Chromosoma (1998) 107:587±594
Springer-Verlag 1998
The chromosomal organization of simple sequence repeats in wheat and rye genomes Angeles Cuadrado*, Trude Schwarzacher John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received: 13 February 1998; in revised form: 18 August 1998 / Accepted: 18 August 1998
Abstract. The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)12, (CAT)5, (AAG)5, (GCC)5 and, in particular, (GACA)4 hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)4 at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae).
Introduction Simple-sequence repeats (SSRs) or microsatellites are a class of repetitive DNA sequences widespread in eukaryotic genomes (Tautz and Renz 1984). They consist of short motifs, 1±5 bp long, repeated in tandem arrays with identical, composite or degenerate motifs. They are not only highly abundant within genomes, but have a high frequency of variation in the number of repeats in differ*Present address: Department of Cell Biology and Genetics, University of Alcala de Henares, Madrid, Spain Edited by: D. Schweizer Correspondence to: T. Schwarzacher e-mail:
[email protected]
ent individuals. In general, microsatellite DNAs have been found at a lower frequency in plants than animals, and it has been estimated that on average a dinucleotide repeat longer than 20 bp in length occurs every 33 kb in plant genomes compared with every 6 kb in mammals (Wang et al. 1994). In humans (GT) motifs dominate (Lagercrantz et al. 1993) while different sequences were found most abundantly in plants, indicating disparities in genome organization, and possibly in maintenance and role of SSRs, in plants and animals. Hybridization of synthetic SSRs to genomic DNA digests has proven useful in the creation of fingerprints that are often specific to single varieties or even individuals (Weising et al. 1989; Schmidt et al. 1993; Depeiges et al. 1995). Members of particular subclass of SSRs, flanked by DNA sequences that are present, ideally, only once in the genome, provide valuable genetic markers (Weber and May 1989; Powell et al. 1996) and are now described by the term `microsatellite markers'. Because of their high abundance and efficiency in detecting variation, microsatellite markers have become a widespread analytical tool in animal, human and plant genetics for identification of individuals, species and varieties; evolution, ecology and population studies; as markers for traits and genes; and in the generation of genetic maps (e.g. Weissenbach et al. 1992; Morgante and Olivieri 1993; Zietkiewicz et al. 1994; Powell et al. 1996; Bryan et al. 1997; Fang and Roose 1997; Röder et al. 1998). Relatively few studies have examined the long-range chromosomal organization and distribution of SSRs using in situ hybridization. In fish and primates (Nanda et al. 1991), clustering of SSRs was shown in different chromosomal regions, such as some heterochromatin, nucleolus-organizing regions and R-bands. In barley and many other species of the tribe Triticeae (Gramineae), in situ hybridization of the polypurine motif (GAA)7, or a GAA-rich repeated DNA clone, resulted in a specific chromosomal distribution pattern that correlated closely with heterochromatic N-bands (Pedersen et al. 1996). Schmidt and Heslop-Harrison (1996) have
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shown that, in beet, each of seven SSRs analysed has a characteristic genomic distribution and motif-dependent dispersion with site-specific amplification on one to seven pairs of centromeres or intercalary chromosomal regions and weaker, dispersed hybridization along chromosomes. Similarly, in chickpea, distribution and intensity of signals varied depending on the SSR motif used as probe for fluorescence in situ hybridization (Gortner et al. 1998). These studies have shown that SSRs are amplified at specific regions of the genome and are possibly incorporated in higher-order repeat structures. As an important class of DNA sequences they must have implications