Oligonucleotide fingerprinting of plant genomes

Electrophoresis 1991,12, 159-169

Kurt Weising Birgit €%eyermad Juliane Ramser’ Gunter Kahl’ Pflanzliche Molekularbiologie, Fachbereich Biologie, Johann Wolfgang Goethe-Universitat Frankfurt Wissenschaftsbereich Genetik, Sektion Biologie, HumboldtUniversitat Berlin

159

Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate “fingerprinting” probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of “DNA fingerprinting” was also introduced into the analysis and characterization of plant genomes, initially by using human or M 13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra- and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety-specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy-to-perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin-based labeling techniques proved to be of similar quality.

1 Introduction A substantial portion of the genomes of higher eukaryotes consists of repetitive DNA belonging to various classes. One class consists of short, tandemly arranged sequence motifs that form long, more or less homogeneous arrays. Polymers made up of very short motifs (2-5 base pairs; bp) are called “simple repetitive sequences” [ 1,2], whereas “minisatellites” are constructed from longer monomers [3, 41. A common feature of tandemly arranged repetitive sequences is their high degree of polymorphism, observed upon comparison of unrelated individual genomes [2-61. Whereas most of this variability results from different copy numbers of the basic motifs (variable number of tandem repeats, VNTR), internal heterogeneity of simple repetitive sequences [2, 71, as well as of minisatellites [ 81, was also observed. Hybridization of restriction-digested genomic DNA against a polymorphic minisatellite or a simple repetitive sequence motif often results in the simultaneous detection of several extremely variable loci and creates a “DNA fingerprint” 14, 5, 9, 101. Most VNTR probes characterized to date are derived from the human genome [3, 4, 111 and some of them were shown to cross-hybridize to a variety of mammalian (e.g. [ 121), avian (e.g. [ 13, 141), and even plant genomes [15,161. Surprisingly, .an internal repeat sequence from bacteriophage M 13 also detects hypervariable loci in a variety of organisms [ 17, 181. Consequently, human minisatellites,M 13repeats, and related sequences have been applied for DNA fingerprinting of a variety of species including humans and other mammals [3,4, 121, birds [13, 141, and, most recently, plants [ 15, 16, 19,201, fungi [21, 221 and even bacteria 1231. Simple repetitive sequences, on the other hand, although providing an almost unlimited source of potential probes for detecting hyperCorrespondence: Dr. Kurt Weising, Pflanzliche Molekularbiologie, Fachbereich Biologie, Johann Wolfgang Goethe-Universitat, Siesmayerstr. 70, DW-6000 Frankfurt/M, Germany Abbreviations: bp, base pair; CTAB, cetyltrimethylammonium bromide; TAE, Tris-acetate-EDTA buffer; TBE, Tris-borate-EDTA buffer; TE, Tris-EDTA buffer; VNTR,variable number of tandem repeats 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

variability [6,241, were not yet extensively used as probes for creating DNA fingerprints. Successful fingerprinting with probes complementary to simple repetitive motifs has been reported for humans [9,10,25,261, rodents [27,281, fish 129, 301, and yeast [311. Epplen and colleagues [9,101 developed a new technique of hybridizing labeled oligonucleotide probes to electrophoretically separated genomic DNA fragments fixed in dried agarose gels. This method has several advantages over the conventional blotting techniques: it is faster; prehybridization steps can be omitted, and a “ 100 % transfer efficiency” is obtained. Techniques for nonradioactive fingerprinting of the human genome with biotinylated or digoxigenated oligonucleotides have also been developed [ 10, 32-341.

In cooperation with Epplen’s group, we extended the use of oligonucleotide probes for DNA fingerprinting to the plant kingdom([35-37], thisreport). Wecoulddemonstrate theubiquitous presence of simple repetitive motifs in plant and fungal genomes. Oligonucleotide probes specific for these motifs were able to detect various levels of inter- and intraspecific polymorphism, depending on the combination of species and probe. We tested and optimized a fast and easy-to-perform DNA isolation procedure suitable for a large variety of plant and fungal species, and investigated the effects of different DNA purification protocols. Finally, we could demonstrate the applicability of nonradioactively labeled oligonucleotide probes for plant DNA fingerprinting.

2 Materials and methods 2.1 Plant material The plant species, cultivars and accessions used in our studies originated from the following sources. Fungi: in vitro grown mycelia of Phycomyces blakesleeanus were kindly supplied by Dr. T. Rausch (University of Frankfurt, Germany). Coprinus cornatus fruit bodies were collected in a forest near Frankfurt. Algae: Oedogonium spec. was collected from a pond at the Botanical Garden (University of Frankfurt). Stenogramtne 0 173-0835/91/02-302-3-0159 $3.50+.25/0

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interrupta was grown in a sea-water aquarium at the Botanical Institute, (UniversityofFrankfurt) and was kindly supplied by Prof. Dr. A. Ried. Higher plants: Banana plants (Musa acuminata) were harvested at the Palmengarten (Frankfurt). Lettuce (Lactuca sativa) was purchased at a local market. Dihaploid potato lines (Solanum tuberosum) were obtained from the Institute for Resistance Genetics (Griinbach, Germany). Different accessions of chickpea (Cicer arietinum), lentil (Lens culinaris), and wild barley (Hordeum spontaneum) were obtained from the collection of the ICARDA Institute (Aleppo, Syria). Different cultivars of rapeseed (Brassica napus) were provided by the Research Center for Plant Protection (Lyngby, Denmark). Two different lines of sugar beet (Beta vulgaris, var. altissima) were obtained from Dr. R. Melzer, Akademie der Landwirtschaft (Kleinwanzleben, Germany). Different species ofthe genus Nicotiana, different tobacco varieties (Nicotiana tabacum), as well as all other plant species used in the present study (mosses, ferns, gymnosperms and angiosperms) were from the Botanical Garden (University of Frankfurt). 2.2 DNA isolation and purification Among the different extraction procedures that we tested 138-4 1I, a modified cetyltrimethylammonium bromide (CTAB) procedure based on the protocols of Saghai-Maroof et al. [39] and Doyle and Doyle [41] turned out to be the method of choice for obtaining good quality total DNA from a large variety of plant species (Table 1). Subsequent purification steps were usually performedprior to restriction digestion of DNA in order to remove RNA and other contaminating substances.

