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
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terns in different species. Initially Jeffreys and Morton [241 used both probes in dogs and cats; subsequently, Georges et al. [25l generated DNA fingerprints in cattle, horse, swine, and dog with a polymer of “Jeffreys core-sequence”. Fingerprints in cattle were also obtained with the original Jeffreys probe, 33.6, as well as with abovinecloneisolatedfrom a genomic library by colony hybridization 1261. “Jeffreys probes” were also used to obtain fingerprints in different species of poultry [271 and mice [281. In the mouse, a clone (mo-1) isolated from a murinegenomiclibrary by screening with“Jeffreys core-sequence” was also found to produce hypervariable banding patterns [291. Two further minisatellite probes cloned from genomic DNA of bird by colony hybridization with probes 33.6 and 33.15 gave highly variable banding patterns in birds as well as in some mammals, including mice [30]. Vassart et al. [22] described the use of DNA from bacteriophage M13 to obtain multilocus VNTRpatterns in man and in cattle. A more detailed investigation of different cattle, pigs, horses, and dogs was carried out by Georges et al. [251. They found the M13 probe to be highly informative in all the species investigated. M 13 was also informative in the chicken [3 1, 321. A further murine probe (pSP642.5GI) used by Georges et al. [211 to fingerprint domestic animals is a genomic clone related to the Drosophila “Per” gene which had also been applied to DNA fingerprinting in man. This probe was reported to be informative in dog, swine, and horse [251. In cattle this probe displayed Y chromosome-specific hybridization patterns [33l, thus providing amarker to monitor male genealogy. Further, the probe pINS3 10,containing a human minisatellite on the 5’ end ofthe insulingene [ 161, also yieldedcomplex banding patterns [341. Additional human minisatellite probes, initially developed by Nakamura el al. [51 using different oligonucleotides specific to known consensus sequences, were used to obtain fingerprints in cattle (probe EFD134.7) 1341, pig (probes pYNH24 and pYNA23) [35l, and horse (probe pCMM68) [361. Coppieters et al. [37] discovered a pig minisatellite suitable for multilocus fingerprinting of swine, horse, and rabbit. In addition, this clone revealed single-locus VNTR patterns if used under highly stringent hybridization conditions. Perret et al. [331 have also reported some locus-specific bovine probes, cloned by screening a male-specific sequence.
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Southern blot experiments, using as a probe a genomic clone containing (GATA),sequences derived from Drosophila, display hypervariable banding patterns in humans [54]. Twelve individuals were investigated but no information was given about the mean number or the inheritance of bands. A Bkm clone was also used to produce DNA fingerprints in rainbow trout [481. Ali et a. [ 561 used different oligonucleotide probes specific to sqr sequences, thereby establishing the oligonucleotide fingerprinting technique. Schafer et al. [381 improved the technique of oligonucleotide fingerprinting using a variety of additional synthetic probes consisting of repeat units of 2 to 4 base pairs (bp). So far, str sequences have been found in every eucaryotic genome, but they display species-specific occurrence and distribution [571. Appropriate str sequences can therefore be applied to demonstrate genomic variations in any eucaryotic individual [58] and were recently used in different genera such as plants (Beyermann et al., submitted; [59,601), ciliates, grasshoppers and leek (J. Epplen, personal communication), poeciliid fish [611,mice [46,57,62I,Rhesus monkeys [63l, and cattle [64,651. This paper deals with informative oligonucleotide probes for different domestic animals. Several different oligonucleotides specific to str sequences were used for the hybridization of DNAs of unrelated individuals of different species. Fingerprints from informative probes were investigated further in order to obtain data for their practical application in domestic animals, e.g., the mean number of polymorphic bands per individual or the inheritance of fragments.
2 Materials and methods
Blood samples were taken from individualsof different species of domestic animals (cattle, sheep, goat, pig, horse, dog, and chicken). Animals from different breeds (see legend to Table 2) were included to obtain high genetic diversity, which facilitates the selection of probes. Peripheral blood was collected in 0.2 x volume of citrate iwlution (0.2 % w/v NaC1, 0.8 % w/v trisodium citrate x 2H,O). DNA was isolated, esA second class of hypervariable loci comprises tandemly sentially as described by Schmidtke et al. [661. DNA (5-10 repeated, short (< 10 bp) simple-sequence motives, e.g., pg) from each animal was digested overnight with 5 U/pg . . .CAC CAC CAC.. . or . .GT GT GT.. ., which were DNA of different restriction enzymes (Hinfl,HaeII1,AluI) as therefore termed simple tandem repeats (str) [381 or, in recommended by the manufacturer, in the presence of 4 mM analogy with the expression minisatellites, “microsatellites” spermidine and 0.1 mg/mL bovine serum albumin. Before [391. Such str sequences were first described by Skinner et al. loading onto the gel, all DNA samples were extracted twicein [40] in the DNA of a hermetic crab. Subsequent investigations phenol-chloroform, precipitated in ethanol, and either disfocused on specific aspects of str biochemistry and biology, solved in TAE (40 mM Tris, 12 mM sodium acetate, 2 mM e. g., the sex-specific organization of str sequences in eu- EDTA, pH 8.3) or TBE buffer (89 mMTris, 89 mM borate, 2.5 caryotic genomes [26, 41-481, the transcription of these mM EDTA, pH 8.2). This was done to minimize background repetitive sequences [49-541, and the possible functions of signals, due to hybridization of the oligonucleotide to protein (GT)” sequences in the regulation of transcription [551. contamination from the restriction digestion. The DNA fragments were separated on 0.7-0.8 % agarose gels in TAE or The first str (to obtain DNA fingerprints) consisted of simple TBE buffer for up to 50 h at 1.5 V/cm. Then the gels were dried quadruplet repeat (sqr) sequences consisting of GATA/ on filter paper (Schleicher & Schiill, GB002) on avacuum-gel GACA repeats. Such sequences were initially found in the dryer for 30 min at room temperature and, in addition, for 60 form of sex-specific satellite DNA of female (heterogametic) min at 60 OC. DNA fragments were denatured for 30 min in colubrid snakes (therefore called Bkm, from banded krait 0.5 M NaOH/O. 15 M NaCl and neutralized for 30 min in 0.5 M minor satellite) [411. The identification of these GATA/ Tris/O.lS M NaC1, pH 8.0, at room temperature. Prior to GACA sequences was obtained by sequencing of W hybridization, gels were equilibrated in 6 x SSC (0.9 M NaC1, chromosome-specific DNA of the snake Elaphe rudiata [43]. 0.09 M sodium citrate). Labeling as well as hybridization of
.