for genome organization and evolution. In this paper, we aim to characterize the chromosomal distribution of regions enriched in ten SSRs of di-, triand tetra-nucleotide motifs in the genomes of rye (Secale cereale L.) and hexaploid bread wheat (Triticum aestivum L.) closely related species belonging to the tribe Triticeae. We discuss the application of in situ hybridization using SSR probes for the identification of chromosomes and the characterization of genomes between phylogenetically related species, the correlation of SSRs to heterochromatin, and their significance for genome organization and evolution. Materials and methods Rye, S. cereale L. Petkus and Riodeva (2n=14, genomic constitution RR), bread wheat, T. aestivum L. Chinese Spring (2n=6x=42, genomic constitution AABBDD) and triticale, Triticosecale Whittmack Lasko (2n=6x=42, genomic constitution AABBRR) were used. Preparation of root-tip chromosomes followed Schwarzacher et al. (1989) and that of pachytene chromosomes followed Cuadrado et al. (1997). Briefly, excised root tips treated for 24 h with ice-cold water and anthers at pachytene were fixed in 3:1 (v/v) 100% ethanol:glacial acetic acid. The material was enzymatically digested and meristematic or meiotic cells squashed in 45% acetic acid on a glass slide. The synthetic oligonucleotides (AC)8, (AG)12, (AAG)5, (AAC)5, (CAC)5, (CAT)5, (GGC)5, (GACA)4, (GATA)4 and (GGAT)4 were end-labelled with biotin-11-dUTP or digoxigenin-16-dUTP (Boehringer Mannheim) by terminal transferase (Boerhinger Mannheim) following the manufacturer's instructions. Sheared total genomic DNA from rye was labelled with digoxigenin-16-dUTP by nick translation. The clone pSc119.2 containing a 120 bp tandem repeated sequence unit from rye (Bedbrook et al. 1980) was labelled with biotin-16-dUTP by nick translation. In situ hybridization followed Heslop-Harrison et al. (1991) and Schmidt and Heslop-Harrison (1996) with the following modifications. The hybridization mixture contained 5SSPE (0.9 M NaCl, 50 mM NaH2PO4, 5 mM EDTA, pH 7.4), 5Denhardt's solution [1 g/ml Ficoll, 1 g/ml polyvinylpyrrolidone, 1 g/ml bovine serum albumin (BSA) in water], 0.5% sodium dodecyl sulphate, 50 g/ml denatured Escherichia coli DNA and 1±2 pmol labelled SSR probe. Chromosomes and probe were denatured together for 5 min at 70 C and hybridization was carried out at 37 C overnight. Slides were washed in 6SSC (900 mM NaCl, 90 mM sodium citrate adjusted with HCl to pH 7) three times for 30 min at room temperature, followed by a stringent wash in 6SSC at the respective duplex stability temperature (Tm)±5 C for 1±2 min. The Tm was calculated for each probe following Wallace et al. (1981) using 4 Cnumber of G and C bases+2 Cnumber of A and T bases. Washing temperature was 35 C for (AAC)5, (AAG)5, (CAT)5 and (GATA)4, 43 C for (AC)8, (GACA)4 and (GGTA)4 and 45 C for (AG)12, (CAC)5 and (GGC)5. Pairs of SSRs
with the same Tm were differentially labelled (biotin versus digoxigenin) and used simultaneously in double-target in situ hybridization experiments. For detection of signals, slides were incubated in 1 g/ml fluoresceinated anti-digoxigenin (Boehringer Mannheim), 0.025 g/ ml streptavidin-Cy3 (Sigma) in 5% (w/v) BSA in 6SSC, stained with 4',6-diamidino-2-phenylindole and mounted in antifade (see Heslop-Harrison et al. 1991). Probe hybridization sites were visualized with an epifluorescence microscope using single, double and triple excitation with UV, blue and green light. Photomicrographs were taken on Fujicolor Super HG 400 print film. After examination and photography of metaphases, coverslips were taken off carefully and the hybridization procedure was repeated (Heslop-Harrison et al. 1992) using a new combination of SSR motifs or 60±100 ng per slide labelled pSc119.2 or 100±200 ng per slide labelled total genomic rye DNA. For the figures, negatives were digitized to PhotoCD and processed using Adobe Photoshop with only those functions that are applied equally to all pixels in the image.