2.2.1 Preparation of crude DNA Up to 3 g of fresh or 0.5 g of lyophilized plant material was ground in liquid nitrogen and dispersed in 15 mL of prewarmed (60" C) CTAB isolation buffer (2 % w/v CTAB, 1.4 M NaCl, 0.2 % 2-mercaptoethanol, 20 mMEDTA, 100 mMTrisHCl, pH 8.0). After 30 minofincubation at 60" C, the solution was extracted once with chloroform-isoamyl alcohol (24: 1) and centrifuged for 10 min and 10000 ga t room temperature. The aqueous phase was transferred to a new tube and nucleic acids were precipitated by adding 0.6 volumes of cold isopropanol. Precipitates were collected by centrifugation (30 min, 10 000 g, 4 "C) and washed once with 20 mL of washing buffer (76 % ethanol, 10 mM ammonium acetate) under gentle agitation for at least 20 min. Samples were then centrifuged as

Table I. Synopsis of results of DNA isolation from different species of fungi and plants using the modified CTAB procedurea). Species Fungi Phycomyces blakesleeanus (Zygomycetes) Saccharomyces cereuisiae (Ascomycetes) Ascochyta rabiei (Ascomycetes) Coprinus comatus (Basidiomycetes) Nematoloma sublateritium (Basidiomycetes) Algae Oedogonium spec. (Chlorophyceae) Spirogyra spec. (Chlorophyceae) Stenogramme interrupta (Rhodophyceae) Fucus serratus (Phaeophyceae)

Resultsb)

Liverworts and Mosses Marchantiapolymorpha (Hepaticae) Polytrichum formosum (Musci) Ferns Equisetum aruense (Equisetatae) Dodia caudata (Filicatae) Polypodium vulgare (Filicatae) Osmunda vulgaris (Filicatae) Nephrolepis exaltata (Filicatae) Gymnosperms Ephedra distachya (Gnetatae) Gingko biloba (Gingkoatae) Taxus baccata (Pinatae) Taxodium distichum (Pinatae) Thuiopsis dolabrata (Pinatae) Juniperus communis (Pinatae) Cryptomeria japonica (Pinatae) Monocotyledonous angiosperms Echirzodorus osiris (Alismataceae; Alismatidae) Musa acuminata (Musaceae; Liliidae) Hordeum vzdgare (Poaceae; Liliidae) Hordeum spontaneum (Poaceae; Liliidae) Asparagus densiporus (Asparagaceae; Liliidae) Dioscorea bulbijera (Dioscoreaceae; Liliidae) Cocos nucijera (Arecaceae; Arecidae) Chamaedorea cataracterum (Arecaceae; Arecidae) Monstera deliciosa (Araceae; Arecidae) Dicotyledonous angiosperms Persea americana (Lauraceae; Magnoliidae) Laurus nobilis (Lauraceae: Magnoliidae) Helleborus niger (Ranunculaceae; Magnoliidae) Urtica dioica (Urticaceae; Hamameliidae) Ficus benjamina (Moraceae; Hamameliidae) Humulus lupulus (Cannabinaceae; Hamameliidae) Aruncus silvester (Rosaceae; Rosidae) Cicer arietinum (Fabaceae; Rosidae) Lens culinaris (Fabaceae; Rosidae) Lens orientalis (Fabaceae; Rosidae) Lens nigricans (Fabaceae; Rosidae) Hippophae rhamnoides (Eleagnaceae; Rosidae) Aucuba japonica (Cornaceae; Rosidae) Simmondsia sinensis (Buxaceae; Rosidae) Macaranga hulleti (Euphorbiaceae; Rosidae) Macaranga triloba (Euphorbiaceae: Rosidae) Macaranga tanarius (Euphorbiaceae: Rosidae) Macaranga hypoleuca (Euphorbiaceae; Rosidae) Camellia sinensis (Theaceae; Dilleniidae) Brassica oleracea (Brassicaceae: Dilleniidae) Brassica napus (Brassicaceae; Dilleniidae) Brassica campestris (Brassicaceae: Dilleniidae) Bryonia dioica (Cucurbitaceae; Dilleniidae) Silene alba (Caryophyllaceae; Caryophyllidae) Silene dioica (Caryophyllaceae; Caryophyllidae) Beta vulgaris (Chenopodiaceae: Caryophyllidae) Rumex acetosella (Polygonaceae; Caryophyllidae) Nicotiana tabacum (Solanaceae; Asteridae) Nicotiana sifvestris (Solanaceae; Asteridae) Nicotiana acuminata (Solanaceae; Asteridae) Nicotiana otophora (Solanaceae; Asteridae) Nicotiana glutinosa (Solanaceae; Asteridae) Nicotiana paniculata (Solanaceae; Asteridae) Lycopersicum esculentum (Solanaceae; Asteridae) Lycopersicum hirsutum (Solanaceae; Asteridae) Solanum tuberosum (Solanaceae; Asteridae) Helianthus annuus (Asteraceae; Asteridae) Lactuca sativa (Cichoriaceae;Asteridae) a) See Section 2.2 b) +, high molecular weight DNA obtained -, no DNA obtained, or DNA largely degraded (+), very low yield, or DNA partially degraded *, DNA obtained from fruit, but not from leaf tissue

(+I + +

+ + + (+I -

(+)

-

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(+)

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+ + +

+ -

+ + (+I + -

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+ + +

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Oligonucleotide fingerprinting of plant genomes

16 1

described above, the pellets were dried under vacuum and dis- red to a new tube, and DNA precipitated by the addition of solved in an appropriate volume of Tris-EDTA buffer (TE: 2.5 volumes of ethanol. After centrifugation, DNA was dis10 mM Tris-HC1, 1 mM EDTA, pH 8). These crude DNA solved in TE and stored at 4 OC. preparations contained various amount of RNA and polysaccharides. 2.3 Restriction enzyme digestion and gel electrophoresis