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oligonucleotide probes was carried out as described by Schafer et al. [381. Hybridization temperatures for the different probes were: 27 OC for (AT),, 35 OC for (GATA),, 43 “C for (CT),, (GA),, (GAA),, (GACA),, (GGAT),, and (GT),, 45 “C for (GTG)5 and (TCC),, 47 “C for (GATA),)(GACA)(GATA>,, and 59 OC for (GC),.
3 Results At least two unrelated individuals of each species were investigated to generate information about the different simple repetitive sequence exhibiting length polymorphisms resolvable by agarose gel electrophoresis. Out of 92 principally different str motifs up to 4 bp long (calculated as 4” ‘+2, n12, where n is the length of the simple repeat motif; see [ 57]), a number of oligonucleotides composed of str sequences (see Section 2) was selected with regard to availability for the hybridization experiments. The resulting autoradiograms were analyzed with respect to occurrence, intensity, and number of polymorphic bands. As in other eucaryotic genomes, notable stretches of str sequences, also exist in the genome of investigated domestic animals. In each species, different oligonucleotide probes yield highly polymorphic banding patterns, as listed in Table 2. For practical applications a larger number of animals was investigated to determine the mean number of polymorphic bands per individual and band sharing rates. In cattle, unrelated individuals from three differente breeds were investigated. Figure 1 depicts the DNA fingerprints of 6 unrelated cattle from the Holstein Friesian, Red Pied, and Simmental breeds, as obtained with oligonucleotide probe (CAC),. To prove somatic stability of the fingerprint bands, DNAs isolated from different tissues(blood, muscle, skin) and semen were digested with restriction enzymes and hybridized with (GTG)5 (unpublished results). Furthermore, fingerprint bands in cattle are transmitted from parents to offspring according to Mendelian laws. Within 9 complete families, 52 out of 53 fragments of the DNA fingerprints of the calves could be traced to one of the parents, using Hinfl-digested DNA hybridized with (GTG), [641. Table 2. Simple tandem repeat specific oligonucleotides informative in domestic animals
-
Species
Number of probed investigated
Informative probesb)
a) For list of probes used see Section 2. b) Oligonucleotide probes revealing at least 4 polymorphic bands per individual if hybridized to DNA digested with the restriction enzyme Hinfl. Gels were prepared using DNA of (i) three unrelated Holstein Friesian cattle, (ii) three unrelated of the Merino sheep, (iii) goats of three different breeds (“Deutsche Edelziege”, “Burenziege”, “Heidschnucke”), (iv) three pigs of the German Landrace breed, (v) three unrelated German halfbreeds (“Hannoveraner”), (vi) three Beagles and (vii)individualsofthree chicken strains see legend to Fig. 2).
Figure I . DNA fingerprints of cattle of different breeds with (CAC),. DNA (10 pg) of each cattle were digested with Hinfl, electrophoresed on a 0.7 % agarose gel and hybridized with (CAC), as described in Section 2. Lanes show fingerprints of cattle of the Holstein Friesian (l), (2), the Red Pied (3), (5) and the Simmental breed (4), (6). Molecular weight markers are given in kilobases of length.
