

Detection of genetically unstable loci in parthenogenic families of lizards of theLacerta genus by DNA fingerprinting

Two parthenogenic families of unisexual species of Caucasian rock lizards of genusLacerta, L. armeniaca andL. unisexualis, were analyzed by DNA fingerprinting. Inheritance of M13 minisatellite and of (GACA) _n , (GATA) _n , and (TCC) _n microsatellite loci in the first generation of the lizards was studied. M13, (GACA) _n , and (TCC) _n loci in the families ofL. armeniaca were strictly inherited, as well as M13 and (GACA) _n loci in the families ofL. unisexualis: each DNA fragment in the fingerprint patterns of progeny could be detected in the maternal pattern. However, when a (TCC)₅₀ microsatellite probe was applied in the study ofL. unisexualis families, specific DNA fragments with altered mobility were revealed in the progeny patterns, and the frequency of such events was rather high. It might be hypothesized that some of the (TCC) _n loci inL. unisexualis genome are highly mutable. Hence, the family analysis allowed us to demonstrate experimentally the presence of genetically unstable loci in genomes of parthenogenic species of vertebrates. The nature and mechanism of the instability of these loci in parthenogenesis remain obscure.     

RFLP analyses of severalBrassica representatives

Molecular variability was determined between, 17Brassica napus cultivars, 8 new selections 4 F₁ hybrids, 5 cultivars ofB. oleracea, B. campestris andB. peruvianum with 4 rDNA probes fromLycopersicon esculentum, 3 probes from a PstI library ofB. oleracea, 3 probes from EcoRI libraries ofB. napus andB. oleracea and the acetolactatsynthase gene fromNicotiana tabacum. Sporadic interspecific and rare intraspecific variability was detected. Contribution presented to the 5^th Czechoslovak Seminar “Plant Gene Engineering”, organized by the Institute of Plant Molecular Biology in České Budějovice, 2–13 September 1991.     

Analysis of VNTR loci in fish genomes using synthetic oligodeoxyribonucleotide probes

A set of synthetic oligodeoxyribonucleotide (oligo) probes, OAT18, OMS1 and OAT24 carrying the (TGG)₆, (GGAT)₄ and (GACA)₆ repeat motifs, respectively, was used to analyze the variable number tandem repeat (VNTR) loci in the genomes of Oncorhyncus mykiss (rainbow trout; family Salmonidae), Oreochromis mossambicus and Oreochromis niloticus (both tilapia belonging to family Cichlidae). Of all the oligos and enzymes (AluI, MboI, HaeIII and HinfI) used, the OAT18/HaeIII combination was found to be most informative for detecting DNA fingerprinting in rainbow trout, while the OMS1/MboI combination gave the most informative pattern for the Or. niloticus genome. In the rainbow trout genome, all three repeat loci were hypervariable, revealing varying degrees of polymorphism as compared to tilapia genomes. Startlingly, the OAT24 probe did not cross-hybridize with Or. mossambicus and lamprey salmon (Lampetra japonica) although GACA repeats have been reported to be evolutionarily conserved in all eukaryotes studied thus far. Cluster analysis with respect to GGAT repeat loci revealed that Or. niloticus diverged from Or. mossambicus before the separation of On. mykiss, suggesting the relatively recent evolution of these loci in rainbow trout, compared to the tilapia genomes. These highly informative probes will find application in various genetic studies of fishes.     

Heterogeneities in the distribution of (GACA)n simple repeats in the karyotypes of primates and mouse

Summary Tandemly organized simple repetitive sequences are widespread in all eukaryotes. The organization of the simple tetrameric (GACA)_n sequences at chromosomal loci has been investigated using in situ hybridization with chemically pure oligonucleotide probes. Both biotin- and digoxigenin-attached (GACA)₄ probes reveal specific hybridization signals over the short arms of all acrocentric autosomes in man. In the other examined primates the NOR-bearing autosomes could be detected by in situ hybridization with (GACA)₄, and a major concentration of the GACA simple repeats could be observed on the Y chromosome in the gibbon and mouse; the hybridization site in the gibbon Y chromosome coincides particularly with the silver-stainable NOR. In the past, accumulations of (GACA)_n sequences were demonstrated mainly on vertebrate sex chromosomes. Therefore, the organization of GACA simple sequences is discussed in the context of their evolutionary potential accumulation and the possible linkage with the primate rDNA loci.     

Evaluation of RAPD, ISSR and AFLP Markers for Characterization of the Loose Smut Fungus Ustilago tritici

Random Amplified Polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) profiling were evaluated for assessing the extent of genetic variation among the isolates of Ustilago tritici (Pers.) Rostr., which causes the loose smut disease of wheat.Thirty random decamer primers, six random primer pairs, four SSR primers such as (GACA)₄, (GATA)₄, (CAA)₅ and (GTG)₅ and nine combinations of AFLP selective primers were used to characterize nine isolates of the fungus. These isolates were collected from infected earheads of seven commercial wheat cultivars grown at eight different locations in Haryana, which is a major wheat growing state in the North-West Plain Zone of India. The RAPD and ISSR primers generated 21 0 scorable amplified fragments, all of which were monomorphic among the isolates.The AFLP primer combinations generated 239 fragments out of which 193 were polymorphic. All the isolates could be precisely differentiated from each other employing AFLP and grouped into two distinct clusters.The molecular classification partly corresponded with geographic distribution and host origin of the isolates. AFLP profiling was found superior to RAPD and ISSR and can be effectively utilized for further characterization of loose smut pathogen.