2.2.2 DNA purification procedures The following methods were used alternatively.

2.2.2. I CsCl density gradient centrifugation DNA samples were transferred to an ultracentrifuge tube and mixed with two volumes of gradient buffer (6.6 M CsC1,50 mM Tris-HC1, pH 8, 10 mM EDTA, 150 pg/mL ethidium bromide). CsCl density gradients were run at 16 "C for 48 h at 40 000 rpm in a 70.1 Ti rotor, or for the same time at 32 000 rpm in an SW40 swing-out rotor. DNA bands were removed using a syringe attached to a wide-gauge needle, and ethidium bromide extraction with TE-saturated n-butanol was performed within the syringe [421. The final aqueous phase was diluted with 2 volumes of TE (to avoid co-precipitation of CsCl), and DNA was precipitated with 2 volumes of ethanol. After centrifugation (10 000 g; 30 min, 4 "C), pellets were washed in 70 % ethanol, dried and redissolved in an appropriate volume of TE.

Usually, 5 pg of DNA from each sample were digested with AZuI, MboI, HinfI or TuqI according to the supplier's recommendations. The samples were loaded on 0.7 to 1.2 % agarose gels in TAE (40 mM Tris-HC1, 20 mM sodium acetate, 1 mM EDTA, pH 7.8) or TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA, pH 8.0), and run at 1-2 V/cm for 24-48 h. After staining with ethidium bromide and photographing, the gels were denatured and neutralized according to [ 91, Then they were either dried on a vacuum gel dryer (for hybridization to radioactively labeled oligonucleotides), or blotted onto a nylon membrane (for hybridization to digoxigenated oligonucleotides).

2.4 Probes and hybridization procedures

Dried gels were hybridized to 32P-endlabeled (GACA)4, (GATAI47 (GTG)57 (CA),, (TCC),, (GGAT)47 or (CT), probes essentially as described [91. Hybridization and stringent washes were performed at T,-5 "C. In most cases, gels were used repeatedly for several different probes. Before hybridizing to a new probe, gels were regenerated by incubation in 5 mMEDTAat60"C (2 x 15min).HybridizationofDNAim2.2.2.2 Chromatography on Quiagen-tip 20 mobilized on nylon blots (Hybond N, Amersham-Buchler) to RNA was removed by adding heat-treated RNase A to a final the digoxigenated (GATA), probe, as well as washing and concentration of 10 pg/mL and incubating the samples for 30 staining procedures, were performed according to the min at 37 "C. Purification of high molecular weight DNA was protocol ofzischler et uZ. [341.The hybridization temperature then performed by ion exchange chromatography on Quiagen was 30 "C (T, - 10" C). columns (Diagen, Diisseldorf, Germany) using buffers and the procedure according to the supplier's instructions for the preparation of bacterial plasmid DNA. A 20 mg capacity 3 Results and discussion column was used. Eluted DNA samples were precipitated with 0.5 volumes of isopropanol, centrifuged, and dissolved in 3.1 Plant DNA isolation and purification TE. It is not a secret for people working on plants that the preparation of DNA from plant tissues is sometimes a bit frustrating. 2.2.2.3 Spun-columnchromatographyon CL-6B Sepharose In fact, we experienced a series of problems concerning the isolation and purification ofhigh molecular weight DNA from CL-6B Sepharose (supplied in 20 % ethanol) was washed a variety of plant species. These problems included partial or three times with TE and stored in the same buffer at 4 OC. total DNA degradation, co-isolation of viscous polysacColumns were prepared in 2 mL reaction tubes, the bottoms of charides, poor accessibility of DNA to restriction enzymes, or which had been perforated. A cushion of 100 pL of glass poor yield. Though it is almost impossible to supply isolation beads, suspended in TE, was overlaid with 1 mL of the protocols optimally suited for each species, we nevertheless Sepharose matrix, and the columns were inserted into 14 mL tried to find a single basic isolation procedure suitable for most disposable centrifugation tubes. After removing residual TE species, to obtain at least a crude preparation of total nucleic by a short centrifugation step (3 min, 1600 rpm, JS-13 rotor, acids that may be further refined for problematic species. Beckman centrifuge), the dry columns were inserted into new 14 mL centrifugation tubes. RNase A-treated DNA samples To that end, we tested several isolation methods, preferentially dissolved in TE were applied to the column, and centrifuged minipreparations suitable for large sample numbers [38-4 11. through the Sepharose matrix (2 min, 1600 rpm;JS-13 rotor). Among these, we favored a modified version of Doyle and Purified DNA was collected from the bottom of the centrifuge Doyle's protocol [411, as outlined in Section 2.2, for several tubes and stored at 4 OC. reasons. (i) The method is fast: the basic four steps that have to

2.2.2.4 Ammonium acetate treatment DNA samples dissolved in TE were brought to a final concentration of 2.5 M ammonium acetate and incubated for 20 min on ice. Precipitated RNA was then removed by centrifugation (30 min, 10 OOOg, 4 "C), the supernatant transfer-

be carried out require a minimum of time and working capacity. (ii) The method can be scaled down to as little as 0.0 1-0.1 g of starting material (B. Beyermann, unpublished results). (iii) Good yields of DNA (between 0.1-0.5 mg/g fresh weight) were routinely obtained from most tissues. (iv) The basic protocol of the method worked reproducibly with a large variety of lower and higher plants (Table 1). Note, however,