As in mammalian species, oligonucleotide finterprinting was also used for the analysis of poultry. Here the colony-bred strains CC and CB were compared with the random-bred strain CH. Originally, the Prague strains CB and CC had been regarded as inbred isogenic 1671. Later they were kept for colony breeding in Innsbruck. Figure 2 shows Hinfl-digested and electrophoretically separated DNA of male and female specimen after hybridization with several oligonucleotide probes. The three oligonucleotides, (CAC),, (GGAT),, and (GT),, yielded a differentiating banding pattern in the strains CB, CC, and CH. Up to 30 fragments with a size of 2 kilobases (kb) to >25 kb were observed for each of the three oligonucleotides. Probe (GA), was less discriminating as compared to the other three simple repetitive oligonucleotides. Only a few bands could be demonstrated in the molecular weight range of 4 kb. However, the latter oligonucleotide distinguished the sexes. A (GA), band with a molecular weight of about 24 kb could only be observed in hens. This is due to accumulation of (GA)” sequences on the W chromosome of females. Even more pronounced differencesbetween the sexes can be obtained by using oligonucleotide (TCC), (I. Nanda et al., this issue). Using oligonucleotides (CAC)S, (GGAT),, and (GT),, differences between strains CB and CC could be observed (see arrows in Fig. 2). Extensive family studies have shown that the fingerprint bands with all the aforementioned probes are inherited according to Mendelian laws (I. Nanda et al., unpublished data).
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Figure 2. DNA fingerprints of chicken of thestrainsCC (C),CB(B)andCH(H) [671 with different oligonucleotideprobes. HinfI digested DNAs of a male and a female of each strain were hybridized with oligonucleotides (CAC),, (GA),, (GGAT),, and (GT), as described in Section 2. Molecular weight markers are given in kilobases of length.
4 Discussion
4.2 Applications
4.1 Oligonucleotide fingerprinting
From the properties of DNA fingerprints (e.g.,demonstration of highly polymorphic, heritable characters from different sources of tissues, independent of age, sex, or most environmental influences) a broad range of applications arises in domestic animals. As in man, identification of individuals is also important in domestic animals, e.g., for the control of semen used in artificial insemination, the identification of cell lines, and chimeras [32,761. Furthermore, DNA fingerprinting, e. g. using skin samples, allows the diagnosis of zygosity even in species showing blood cell chimerism due to placental anastomoses. Paternity checks are performed in domestic animals in forensic cases (if paternity has been disputed by the owner of an animal) or for special breeding purposes, e.g.,for calves born after embryo transfer.
Oligonucleotide fingerprinting provides a powerful tool to demonstrate genetic variability in man, animal species, and plants. Here we investigated several species of domestic animals. At least two oligonucleotide probes were found to be informative in the investigated species although in some species only a relatively small number of probes was examined. Oligonucleotide (TG),, was recently reported to reveal highly variable banding patterns in the horse, sheep, and chicken 1681. These results confirm previous data that simple repetitive DNA is ubiquitously dispersed over all eucaryotic genomes and promise the rapid introduction of DNA fingerprintingin additional species of domestic animal such as fish (carps, salmonids [611; J . T. Epplen and M. Schartl, unpublished data) or honeybees.
DNA fingerprinting should also prove to be useful in investigating the phylogeny and genetic structuring of populaThe use of oligonucleotidefingerprinting provides some tech- tions in particular cases, although some doubt has been raised nical advantages that are important for practical individual- about the reliability of calculating more distant relationships ization studies: (i) Oligonucleotideprobes are chemically syn- [771. The control of inbred lines [781 and determination of thesized, thus bypassing problems concerning alteration of genetic relationships [3 1, 791 has already been realized by repetitive sequences which may occur if highly repetitive DNA fingerprinting. Thus oligonucleotide fingerprinting can sequences are cloned [ 69,701. (ii)Hybridization directly in the be used as a powerful tool in the analysis of genetic relationgel is much faster and avoids loss of DNA as compared to ships. In poultry, the highly discriminating oligonucleotides Southern blot procedures [7 11. (iii) Appropriate conditions (CAC)S, (GGAT)*, and (GT), have enough discriminatory constrain hybridization if sequences arenot perfectly matched potential to differentiate between individual chickens as well [72]. Thus reproduciblefingerprints are obtained. (iv) The use as all strains. Furthermore, the genetic make-up of inbred of nonradioactively labeled oligonucleotides, recently devel- chickens can be evaluated [80]. By establishing a fingerprint oped for hybridization directly in the gel [73-751, enables database for farm animals, genetic relationships can be DNA fingerprinting to be introduced into nonspecialized verified. Such data may be useful, e. g.for selecting lines or individuals in cross-breeding programs. laboratories.
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4.3 Genetic linkage analysis
DNA fingerprinting in domestic animals
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[ill Jeffreys, A. J., Wilson,V., Thein, S. L., Weatherall, D. J. and Ponder,