Results Metaphase chromosomes from wheat (ABD genomes), rye (R genome) and triticale (ABR genomes) were identified individually and to their genome of origin by morphology, and in many cases by re-probing preparations with rye genomic DNA and/or the repeated sequence probe pSc119.2 (e.g. Fig. 1c, d). In triticale, this allowed positive identification of all 21 chromosome pairs, and in rye, of all seven pairs (see Cuadrado et al. 1995; Figs. 1, 2b, d and e). In wheat, pSc119.2 identified all seven B genome chromosome pairs and pair 4A and sometimes pairs 5A, 2D, 3D, 4D and 5D (see Mukai et al. 1993; Figs. 1, 2a, c and 3). We used ten synthetic SSRs as probes for single- and double-target fluorescence in situ hybridization. Differences were seen in the abundance and localization of motifs between the different genomes, although a general distribution pattern emerged (Figs. 1±3, Table 1). In all four genomes, most motifs showed dispersed hybridization extending over the length of the chromosome arms [e.g. (AAG)5 Fig. 1a, (CAT)5 Fig. 1b]. Often, a higher density of signal, or series of bands, was visible at the pericentromeric regions, particularly in the B genome chromosomes, in the long arm of chromosome 4A and in most rye chromosomes using (AG)12, (CAT)5, (AAG)5, (GCC)5 and in particular (GACA)4 (Table 1). Some chromosome arms of rye and wheat showed additional weak sub-telomeric signals (e.g. Figs. 1±3). The remaining A genome chromosomes and all D genome chromosomes showed only few and weak bands (Table 1, Figs. 1, 2a, c and 3). To analyse the relationships of the SSR distributions to each other we carried out both simultaneous and sequential hybridization experiments and used pachytene chromosomes, which are some ten times longer than root-tip metaphase chromosomes, for higher resolution (data not shown). While signal from different probes was often found in similar locations it was rarely entirely co-localized. The SSR sequence (GACA)4 was the only motif we used that gave a distinct distribution pattern on chromosomes from the R and B genomes, as well as chromosome
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Fig. 1a±d. A root-tip metaphase of hexaploid triticale Lasko (ABR genome constitution) after in situ hybridization with digoxigenin-labelled (AAG)5 detected by green fluorescence (a) and biotin-labelled (CAT)5 detected by red fluorescence (b). The preparation was subsequently reprobed with total digoxigenin-labelled rye DNA (green, c) and biotin-labelled pSc119.2 (red, d, two chromosomes at the top right corner are missing from the micrograph due to an internal fault in the microscope). All chromosomes were identi-
fied using chromosome morphology, total genomic rye DNA and the pSc119.2 distribution pattern; one chromosome 6B is missing from this preparation. The simple sequence repeat (SSR) sites are located near the centromeres of all B genome chromosomes, chromosome 4A and some rye chromosomes. Note that the (AAG)5 (a) and (CAT)5 (b) distribution patterns are similar, but not identical. Bar represents 10 m
4A (Figs. 1 to 3). In these chromosomes, a rich pattern of intercalary and some telomeric bands was observed while a few, weak signals were present in other chromosomes of the A and D genome in some metaphase plates. The (GACA)4 distribution pattern of Chinese Spring, taken from three metaphases, was superimposed onto the standard wheat karyotype of Gill et al. (1991) (Fig. 3) and shown to resemble, with only minor differences (see Discussion), the N-banding pattern of wheat chromosomes. The B genome chromosomes from the triticale variety Lasko showed some additional minor changes, notably in chromosome 7B, probably because of intra-genomic polymorphisms. In rye Riodeva, 29 mostly intercalary and pericentromeric bands could be identified after probing with (GACA)4 (Fig. 2d, e), which did not correlate with any known banding pattern (see Discussion). In rye Petkus (not shown), with some minor exceptions, the same bands were identified, although in some cases, notably in chromosome 6R, with different relative strengths. Because of the distinctive distribution pattern,
we used the (GACA)4 hybridization signal to construct a karyotype of rye Riodeva chromosomes (Fig. 2e) using the chromosome arm length information and pSc119.2 karyotype from Cuadrado et al. (1995) and combining the (GACA)4 hybridization sites analysed in five metaphase spreads. Discussion The distinctive hybridization patterns of various SSR motifs presented here, showing characteristics specific to both chromosomes and genomes, are of significance for understanding the evolution of the genome, and are applicable as markers for chromosome identification. The SSR motifs were chosen (Table 1) to have up to 75% homology with each other. Both theoretical considerations of hybridization stringency and the motif-specific hybridization patterns observed [e.g. different results with (AC)8, (AG)12 and (CAGA)4; see Table 1] demonstrate that the
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Fig. 2a±e. Root-tip metaphase chromosomes of wheat Chinese Spring (a, c) and rye Riodeva (b, d) after 4',6-diamidino-2-phenylindole (DAPI) staining (a, b) and in situ hybridization with biotinlabelled (GACA)4 detected by red Cy3 fluorescence (c, d). The distribution pattern with (GACA)4 to wheat chromosomes (a, c) resembles the N-banding pattern and all B genome chromosomes and chromosomes 4A could be identified. One chromosome 1B was obscured lying off the metaphase. The karyotype (e) of rye shows one
chromosome of each type (chromosomes 1±7 of b and d, 1©±7© being the other homologues) stained with DAPI (left), (GACA)4 in situ hybridization signal (middle) and a schematic representation of the banding pattern averaged from five metaphase plates (right). 29 sites (red) with many intercalary locations are described, and identify all 14 chromosome arms. The gap on the short arm of chromosome 1R denotes the nucleolus-organizing region. Bar represents 15 m in a, c and 10 m in b, d
conditions used here are distinct enough that non-specific hybridization or cross-hybridization of probes can be ruled out. It is likely that fluorescence in situ hybridization with SSR oligomers detects some of the single- and low-copy microsatellite loci that are relatively uniformly distributed within plant genomes (Lagercrantz et al. 1993; Wang et al. 1994; Röder et al. 1998). Organized into the higher order metaphase structure, the 20±100 bp microsatellite sites might be localized closely enough to be responsible for the weak but overall dispersed fluorescence that we observed with most motifs to all chromosomes (Figs.