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that some problems occurred with several species of fungi, algae, and especially with leaf material from gymnosperms. For these problematic species, the experimentist should refer to more specialized protocols designed for fungi [43-451 and gymnosperms [461. The DNA of most species investigated so far, which was isolated according to the basic protocol, could be used right away. Sometimes it proved to be necessary to include further purification steps. Originally, we routinely applied CsCl gradient centrifugation for the preparation of highly purified DNA. As an example, Fig. 1A shows that tobacco leaf, root, stem, and petal DNA purified by this technique is of high molecular weight, free from RNA, and readily accessible for restriction enzymes. However, CsCl gradient centrifugation is costly and time-consuming. Therefore, we tested three alternative purification schemes: (i) RNase treatment followed by ion exchange chromatography on Quiagen-tip 20, (ii) RNase treatment followed by spun-column chromatography on Sepharose CL-6B, and (iii) removal of RNA by precipitation with ammonium acetate. Crude DNA preparations derived from tomato leaf and stem tissue were divided into four equal aliquots, and each aliquot was purified according to one of the different procedures. Three parameters were considered: (i) characteristics of D N A (molecular weight, accessibility for restriction enzymes, purity and yield), (ii) appearance of banding patterns after hybridization to oligonucleotide probes, and (iii) economy of the methods. The results are summarized in Fig. 1. High molecular weight DNA was obtained by all four techniques (Fig. 1 A, B). However, some smearing, indicative of partial degradation, was observed upon ammonium acetate and Quiagen treatment (Fig. IB, lanes a, b, g, h). DNA was gen-

Electrophoresis 1991,12. 159-169

erally accessible to digestion by restriction enzymes (Fig. 1A, lanes e-h; Fig. lC, lanes a, b). With the exception of ammonium acetate treatment, the obtained DNA preparations were sufficiently free of RNA. Since up to 40 % of DNA coprecipitated with the RNA using the ammonium acetate method, we consider this purification step not to be selective enough to be recommendable. Banding patterns upon hybridization to oligonucleotide probes were similar for all purification techniques. An example is shown in Fig. 1C: although a slight smear indicative of degradation was observed in undigested, Quiagen-purified DNA, the quality of the banding pattern observed after TaqI digestion and hybridization to (GATA), was comparable to that obtained with CsCIpurified DNA. With the exception of the ammonium acetate technique, these experiments show that the different purification procedures yield comparable results. Thus, for routine work we recommend using the cheapest and fastest method. In our hands, this is the Sepharose technique, which has been successfully applied to the isolation of potato, tobacco, tomato and sugar beet DNA, and that yielded reproducible results in screening a large progeny of a tomato crossing experiment (B. Beyermann, unpublished).

3.2 Plant DNA fingerprinting with oligonucleotide probes 3.2.1 The occurrence of simple repetitive sequences in plant genomes To get a fair idea of the distribution of simple repetitive sequence motifs within the plant kingdom, we screened Hinfldigested DNA from a large number of plant species for the presence and organization of various simple repetitive sequences by in-gel hybridization to the corresponding oligoFigure I . Electrophoretic analysis of DNA purified by different procedures (for details see Section 3.1). D N A was isolated from different tissues of tobacco (A) or tomato (B, C). (A) Ethidium bromide staining pattern of tobacco DNA isolated from leaves (a, e), stems (b, f), roots (c, g), and petals (d, h) according to the CTAB protocol outlined in Section 2.2.1, followed by a CsCl purification step. (a) to (d) undigested DNA: (e) to (h) EcoR1digested DNA. Intact lambda D N A was included in the restriction assay to check for complete digestion. (B) Ethidium bromide staining pattern of tomato DNA isolated from leaves (a, c, e, g) and stems (b, d, f, h) and purified according to different protocols: (a), (b) ammonium acetate precipitation, (c), (d) CsCl centrifugation, (e), (f) spun-column chromatography on Sepharose, (g), (h) ion exchange chromatography on Quiagen. (C) Ethidium bromide staining pattern of Tuql-digested tomato leaf DNA (a, b) and its hybridization to the (GATA), oligonucleotide probe. Comparison of results obtained with theQuiagen(a,c)andtheCsCI centrifuoation (b, d) procedure.

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IGATA

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Y

L

Fungi

Mosses

--.

Algae

Ferns

Gymnosperms

Dicots

Monocots

Angiosperms Figure 2. Screeningfor the occurrence of simple repetitiveDNA motifs in genomes offungi, andlower and higher plants. DNA was digested’withHinfI and separated on 1 % agarose gels in TBE buffer (5 pg/lane). Dried gels were hybridized to the 32P-labeledoligonucleotide probes (GATA), (upper panel), (GAC A), (medium panel), and (TCC), (lower panel). Positions of molecular weight markers are indicated in kilobases (kb). Fungi: (a) Phycomyces blakesleeanus, (b) Coprinus comatus. Algae: (c)Oedogonium spec., (d) Stenogramme interrspta. Mosses: (e)Polytrichumformosuni. Ferns: (f)Equisetum arvense, (9) Polypodium vulgare, (h) Osmunda regalis, (i) Dodia caudata. Gymnosperms: (k) Juniperus communis. Monocots: (1) Echinodorus osiris, (m) Hordeum spontaneum (accession 4 1.3), (n)Musa acuminata, (0)Asparagus densiflorus, (p) Chamaedorea cataracterum. Dicots: (4)Helleborus niger, (r) Silene alba, (s) Rumex acetosella, (t)Urticadioica, (u) Humulus lupulus, (v) Ficus benjamina, (w) Lens culinaris (accession ILL 5989), (x) Cicer arietinum (accession ILC 1250),(y) Simmondsia sinensis, (z) Aruncus dioicus, (a’) Brassica napus var. oleifera (cv. Line), (b’) Camellia sinensis, (c’) Solanum tuherosum (dihaploid line 82.452 I), (d’) Nicotiana tabacum var. atropurpurea, (e‘) Lactuca sativa.