B. A. J., Am. J . Hum. Genet. 1986,39, 11-24.
Apart from these applications, fingerprinting can be utilized to [ 121 Wong, Z., Wilson, V., Jeffreys, A. J. and Them, S. L., Nucleic Acids Res. 1986,14,4605-4616. establish genetic linkage maps. Relatively littleis known about [131 Singer, M. F., Int. Rev. Cyfol. 1982, 76, 67-1 12. the genetic maps of domestic animals (e.g. [81,821). VNTR [141 Schacker, U., Schneider, P. M., Holtkamp, B., Bohnke, E., Fimmers, probes are expected to be a powerful tool to increase the R., Sonneborn, H. H. and Rittner, C., Forensic Sci. Znt. 1989, 44, number of available genetic markers. In human metaphase 209-224. chromosomes, the (CAC)d(GTG), loci coincide with the R [151 Higgs, D. R., Goodbourn, S. E. Y., Wainscoat, J. S., Clegg, J. B. and bands and are therefore spread over the whole genome [731. Wetherall, D. J., Nucleic Acids Res. 1981,9,4213-4214. This is an advantage for linkage analysis as compared to [ 161 Bell,G.I.,Selby,M. J.andRutter, W. J., Nature 1982,295,3 1-35. minisatellite loci, which are reported to be localized on [ 171 Knott, T. J., Wallis, S. C., Pease, R. J., Powell, L. M., Scott,J., Nucleic Acids Res. 1986,14,9215-9216. telomeric regions [83,841. In contrast to man, large pedigrees can be designed in domestic animals, enabling efficientlinkage [181 Cooke, H. and Smith, B. A., Cold Spring Habor Symp. Quant. Biol. 1986,51,213-219. studies. These can be used to identify disadvantageous genes [191 Inglehearn, C. F. and Cooke, H. J., Nucleic Acids Res. 1990, 18, (“defect genes”) or to monitor advantageous genes. Even 471-476. more complex traits can be investigated by linkage analysis [20] Fowler, J. C . S., Drinkwater, R., Burgoyne, L., Skinner, J. D., Nucleic using multilocus VNTRs [341. By cloning asingle band, which Acids Res. 1987,15,3929. has been shown to be linked to the character investigated, out [211 Georges, M., Cochaux, P., Lequarre, A.-S., Young, M. W. and Vassart, G., Nucleic Acids Res. 1987,15, 7 193. of a whole set of fingerprint patterns [851, it is possible to develop multiallelic marker systems. Finally, this may be a [22] Vassart, G., Georges, M., Monsieur, R., Brocas, H., Lequarre, A. S., Cristophe, D., Science 1987,235,683-684. step towards isolating candidate genes for complex inherited characteristics such as the litter size in pigs, or resistance to [231 Fowler, J. C. S., Gill, P., Werret, D. J. and Higgs, D. R., Hum. Genet. 1988,79,142-146. disease. Multilocus VNTRs are useful tools for linkage [241 Jeffreys, A. J. and Morton, D. B., Anim. Genet. 1987,18, 1-15. analysis in domestic animals until the genetic map is com- [251 Georges, M., Lequarre, A.-S., Castelli, M., Hanset, R. and Vassart, pleted. Nevertheless, single-locus VNTRs that flank imporG., Cytogenet. Cell Genet. 1988,47, 127-131. tant coding regions will be the goal of animal breeders because [261 Kashi, Y., Iraqui, F., Tikochinski, Y., Ruzitsky, B., Nave, A., they greatly facilitate the broad application of linkage studies Beckmann, J. S., Friedmann, A., Soller, M. and Gruenbaum, Y., Genomics 1990,7,3 1-36. in breeding programs. For example, it may be reasonable to use probes linked to quantitative trait loci as well as probes [271 Hillel, J., Plotzky, Y., Haberfold, A., Lavi, U., Cahaher, A. and Jeffreys, A. J.,Anim. Genet. 1989,20, 145-155. suitable to investigate important genes directly (e.g., the milk A. J., Wilson, V., Kelly, R., Taylor, B. A. and Bulfield, G., [281 Jeffreys, proteins in cattle [861) for marker assistant selection [871, or Nucleic Acids Res. 1987, 15, 2823-2836. combined with Y-specific probes [33, 881 to investigate [291 Kominami, R., Mitani, K. and Muramatsu, M., Nucleic Acids Res. preimplantation embryos. 1988,16, 1197. We thank Dr. Indrajit Nanda f o r providing the data on chicken Jingerprints, Dr. Karel Hala for having provided the chicken blood and Dr. Linington for critically reading the manuscript. The oligonucleotide probes are subject to patent applications. Commercial enquiries should be directed to Fresenius AG, Oberursel, Germany. Received July 5, 1990
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Electrophoresis 1991,12, 169-174 1701 Wong, Z., Wilson, V., Thein, S. L., Weatherall, and Ponder, B. A. J., Ann. Hum. Genet. 1987,51,269-288. [711 Reed, C. K. and Mann, D. A., Nucleic Acids Res. 1985, 13, 7207-722 1. [721 Itakura, K.,Rossi, J. J. and Wallace, R. B.,Annu.Rev.Biochem. 1984, 53,323-356. [731 Zischler, H., Nanda, I., Schafer, R., Schmid, M., Epplen, J. T., Hum. Genet. 1989,82,227-232. 1741 Zischler, H., Schafer, R. and Epplen, J. T., Nucleic Acids Res. 1989, 17, 4411. [751 Schafer, R., Zischler, H. and Epplen, J. T., Nucleic Acids Res. 1988, 16,9344. [761 Farber, C. M., Georges, M., De Bock, G., Verhest, A., Simon, P., Verschraegen,M. andVassart,G., Hum. Genet. 1990,82,197-198. [771 Lynch, M., Mol. Biol. Evol. 1988,5,584-599. [781 Kurz, T. W., Montano, M., Chan, L. and Kabra, P., Hypertension 1989,I3,188-192. [791 Gilbert, D. A., Lehman, N., O’Brien, S. J. and Wayne, R. K., Nature 1990,344,764-767. [801 Epplen, J. T., Kamrnerbauer, C., Steimle,V., Zischler, H., Albert, E., Andreas, A., Hala, K., Nanda, I., Schmid, M., RielJ, 0. and Weising, K. in: Radola, B. J. (Ed.), Electrophoresis Forum ’89, Technische Universitat Miinchen 1989, pp. 175-186. [811 Fries, R. and Ruddle, F. H., in: John, J. S. (Ed.), 10th Bebville Symposium in Agricultural Research: Biotechnology for Solving Agricultural Problems 1986,IO, 19-37. [821 Fries, R., Beckmann, J. S., Georges, M., Soller, M. and Womak, J., Anim. Genet. 