1±3, Table 1; compare also the results in beet, Schmidt and Heslop-Harrison 1996). However, the strong hybridization sites seen at distinct locations along the chromosomes probably represent regions where SSRs are included within larger, tandemly repeated units or where SSRs are present as very large perfect or degenerate arrays. Simple sequence repeat distribution patterns Although the hybridization pattern varied depending on the sequence of the oligomer used, a typical picture of
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Fig. 3. Schematic drawing of (GACA)4 distribution patterns on wheat Chinese Spring. Hybridization sites from three metaphases were overlayed onto the standard karyotype of Gill et al. (1991) and numbers indicate band designations. Sites that were not seen in all cases are drawn in grey. Nucleolus-organizing regions on the short arms of chromosome 1B and 6B are shown as gaps
SSR distribution between the four genomes of wheat and rye emerged (Table 1). In the R genome, bands were found in all seven chromosomes at mainly intercalary sites. In wheat, the strongest hybridization signal and majority of bands were detected on the pericentromeric region of all seven chromosomes of the B genome and chromosome 4A while the remaining chromosomes of
the A and D genome showed only a few weak bands (Fig. 3). The anomalous composition of chromosome 4A and its close relationship to the B genome is well known (Gill et al. 1991). In general, the SSR motif distribution in wheat and rye, with many intercalary and only minor telomeric sites (unlike the heterochromatin banding pattern, see later), is very similar, indicating that their
592 Table 1. Distribution of ten SSRs among the genomes of wheat and rye Type
A genome
B genome
D genome
R genome
(AC)8
weak dispersed
weak dispersed + diffuse pericentromeric sites
weak dispersed
weak dispersed + few weak sites
(AG)12
on 4 A only
weak + some pericentromeric sites
very weak
dispersed + few sharp sites
(CAC)5
dispersed, no clear pattern (strongest on B genome)
(CAT)5
dispersed + sites on 4 A
strong dispersed + pericentromeric sites (not as clear as GACA)
weak dispersed
dispersed + few weak sites
(AAG)5
weak dispersed + sites on 4 A
weak dispersed + very few sites
weak dispersed + few weak sites
(AAC)5
similar to AC
weak dispersed + strong pericentromeric and some telomeric sites
(GGC)5
dispersed
dispersed + a few clear, but weak pericentromeric bands
dispersed
dispersed + few clear bands
(GACA)4
dispersed + few clear bands, strong on 4 A
dispersed + many clear pericentromeric, intercalary and some telomeric sites (N-bands)
dispersed + very few clear bands
dispersed + clear pericentromerc and intercalary sites
(N-bands)
(N-bands +)
(N-bands) (GATA)4
dispersed, no clear bands (strongest on B genome)
(GGAT)4
dispersed + a few bands (strongest on B genome)
observed organization may be an ancient feature of the wheat and rye, and possibly all Triticeae, genomes. In all three wheat genomes, the (GACA)4 and other SSR distribution patterns (Figs. 1, 2a, c and 3, Table 1) corresponded well with the N-band pattern as described by Gill et al. (1991): minor differences involved N-bands in the telomeric position of chromosome 6BS and 7AL that were not found by in situ hybridization with (GACA)4, and (GACA)4-positive bands near the nucleolus-organizing regions on chromosome 1B and 6B that are C-band, but not N-band positive. Also, closely spaced N-bands often appeared as large blocks after SSR in situ hybridization. N-banding, a technique similar to Giemsa C-banding, results from treatment of chromosome preparations with 1 M NaH4PO4, pH 4.15 at 94 C before staining with Giemsa and has been suggested to reveal only heterochromatic polypyrimidine/polypurine DNA sequences (Gill et al. 1991). Pedersen et al. (1996) concluded that as the chromosomal location of the GAA satellite coincides very well with the heterochromatin stainable by N-banding in barley, rye and wheat and possibly the whole tribe Triticeae in general, GAA is indeed the major component of N-bands. Our results in wheat, using ten different SSR motifs with different base pair compositions, of which many are located in N-band regions, indicate that the polypyrimidine/polypurine conformation of the DNA alone is not responsible for N-banding, but that the organization and repetitive nature of the sequences and possibly the proteins associated with such repeats within N-band regions might play a role. For example, banding produced by a rapidly renaturing fraction of DNA also results in N-banding (Gerlach and Peacock