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nucleotide probes. Representative results obtained with probes (GATA)4,(GACA)4, and (TCC)Sare shown in Fig. 2. Some motifs are so highly abundant in some species that a smear results on the autoradiograms (e.g., GATA repeats in the green alga Oedogonium and the fern Osmunda regalis, GACA repeats in the red alga Stenogramme, and TCC repeats in the shave-grass, Equisetum arvense). Even after short exposure times, no distinct bands could be seen in these cases. It is obvious that these probe/species combinations are not good for DNA fingerprinting. Other motifs occur relatively rarely in some species,faint bands only being observedupon prolonged exposure of the autoradiograms (e.g., TCC repeats in most species). In many cases, however, distinct banding patterns were revealed by the probes, especially with (GATA)4. If clear-cut patterns were observed, the respective probe was regarded a good candidate for fingerprint analysis of the respective species. To summarize the results ofthese experiments (Fig. 2, and unpublished data), we could show that all simple sequences tested (CA-, CT-, GATA-, GACA-, GTG-, GGAT- and TCC-multimers) are present and repetitive to various extents throughout the plant kingdom. For their application to DNA fingerprinting, the optimal probe/species combinations that give distinct banding patterns have to be developed empirically. These results confirm and extend recent data obtained by hybridizing human and M 13-derived minisatellites to plant and fungal genomes 15, 16, 18-221, or by hybridizing poly(GT) to yeast DNA 13 I]. Obviously, minisatellites and simple repetitive motifs are ubiquitous constituents of eukaryotic genomes including plants and fungi, although the number and organization of these motifs varies considerably among species. Similar observations have been made in studies on animal genomes [2].

crossing experiment between different cultivars, the bands were inherited in a 3: 1 ratio, suggesting a homozygous allelic distribution in the parents. Taken together, these results suggest a high level of homozygosity within a rice cultivar (which is not surprising, since cultivated rice is self-fertilized). Using the M 13 probe, considerable genetic variation was observed in apple, raspberry, cherry, tomato, poplar and several other plant species [ 19,20,5 1-54]. Obviously, minisatellites provide useful tools for revealing genetic polymorphisms in plants (Table 2). These tools were already applied for, e.g., variety identification in rice [151 and blackberries [521, population studies on raspberries and blackberries 1531, and paternity testing of apple trees 1541. In order to expand the collection of informative probes, we introduced selected oligonucleotide probes complementary to simple repetitive motifs to plant DNA fingerprint analyses (135-371, this study). Probe/species combinations yielding distinct hybridization patterns were chosen for these studies. Figure 3 shows the results obtained with the (GTG)5 probe hybridized to various DNA samples from plants of the genus Nicotiana. To detect the distinguishing capacity of the probe, we compared (i) several species of thegenusNicotiana (lanes a Table 2. Probes used for DNA fingerprinting plant and fungal genomes Probes Human minisatellites (33.6; 33.15) Colletotrichum gloeosporioides Oryza sativa Oryza glaberrima Populus deltoides PoDulus tremuloides ~

~~

The functional significance of both classes of repetitive DNA, however, ist still far from being clear. Some lines of evidence suggest that minisatellite variability is mainly caused by recombinational processes [41. In fact, minisatellites have been found at sites of meiotic crossing-over 1471, some of their core sequences show similarity to the bacterial recornbinator chi 131, and minisatellites occur at recombinational hot spots in the human genome [481. Simple repetitive sequences, on the other hand, are mainly thought to originate from slippedstrand mispairing during DNA replication or repair [7, 491. The idea was put forward that this kind of repetitive DNA might serve no function per se, but instead reflect internal genomic mechanisms that have the tendency to dynamically produce and delete stretches of these motifs 1I, 6,7,491. The fact that the relative abundance and the organization of specific motifs varies considerably between species and does not bear any obvious correlation to phylogenetic categories ([21, this study) strengthens this hypothesis and suggests that simple sequences may have arisen independently throughout evolution [49, 501.

M 13 repeat Fusarium Pinus torreyana Asimina triloba Polyalthia glauca Populus tremuloides Lycopersicum escuientum Arabidopsis thaliana Linurn bienne Linum usitatissimum Medicago sativa Malus domestica Prunus serotina Rubus (several species)

3.2.2 Inter- and intra-specific variability detected by oligonucleotide fingerprinting

Simple repetitive sequences Saccharomyces cerevisiae Cicer arietinum Lens culinaris Brassica napus Solanurn tuberosum Nicotiana(severa1 species) Beta vulgaris Hordeum vulgare Hordeum spontaneum Musa acuminata

Several authors screened a variety of plant species for the presence and variability of either the human minisatellites33.6 and 33.15 13,51 or the M13 repeat sequence [171. A detailed study, focusing on rice, revealed cultivar-specific patterns upon hybridization to the 33.6 probe, but almost no differences between individuals [ 151. Banding patterns were somatically stable during mitosis. In the F2 generation of a

References

~~

Random potato cDNA and genomic clones Solanurn tuberosum

1221 I191

DO, 541 [201 [20,52,531 [641

Minisatellite-likewheat sequence Trilicum aestivum Random beet cDNA clone Beta vulgaris

[661 1311 [35-37,671 this study

Oligonucleotide fingerprinting of plant genomes

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to d), (ii) several varieties of Nicotiana tabacum (lanes d, e, f, k), (iii) several plants from the same variety (var. Wisconsin 38: lanes f, g, h, and var. atropurpurea: lanes k, I, m), (iv) several organs from the same plant (var. Wisconsin 38: lanes h, i, and var. atropurpurea: lanes m to p). As is evident, hybridization patterns of DNA from different organs of the same plant, and from different plants of the same variety, are virtually indistinguishable. This holds true for both varieties tested. Slight differences, however, were observed upon comparison of different tobacco varieties, and gross differences exist between several species of Nicotiana. Similar results, but less complex patterns, were obtained using (GATA),, (GACA),, (TCC),, and (GGAT), probes (not shown). Obviously, simple repetitive motifs can be used for the identification of tobacco varieties and species, but not for the discrimination of individuals. Moreover, the patterns seem to be somatically stable.

a

b

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h

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Figure 3. Inter- and intraspecific variability in the genus Nicotiana (tobac co), as detected by the (GTG), probe. DNA was isolated from different tissues ofindividualplants(seebelow), digestedwith Hinfl andseparatedon a 1 % agarose gel in TAE buffer (5 pg/lane). The dried gel was hybridized to the 32P-labeled oligonucleotide probe (GTG),. Positions of molecular weight markers are indicated in kb. (a) Nicotiana paniculata, leaf, (b) Nicotiana glutinosa, leaf, (c) Nicotiana acuminata, leaf, (d) Nicotiana tabacum, var. Virginia, leaf, (e) Nicotiana tabacum, SRl, leaf, (f) to (i) Nicotiana tabacum, var. Wisconsin 38: (f), (g), (h) leaves from three in dividual plants I, 11,111,(i) root from plant 111(k) to (p) Nicotiana tabacum, var. atropurpurea: (k), (l), (m) leaves from three individual plants IV, V, VI (n), (o), (p) stem, petals, root from plant VT.