1989,20,3-29. [831 Royle, N. J., Clarkson, R. E., Wong, Z. and Jeffreys, A. J., Genomics 1988,3,352-360. [841 Wells, R. A., Green, P. L. and Reeders, S. T., Genomics 1989, 5, 761-772. 1851 Wong, Z., Wilson, V., Jeffreys, A. J.,Thein, S. L.,NucleicAcids Res. 1986,14,4605-4616. [86] Kiihn, C.,DoctoralThesis,TierarztlicheHochschuleHannover,Germany, 1989. 1871 Geldermann, H., Pieper, U. and Roth, B., Theor. Appl. Genet. 1985, 70, 138-146. [881 Leonard, H., Kirszenbaum,M.,Cotinit, C.,Chesme, P., Heyman,Y., Stinnadre, M. G., Bishop, C., Delouis, C., Vaiman, M. and Fellous, M., Theriogenolugy 1987,27,248. 1891 Broad, T., Biotechnulogy - A Major Contributor to New Zealand’s Future Growth, DSIR, Palmerston, New Zealand 1988.
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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
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terns in different species. Initially Jeffreys and Morton [241 used both probes in dogs and cats; subsequently, Georges et al. [25l generated DNA fingerprints in cattle, horse, swine, and dog with a polymer of “Jeffreys core-sequence”. Fingerprints in cattle were also obtained with the original Jeffreys probe, 33.6, as well as with abovinecloneisolatedfrom a genomic library by colony hybridization 1261. “Jeffreys probes” were also used to obtain fingerprints in different species of poultry [271 and mice [281. In the mouse, a clone (mo-1) isolated from a murinegenomiclibrary by screening with“Jeffreys core-sequence” was also found to produce hypervariable banding patterns [291. Two further minisatellite probes cloned from genomic DNA of bird by colony hybridization with probes 33.6 and 33.15 gave highly variable banding patterns in birds as well as in some mammals, including mice [30]. Vassart et al. [22] described the use of DNA from bacteriophage M13 to obtain multilocus VNTRpatterns in man and in cattle. A more detailed investigation of different cattle, pigs, horses, and dogs was carried out by Georges et al. [251. They found the M13 probe to be highly informative in all the species investigated. M 13 was also informative in the chicken [3 1, 321. A further murine probe (pSP642.5GI) used by Georges et al. [211 to fingerprint domestic animals is a genomic clone related to the Drosophila “Per” gene which had also been applied to DNA fingerprinting in man. This probe was reported to be informative in dog, swine, and horse [251. In cattle this probe displayed Y chromosome-specific hybridization patterns [33l, thus providing amarker to monitor male genealogy. Further, the probe pINS3 10,containing a human minisatellite on the 5’ end ofthe insulingene [ 161, also yieldedcomplex banding patterns [341. Additional human minisatellite probes, initially developed by Nakamura el al. [51 using different oligonucleotides specific to known consensus sequences, were used to obtain fingerprints in cattle (probe EFD134.7) 1341, pig (probes pYNH24 and pYNA23) [35l, and horse (probe pCMM68) [361. Coppieters et al. [37] discovered a pig minisatellite suitable for multilocus fingerprinting of swine, horse, and rabbit. In addition, this clone revealed single-locus VNTR patterns if used under highly stringent hybridization conditions. Perret et al. [331 have also reported some locus-specific bovine probes, cloned by screening a male-specific sequence.
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Southern blot experiments, using as a probe a genomic clone containing (GATA),sequences derived from Drosophila, display hypervariable banding patterns in humans [54]. Twelve individuals were investigated but no information was given about the mean number or the inheritance of bands. A Bkm clone was also used to produce DNA fingerprints in rainbow trout [481. Ali et a. [ 561 used different oligonucleotide probes specific to sqr sequences, thereby establishing the oligonucleotide fingerprinting technique. Schafer et al. [381 improved the technique of oligonucleotide fingerprinting using a variety of additional synthetic probes consisting of repeat units of 2 to 4 base pairs (bp). So far, str sequences have been found in every eucaryotic genome, but they display species-specific occurrence and distribution [571. Appropriate str sequences can therefore be applied to demonstrate genomic variations in any eucaryotic individual [58] and were recently used in different genera such as plants (Beyermann et al., submitted; [59,601), ciliates, grasshoppers and leek (J. Epplen, personal communication), poeciliid fish [611,mice [46,57,62I,Rhesus monkeys [63l, and cattle [64,651. This paper deals with informative oligonucleotide probes for different domestic animals. Several different oligonucleotides specific to str sequences were used for the hybridization of DNAs of unrelated individuals of different species. Fingerprints from informative probes were investigated further in order to obtain data for their practical application in domestic animals, e.g., the mean number of polymorphic bands per individual or the inheritance of fragments.
2 Materials and methods
Blood samples were taken from individualsof different species of domestic animals (cattle, sheep, goat, pig, horse, dog, and chicken). Animals from different breeds (see legend to Table 2) were included to obtain high genetic diversity, which facilitates the selection of probes. Peripheral blood was collected in 0.2 x volume of citrate iwlution (0.2 % w/v NaC1, 0.8 % w/v trisodium citrate x 2H,O). DNA was isolated, esA second class of hypervariable loci comprises tandemly sentially as described by Schmidtke et al. [661. DNA (5-10 repeated, short (< 10 bp) simple-sequence motives, e.g., pg) from each animal was digested overnight with 5 U/pg . . .CAC CAC CAC.. . or . .GT GT GT.. ., which were DNA of different restriction enzymes (Hinfl,HaeII1,AluI) as therefore termed simple tandem repeats (str) [381 or, in recommended by the manufacturer, in the presence of 4 mM analogy with the expression minisatellites, “microsatellites” spermidine and 0.1 mg/mL bovine serum albumin. Before [391. Such str sequences were first described by Skinner et al. loading onto the gel, all DNA samples were extracted twicein [40] in the DNA of a hermetic crab. Subsequent investigations phenol-chloroform, precipitated in ethanol, and either disfocused on specific aspects of str biochemistry and biology, solved in TAE (40 mM Tris, 12 mM sodium acetate, 2 mM e. g., the sex-specific organization of str sequences in eu- EDTA, pH 8.3) or TBE buffer (89 mMTris, 89 mM borate, 2.5 caryotic genomes [26, 41-481, the transcription of these mM EDTA, pH 8.2). This was done to minimize background repetitive sequences [49-541, and the possible functions of signals, due to hybridization of the oligonucleotide to protein (GT)” sequences in the regulation of transcription [551. contamination from the restriction digestion. The DNA fragments were separated on 0.7-0.8 % agarose gels in TAE or The first str (to obtain DNA fingerprints) consisted of simple TBE buffer for up to 50 h at 1.5 V/cm. Then the gels were dried quadruplet repeat (sqr) sequences consisting of GATA/ on filter paper (Schleicher & Schiill, GB002) on avacuum-gel GACA repeats. Such sequences were initially found in the dryer for 30 min at room temperature and, in addition, for 60 form of sex-specific satellite DNA of female (heterogametic) min at 60 OC. DNA fragments were denatured for 30 min in colubrid snakes (therefore called Bkm, from banded krait 0.5 M NaOH/O. 15 M NaCl and neutralized for 30 min in 0.5 M minor satellite) [411. The identification of these GATA/ Tris/O.lS M NaC1, pH 8.0, at room temperature. Prior to GACA sequences was obtained by sequencing of W hybridization, gels were equilibrated in 6 x SSC (0.9 M NaC1, chromosome-specific DNA of the snake Elaphe rudiata [43]. 0.09 M sodium citrate). Labeling as well as hybridization of
.
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oligonucleotide probes was carried out as described by Schafer et al. [381. Hybridization temperatures for the different probes were: 27 OC for (AT),, 35 OC for (GATA),, 43 “C for (CT),, (GA),, (GAA),, (GACA),, (GGAT),, and (GT),, 45 “C for (GTG)5 and (TCC),, 47 “C for (GATA),)(GACA)(GATA>,, and 59 OC for (GC),.
3 Results At least two unrelated individuals of each species were investigated to generate information about the different simple repetitive sequence exhibiting length polymorphisms resolvable by agarose gel electrophoresis. Out of 92 principally different str motifs up to 4 bp long (calculated as 4” ‘+2, n12, where n is the length of the simple repeat motif; see [ 57]), a number of oligonucleotides composed of str sequences (see Section 2) was selected with regard to availability for the hybridization experiments. The resulting autoradiograms were analyzed with respect to occurrence, intensity, and number of polymorphic bands. As in other eucaryotic genomes, notable stretches of str sequences, also exist in the genome of investigated domestic animals. In each species, different oligonucleotide probes yield highly polymorphic banding patterns, as listed in Table 2. For practical applications a larger number of animals was investigated to determine the mean number of polymorphic bands per individual and band sharing rates. In cattle, unrelated individuals from three differente breeds were investigated. Figure 1 depicts the DNA fingerprints of 6 unrelated cattle from the Holstein Friesian, Red Pied, and Simmental breeds, as obtained with oligonucleotide probe (CAC),. To prove somatic stability of the fingerprint bands, DNAs isolated from different tissues(blood, muscle, skin) and semen were digested with restriction enzymes and hybridized with (GTG)5 (unpublished results). Furthermore, fingerprint bands in cattle are transmitted from parents to offspring according to Mendelian laws. Within 9 complete families, 52 out of 53 fragments of the DNA fingerprints of the calves could be traced to one of the parents, using Hinfl-digested DNA hybridized with (GTG), [641. Table 2. Simple tandem repeat specific oligonucleotides informative in domestic animals
-
Species
Number of probed investigated
Informative probesb)
a) For list of probes used see Section 2. b) Oligonucleotide probes revealing at least 4 polymorphic bands per individual if hybridized to DNA digested with the restriction enzyme Hinfl. Gels were prepared using DNA of (i) three unrelated Holstein Friesian cattle, (ii) three unrelated of the Merino sheep, (iii) goats of three different breeds (“Deutsche Edelziege”, “Burenziege”, “Heidschnucke”), (iv) three pigs of the German Landrace breed, (v) three unrelated German halfbreeds (“Hannoveraner”), (vi) three Beagles and (vii)individualsofthree chicken strains see legend to Fig. 2).