CGATA14 Lentil

(GACA)d Lentil

Variety-specific patterns were also observed with rapeseed (Brassica napus) using the (GATA), probe (1371, Fig. 4,right panel). Different accessions of lentil (Lens culinaris) hybridized to the same probe also showed variable patterns (Fig. 4, left panel), and two lentil individuals derived from the same accession looked similar (lanes e and f). The results obtained with the lentil/(GATA), combination, however, suffer from an increased background smear. The (GACA), probe (not shown for rapeseed) was less informative: four out of six lentil accessions showed similar patterns (lances c to g), only two accessions diverged (lanes a and b). Different probes usually revealed differential information if hybridized to the same gel. This was not only observed for lentil and rapeseed, but also for sugar beet and chickpea. Figure 5 shows the results of hybridizing the oligonucleotide probes (GATA),, (GTG),, (GACA),, and (CT), to MboI-

(GA TA )G Rapeseed

Figure 4.Intraspecific variability in lentil (Lens culinaris) and rapeseed (Brassica napus, var. oleifera), as detected by the (GATA), and (GACA), probes. DNA was isolated from leaves of individual plants (see below), digested with AluI (lentil)or TaqI (rapeseed), and separatedon 1 % agarosegelsiri TAE buffer (5 pgt'lane). The dried gels were hybridized to the "P-labeled oligonucleotide probes (GATA), (lentil and rapeseed) or (GACA), (lentil). Positions of molecular weight markers are indicated in kb. Lentil: DNA samples from seven individualplants of six lentil accessions were applied to the gel: (a)ILL 5582, (b) ILL 5588, (c) ILL 5700, (d)ILL 5873, (e) ILL 5876, plant I, (f)ILL 5876, plant 11,(g)ILL 5988. Rapeseed: DNA samples fromindividualplantsofeight cultivars were applied to the gel: (a) cv. Topas, (b) cv. Lirama, (c) cv. Optima, (d) cv. Rally, (e) cv. Line, (f) cv. Hanna, (9) cv. Olga, (h) cv. Jet neuf.

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differences in information were observed among different enzymes with a 4 bp recognition sequence (AZuI, MboI, TaqI and Hinfl). This was as expected, since the chosen enzymes were supposed to cut at random sites outside the simple motif clusters. The use of 6 cutters such as EcoRI, however, shifted the mean fragment size to a higher molecular weight range, thereby reducing the resolving power of the gels. The use of 4base recognition enzymes is therefore recommended. We also tested the applicability of nonradioactively labeled oligonucleotides to plant DNA fingerprinting (Fig. 7). DNA from different chickpea individuals was digested with TaqI, split in two equal aliquots, and electrophoretically separated on two agarose gels under identical conditions. One of the gels was dried and hybridized to a 32P-endlabeled(GATA), probe as usual. The other gel was blotted onto a nylon membrane, and the blot hybridized to a digoxigenated (GATA), probe. Figure 7 shows that the banding patterns revealed by both methods are of similar quality. This means that the nonradioactive method can be routinely used in institutions that own no radioisotope laboratories, e.g., plant breeding institutes. However, several drawbacks of the nonradioactive technique should be mentioned. First, strong signals are not easily removed from the membrane 1341, rendering re-use of blots difficult. Second, signal intensities are higher if 32P-labeled oligonucleotides are used (the autoradiogram shown in theleft panel of Fig. 7 represents a short exposure, whereas the staining reaction was performed under saturating conditions; right panel).

Sugarbeet Figure 5 . Intraspecific variability in sugar beet (Beta vulgaris var.

altissima): Different probes reveal different levels of polymorphism. DNA was isolated from leaves of individual plants from two different breeding lines(a, b),digestedwithMboI and separatedona0.7 %agarosegelinTAE buffer (3 pg/lane). The dried gel was consecutively hybridized to the 32P-labeled oligonucleotide probes (GATA),, (GTG),, (GACA),, and (CT),. Positions of molecular weight markers are indicated in kb.

digested DNA from two different breeding lines of sugar beet (Beta vulgaris, var. altissima). Clearly, the (GATA), probe shows the most clear-cut results in terms of banding patterns and distinguishing capacity. Similar results were obtained in an extensive analysis of polymorphism exhibited by different chickpea (Cicer arietinurn) accessions ([35-371, K. Weising, F. Weigand, G. Kahl, J . T. Epplen, in preparation). In this case, hybridization of (GATA), to TaqI-digested chickpea DNA reveals highly variable and accession-specific patterns. In contrast, (GTG), provides no accession-specific information at all, and (GACA), and (GGAT), show only limited heterogeneity. In the case of chickpea, not only accessions but also individuals can be distinguished by some probes. Figures 6 and 7 show that (GATA), and (CA),, but not (GGAT),, reveal differences between individuals belonging to the same accessions. Whereas accession specificity of patterns is more or less conservedin these cases (compare lanes a toe with lanes f to j in Fig. 7), many fragments show slight, but individualspecific differences in mobility. This is most probably caused by allele-specific variable numbers of tandemized simple motifs. The role of the restriction enzyme for obtaining an informative fingerprint in a given combination of probe and species was tested in several cases (not shown). Usually, no significant

Taken together, it can be concluded that the obtained pattern complexity and variability strongly depends on the sequence motif used for hybridization. The optimal combination of probe and species has to be determined empirically for each purpose. Using different probes, species-, variety- or individual-specific patterns can be obtained. In case of medium to strong signal intensities, nonradioactive techniques may be applied. In combination with recent results on DNA fingerprinting and identification ofyeast strains using apoly (GT)probe [3 11,our results show that simple sequences are a useful new tool for DNA fingerprinting of plants and fungi. Taking into account that even random repetitive oligonucleotide probes revealed polymorphisms in the human genome [241, the collection of informative oligonucleotide probes is probably almost unlimited.