Figure I . DNA fingerprints of cattle of different breeds with (CAC),. DNA (10 pg) of each cattle were digested with Hinfl, electrophoresed on a 0.7 % agarose gel and hybridized with (CAC), as described in Section 2. Lanes show fingerprints of cattle of the Holstein Friesian (l), (2), the Red Pied (3), (5) and the Simmental breed (4), (6). Molecular weight markers are given in kilobases of length.
As in mammalian species, oligonucleotide finterprinting was also used for the analysis of poultry. Here the colony-bred strains CC and CB were compared with the random-bred strain CH. Originally, the Prague strains CB and CC had been regarded as inbred isogenic 1671. Later they were kept for colony breeding in Innsbruck. Figure 2 shows Hinfl-digested and electrophoretically separated DNA of male and female specimen after hybridization with several oligonucleotide probes. The three oligonucleotides, (CAC),, (GGAT),, and (GT),, yielded a differentiating banding pattern in the strains CB, CC, and CH. Up to 30 fragments with a size of 2 kilobases (kb) to >25 kb were observed for each of the three oligonucleotides. Probe (GA), was less discriminating as compared to the other three simple repetitive oligonucleotides. Only a few bands could be demonstrated in the molecular weight range of 4 kb. However, the latter oligonucleotide distinguished the sexes. A (GA), band with a molecular weight of about 24 kb could only be observed in hens. This is due to accumulation of (GA)” sequences on the W chromosome of females. Even more pronounced differencesbetween the sexes can be obtained by using oligonucleotide (TCC), (I. Nanda et al., this issue). Using oligonucleotides (CAC)S, (GGAT),, and (GT),, differences between strains CB and CC could be observed (see arrows in Fig. 2). Extensive family studies have shown that the fingerprint bands with all the aforementioned probes are inherited according to Mendelian laws (I. Nanda et al., unpublished data).
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Figure 2. DNA fingerprints of chicken of thestrainsCC (C),CB(B)andCH(H) [671 with different oligonucleotideprobes. HinfI digested DNAs of a male and a female of each strain were hybridized with oligonucleotides (CAC),, (GA),, (GGAT),, and (GT), as described in Section 2. Molecular weight markers are given in kilobases of length.
4 Discussion
4.2 Applications
4.1 Oligonucleotide fingerprinting
From the properties of DNA fingerprints (e.g.,demonstration of highly polymorphic, heritable characters from different sources of tissues, independent of age, sex, or most environmental influences) a broad range of applications arises in domestic animals. As in man, identification of individuals is also important in domestic animals, e.g., for the control of semen used in artificial insemination, the identification of cell lines, and chimeras [32,761. Furthermore, DNA fingerprinting, e. g. using skin samples, allows the diagnosis of zygosity even in species showing blood cell chimerism due to placental anastomoses. Paternity checks are performed in domestic animals in forensic cases (if paternity has been disputed by the owner of an animal) or for special breeding purposes, e.g.,for calves born after embryo transfer.
Oligonucleotide fingerprinting provides a powerful tool to demonstrate genetic variability in man, animal species, and plants. Here we investigated several species of domestic animals. At least two oligonucleotide probes were found to be informative in the investigated species although in some species only a relatively small number of probes was examined. Oligonucleotide (TG),, was recently reported to reveal highly variable banding patterns in the horse, sheep, and chicken 1681. These results confirm previous data that simple repetitive DNA is ubiquitously dispersed over all eucaryotic genomes and promise the rapid introduction of DNA fingerprintingin additional species of domestic animal such as fish (carps, salmonids [611; J . T. Epplen and M. Schartl, unpublished data) or honeybees.
DNA fingerprinting should also prove to be useful in investigating the phylogeny and genetic structuring of populaThe use of oligonucleotidefingerprinting provides some tech- tions in particular cases, although some doubt has been raised nical advantages that are important for practical individual- about the reliability of calculating more distant relationships ization studies: (i) Oligonucleotideprobes are chemically syn- [771. The control of inbred lines [781 and determination of thesized, thus bypassing problems concerning alteration of genetic relationships [3 1, 791 has already been realized by repetitive sequences which may occur if highly repetitive DNA fingerprinting. Thus oligonucleotide fingerprinting can sequences are cloned [ 69,701. (ii)Hybridization directly in the be used as a powerful tool in the analysis of genetic relationgel is much faster and avoids loss of DNA as compared to ships. In poultry, the highly discriminating oligonucleotides Southern blot procedures [7 11. (iii) Appropriate conditions (CAC)S, (GGAT)*, and (GT), have enough discriminatory constrain hybridization if sequences arenot perfectly matched potential to differentiate between individual chickens as well [72]. Thus reproduciblefingerprints are obtained. (iv) The use as all strains. Furthermore, the genetic make-up of inbred of nonradioactively labeled oligonucleotides, recently devel- chickens can be evaluated [80]. By establishing a fingerprint oped for hybridization directly in the gel [73-751, enables database for farm animals, genetic relationships can be DNA fingerprinting to be introduced into nonspecialized verified. Such data may be useful, e. g.for selecting lines or individuals in cross-breeding programs. laboratories.