4 Concluding remarks A variety of probes is now available for plant DNA fingerprinting: human minisatellites, the M 13 repeat, and a probably unlimited collection of simple repetitive sequences (Table 2). The use of these probes has already made possible the genetic characterization of plant varieties, cultivars and accessions ([ 15,20,35-37,52-541, this work), and of pathogenic fungal strains [21, 221. However, the potential of the fingerprinting technique goes far beyond. Some of the most important future applications of this method include: (i) demographic studies on plant populations analogous to similar studies on mammals and birds [ 13, 14,55,561, as already initiated on natural populations of raspberry and blackberry [531, (ii) the genetic

Oligonucleotide fingerprinting of plant genomes

Electrophoresis 1991,12, 159-169

Chickpea

Chickpea (GATA

16 7

Figure 6 . Intraspecific variability in chickpea (Cicer nrietinum): The oligonucleotide probes (GATA), and (CA),, but not (GGAT),, detect polymorphisms among individuals derived from the same accession. DNA was isolated from leaves of individual plants, digested with TaqI, and separated on a 1 % agarose gel in TAE buffer (5 &am). The dried gel was consecutively hybridized to the 32P-labeled oligonucleotide probes (GATA),, (CA),, and (GGAT),. Positions of molecular weight markers are indicated in kb. (a)-(c) Three individuals from accession ILC 82.150. (d)-(f) Three individuals from accession ILC 1250.

Figure 7. Intraspecific variability in chickpea (Cicer nrietinum): Comparison of fingerprints obtained with 32P- and digoxigenin-labeled oligonucleotides. DNA was isolated from leaves of individual plants and cut with TnqI. The restriction-enzyme-digested samples were split into equal aliquotsand separatedon two identical 1 % agarose gels in TAE buffer (2 pg/lane). One of the gels was dried and hybridized to a 3ZP-labeled (GATA), probe. The resulting autoradiogram is shown in the left panel. The other gel was blotted onto a nylon membrane and hybridized to a digoxigenin-labeled (GATA), probe. The resulting staining pattern is shown in the right panel. Positions of molecular weight markers are indicated in kb. (a)-(e) Five individuals from accession ILC 1250. (f)-(k) Five individuals from accession ILC 82.150.

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characterization of cell lines, cell hybrids (as was done in mammalian cell lines; [57]), and chromosome addition lines, (iii) the use in plant breeding programs. This topic will be of most significant importance. It includes the characterization of variability within landraces and cultivars, the assessment of inbreeding [%I, and DNA fingerprinting-assisted gene introgression [ 591. The availability of nonradioactive techniques will be of special importance in that field. Now that a variety of multilocus probes is available for plant DNA fingerprinting, the development of new probes should focus on multiallelic single-locus probes (as identified for humans; e.g. 1111). These will be advantageous for several purposes such as linkage analysis and genome mapping. Single-locus VNTR loci in plants could be identified by (i) screening plant DNA with VNTR sequences known to be single-locusspecific in animals, (ii) cloning hypervariable loci in order to use unique flanking regions as locus-specific hybridization probes, or as locus-specific primers for DNA amplification in the polymerase chain reaction. The latter technique, which specifically amplifies a single locus or a few defined hypervariable loci, has already been successfully applied in animal systems using minisatellite or simple sequence flanking regions [60-631. The isolation of plant-specifichypervariable sequences would also be of interest. To date, most of the information about hyperpolymorphisms in plant DNA is derived from sequences originally found elsewhere in the living world, and plant hypervariable sequences have only been characterized by chance in the course of other studies [64,651.

We appreciate the kind gijii of unlabeled and digoxigenated oligodeoxynucleotides from Dr. J. T. Epplen (Max-PlanckInstitut fur Psychiatrie, Martinsried) and the help of S . Kost in the preparation of thefigures. K . Weising acknowledges a fellowship from Dechema (Frankfurt, Germany). Received July 4, 1990

5 References [I] Tautz, D. and Renz, M., Nucleic Acids Res. 1984 12,4127-4138. [2]Epplen, J. T., J. Hered. 1988,79,409-417. 131 Jeffreys,A.J.,Wilson,V.andThein,S.L.,Nature1985,314,67-73. 141 Jeffreys, A. J., Biochem. Soc. Transact. 1987,15,309-317. I51 Jeffreys,A.J.,Wilson,V.andThein,S.L.,Nature 1985,316,76-79. [61 Tautz, D., Nucleic Acids Res. 1989,17,6463-6471. [71 Tautz,D.,Trick,M. andDover,G.A.,Nature 1986,322,652-656. [8] Jeffreys,A. J.,Neumann,R. andWilson,V., Cell 1990,60,473-485. [9]Ali, S., Muller, C. R. and Epplen, J. T., Hum. Genet. 1986, 74, 239-243. [lo]Schafer, R., Zischler, H., Birsner, U., Becker, A. and Epplen, J. T., Electrophoresis 1988,9,369-374. 1111 Nakamura, Y.,Leppert, M., OConnell, P., Wolff, R., Holm, T., Culver,M.,Martin, C., Fujimoto,E., Hoff, M.,Kumlin,E. and White, R., Science 1987,235,516-522. [12]Jeffreys, A. J. and Morton, D. B., Anim. Genet. 1987,18,1-5. [131 Wetton, J. H., Carter, R. E., Parkin, D. T. and Walters, D., Nature 1987,327,147-149. 1141 Burke, T. and Bruford, M. W., Nature 1987,327,149-152. 1151 Dallas, J. F.,Proc. Natl. Acad. Sci. USA 1988,85,6831-6835. 1161 Rogstad, S. H., Patton, J. C. and Schaal, B. A., Nucleic Acids Res. 1988,16,11378. I 1 71 Vassart, G., Georges, M., Monsieur, R., Brocas, H., Lequarre, A. S. and Christophe, D., Science 1987,235,683-684.

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DNA fingerprinting in domestic animals

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[591 Hillel, J., Schaap, T., Haberfeld, A., Jeffreys, A. J., Plotzky, Y., Cahaner, A. and Lavi, U., Genetics 1990,124, 783-789. [601 Jeffreys, A. J., Wilson, V., Neumann, R. and Keyte, J., Nucleic Acids Res. 1988,16,10053-10071. [611 Boerwinkle, E., Xiong, W., Fourest, E. and Chan, L., Proc. Natl. Acad. Sci. USA 1989,86,212-216. [621 Weber,J. L. andMay,P.E.,Am.J.Hum. Genet. 1989,44,388-396. [631 Litt, M. and Luty, J. A., Am. J. Hum. Genet. 1989,44, 397-401.

Johannes Buitkamp’ Hubert Ammer2 Hermann Geldermann’ ‘Institute for Animal Breeding, University of Hohienheim, Stuttgart *Max-Planck-Institutefor Psychiatry, Martinsried

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[641 Gebhardt, C., Blomendahl, C., Schachtschabel, U., Debener, T., Salamini, F. and Ritter, E., Theor. Appl. Genet. 1989, 78, 16-22. [651 Martienssen, R. A. and Baulcombe, D. C., Mol. Gen. Genet. 1989, 217,401-410. [661 Nagamine,T.,Todd, G. A.,McCann,K. P., Newbury, H. J. and FordLloyd, B. V., Theor. Appl. Genet. 1989,78,847-851. [67] Beyermann, B., Nurnberg, P., Weihe, A., Meixner, M., Epplen, J. T. and Borner, T., Theor. Appl. Genet. in press.

DNA fingerprinting in domestic animals DNAs of several species of- domestic animals digested with the restriction endonucleases HinfI, AluI and HaeIII were hybridized with different synthetic probes. DNA fingerprint patterns were found in each investigated species by at least two of these probes. Furthermore, two probes gave sex-specific banding patterns in the chicken. Some applications of DNA fingerprinting in domestic animals are discussed.

1 Introduction As in man, the analysis of restriction fragment length polymorphisms (RFLPs) is one of the most efficient means of monitoring genetic diversity in domestic animals. The first results on RFLPs in pig, sheep and cattle were published in 1985 11-31. Since then, the number of RFLPs described in domestic animals has increased considerably, but is still low compared to RFLPs in man 141. A higher degree ofvariability due to variable numbers of tandem repeats (VNTR) [51results in multiallelic RFLP patterns that can be observed in certain regions of repetitive DNA. When such banding patterns are specific to a defined DNA locus they are called single-locus VNTRs. Goodbourn et al. I61 had proposed that the families of repetitive sequences existing in the human genome could provide valuable markers for genome analysis. It has since been confirmed that several independent DNA loci share sequence homology, thereby enabling the demonstration of many highly polymorphic DNA loci simultaneously. Such multilocus VNTRs (called “DNA fingerprints” due to the high individual specificity)were studied methodically and applied by Jeffreys and co-workers 17-12]. Jeffreys et al. I71 termed their repetitive sequences “minisatellites”, which are characterized by a tandemly repeated consensus sequence [ 131 of 16-64 nucleotides 171. Some of these minisatellites comprise “families” that are related by core sequences [5,71, characterized by a high degree of homology in their consensus sequences. These homologies allow the cloning of novel VNTR probes by screening genomic libraries, using known minisatellite sequences 17, 141. Furthermore, several mini-

satellite sequences are closely linked to coding regions, such as the human a-globin gene [151, the insulin gene 1161, or the apolipoprotein B gene [171, as well as to subtelomeric regions of the sex chromosomes 118, 191. Some of these minisatellites have been used for DNA fingerprinting in man [7, 14,20-231. Some human VNTR probes can also be used in domestic animals (Table 1) because minisatellite sequences show not only intraspecies, but also substantial interspecies homologies. Probes 33.6 or 33.15 (“Jeffreysprobes”) andrelated sequences are now known to display hypervariable banding patTable 1. Minisatellite probes used in domestic animals Species

Probesa)

Authors

Cat Dog

33.6133.15 33.6133.15 M13; pUCJ; pSP64.2.5RI M13; pUCJ; pSP64.25RI PS3 pYNH24; pYNA23 M13; pUCJ; pa3’HVR64 INS3 10; EFD 134.7 pSP64.2.5RI 33.6; pGBJ4.3a 33.15,M13 M13; pUCJ; pSP64.2.5RI PS3 EE4 1.O 1b, pCMM86 33.6l33.15 mo- 1 PS3 33.6 (chicken, duck, turkey, goose) M13 (chicken)

Jeffreys and Morton I241 Jeffreys and Morton (241 Georges et al. [251 Georges et al. [251 Coppieters et a1.[371 Troyer et a1.[351 Georges et ~1.1251 Georges et a1.[34l Perret et al. [331 Kashi et al. [261 Broad (891 Georges et a1.[251 Coppieters et a1.[371 Broad [89l Troyer ef a1.[361 Jeffreys et a1.[28l Kominami et al. [291 Coppieters et al. [371

Swine

Cattle

Sheep Horse

Mouse

Correspondence: Prof. Dr. Hermann Geldermann or Dr. Johannes Buitkamp, Universitat Hohenheim, Institut fur Tierhaltung und Tierzuchtung, Fachgebiet Tierzuchtung 470, GarbenstraDe 17, W-7000 Stuttgart 70, Germany

Rabbit Poultry

Abbreviations: Bkm, banded krait minor; bp, base pair(s); RFLP, restriction fragment length polymorphism; sqr, simple quadruplet repeat; str, simple tandem repeat; VNTR, variable number of tandem repeats

a) Information about single probes is given in the introduction. b) This probe was used only for commercial purposes and no further information is given.

0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

Hillel et a1.[27] Kuhnlein et a1.[3 11

0173-0835/91/02-302-3-0169 %3.50+.25/0