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4.3 Genetic linkage analysis
DNA fingerprinting in domestic animals
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[ill Jeffreys, A. J., Wilson,V., Thein, S. L., Weatherall, D. J. and Ponder,
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Apart from these applications, fingerprinting can be utilized to [ 121 Wong, Z., Wilson, V., Jeffreys, A. J. and Them, S. L., Nucleic Acids Res. 1986,14,4605-4616. establish genetic linkage maps. Relatively littleis known about [131 Singer, M. F., Int. Rev. Cyfol. 1982, 76, 67-1 12. the genetic maps of domestic animals (e.g. [81,821). VNTR [141 Schacker, U., Schneider, P. M., Holtkamp, B., Bohnke, E., Fimmers, probes are expected to be a powerful tool to increase the R., Sonneborn, H. H. and Rittner, C., Forensic Sci. Znt. 1989, 44, number of available genetic markers. In human metaphase 209-224. chromosomes, the (CAC)d(GTG), loci coincide with the R [151 Higgs, D. R., Goodbourn, S. E. Y., Wainscoat, J. S., Clegg, J. B. and bands and are therefore spread over the whole genome [731. Wetherall, D. J., Nucleic Acids Res. 1981,9,4213-4214. This is an advantage for linkage analysis as compared to [ 161 Bell,G.I.,Selby,M. J.andRutter, W. J., Nature 1982,295,3 1-35. minisatellite loci, which are reported to be localized on [ 171 Knott, T. J., Wallis, S. C., Pease, R. J., Powell, L. M., Scott,J., Nucleic Acids Res. 1986,14,9215-9216. telomeric regions [83,841. In contrast to man, large pedigrees can be designed in domestic animals, enabling efficientlinkage [181 Cooke, H. and Smith, B. A., Cold Spring Habor Symp. Quant. Biol. 1986,51,213-219. studies. These can be used to identify disadvantageous genes [191 Inglehearn, C. F. and Cooke, H. J., Nucleic Acids Res. 1990, 18, (“defect genes”) or to monitor advantageous genes. Even 471-476. more complex traits can be investigated by linkage analysis [20] Fowler, J. C . S., Drinkwater, R., Burgoyne, L., Skinner, J. D., Nucleic using multilocus VNTRs [341. By cloning asingle band, which Acids Res. 1987,15,3929. has been shown to be linked to the character investigated, out [211 Georges, M., Cochaux, P., Lequarre, A.-S., Young, M. W. and Vassart, G., Nucleic Acids Res. 1987,15, 7 193. of a whole set of fingerprint patterns [851, it is possible to develop multiallelic marker systems. Finally, this may be a [22] Vassart, G., Georges, M., Monsieur, R., Brocas, H., Lequarre, A. S., Cristophe, D., Science 1987,235,683-684. step towards isolating candidate genes for complex inherited characteristics such as the litter size in pigs, or resistance to [231 Fowler, J. C. S., Gill, P., Werret, D. J. and Higgs, D. R., Hum. Genet. 1988,79,142-146. disease. Multilocus VNTRs are useful tools for linkage [241 Jeffreys, A. J. and Morton, D. B., Anim. Genet. 1987,18, 1-15. analysis in domestic animals until the genetic map is com- [251 Georges, M., Lequarre, A.-S., Castelli, M., Hanset, R. and Vassart, pleted. Nevertheless, single-locus VNTRs that flank imporG., Cytogenet. Cell Genet. 1988,47, 127-131. tant coding regions will be the goal of animal breeders because [261 Kashi, Y., Iraqui, F., Tikochinski, Y., Ruzitsky, B., Nave, A., they greatly facilitate the broad application of linkage studies Beckmann, J. S., Friedmann, A., Soller, M. and Gruenbaum, Y., Genomics 1990,7,3 1-36. in breeding programs. For example, it may be reasonable to use probes linked to quantitative trait loci as well as probes [271 Hillel, J., Plotzky, Y., Haberfold, A., Lavi, U., Cahaher, A. and Jeffreys, A. J.,Anim. Genet. 1989,20, 145-155. suitable to investigate important genes directly (e.g., the milk A. J., Wilson, V., Kelly, R., Taylor, B. A. and Bulfield, G., [281 Jeffreys, proteins in cattle [861) for marker assistant selection [871, or Nucleic Acids Res. 1987, 15, 2823-2836. combined with Y-specific probes [33, 881 to investigate [291 Kominami, R., Mitani, K. and Muramatsu, M., Nucleic Acids Res. preimplantation embryos. 1988,16, 1197. We thank Dr. Indrajit Nanda f o r providing the data on chicken Jingerprints, Dr. Karel Hala for having provided the chicken blood and Dr. Linington for critically reading the manuscript. The oligonucleotide probes are subject to patent applications. Commercial enquiries should be directed to Fresenius AG, Oberursel, Germany. Received July 5, 1990
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