1980) and could correlate with the renaturation properties of SSRs (Traut 1991). We did not find any relationship between the (GACA)4 chromosomal distribution and other banding patterns reported for rye. Three intercalary N-bands, one each on chromosome 2RL, 3RS and 6RL, correspond to intercalary C-bands (Schlegel and Gill 1984), polypyrimidine tract DNA (Appels et al. 1978) and (GAA)7 distribution at high stringency (Pedersen et al. 1996). The (GACA)4 motif under the conditions used here detected these and many more bands (Fig. 2d, e). In general, the SSR signal was excluded from the prominent sub-telomeric C-band-positive heterochromatin (Figs. 1, 2d, e) that is the site of the major repetitive satellite sequence families found in rye pSc74, pSc119.2, pSc200 and pSc250 with repeat units of 120, 350 and 480 bp (Bedbrook et al. 1980; Cuadrado et al. 1995; Vershinin et al. 1995). As probes for in situ hybridization, (GACA)4 allowed the unequivocal identification of all the chromosomes of the B and R genome and has many advantages over Giemsa C-banding. Hence, with its many intercalary hybridization sites and the ease of using it in multitarget experiments, the SSR distribution will be useful for physical mapping projects and for the detection of alien material in breeding lines and chromosomal rearrangements. Use of SSRs in conjunction with other repetitive probes will result in a saturated physical map of the Triticeae genomes and the possibility of analysing cytogenetically small sections of the chromosomes. This will expand our ability to analyse, understand and manipulate the genomes of wheat and rye with unprecedented precision and efficiency.
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Organization of SSR-enriched regions The fact that different SSR motifs were often found at similar, although not identical, physical positions may mean that various motifs are clustered in closely adjacent or interspersed blocks. This agrees with the molecular characterization of various SSR-containing clones in wheat, in which perfect repeats and compound SSRs, consisting of two or three adjacent blocks of different repeat types were found frequently (Bryan et al. 1997; Röder et al. 1998). Similarly, clones of GAA satellite repeat DNA from barley and wheat, although primarily composed of GAA repeats, also included other triplets, such as GAG and GAC (Dennis et al. 1980; Pedersen et al. 1996). The fact that in wheat and rye (GACA)4 revealed most sites after in situ hybridization to chromosomes and that most SSR motifs gave similar patterns (Table 1, Figs. 1±3) is in contrast to the findings of Schmidt and Heslop-Harrison (1996) for sugar beet: (CA)n seems to be most abundant and different SSR motifs showed specific and contrasting distribution patterns, indicating that each motif is amplified and distributed independently. Similarly, in chickpea, the SSRs (CA)8 and (GATA)4 have been found to be confined to heterochromatic regions while others, (A)16, (TA)9 and (AAC)5, showed a euchromatic distribution with centromeric regions being largely excluded (Gortner et al. 1998). Have several SSR motifs in wheat and rye been amplified together? Or are there specific regions in the large Triticeae chromosomes that are suitable for SSR amplification? It is possible that heterochromatic bands (in particular N-bands) might constitute such chromosomal organization, at least, in wheat. In rye, however, the major C-bands seem to be devoid of SSRs and there are few detectable Nbands, indicating that heterochromatin is organized differently in wheat and rye and might have evolved at different times. In vitro experiments suggest that slippage replication is responsible for small-step expansion of SSR stretches and could constitute a major mechanism for DNA sequence evolution of not only short but also long (more than 30 kb) SSR regions (Levinson and Gutman 1987; Schlötterer and Tautz 1992; Hancock 1996). However, large mutational steps are also possible and are presumably generated by processes other than slippage, such as unequal crossover (Levinson and Gutman 1987; McMurray 1995) or when genes involved in DNA mismatch repair are defective (Strand et al. 1993). It is very likely that expanding SSR stretches become compound or interrupted and that they are incorporated in or associated with higher order repeated sequence motifs, as described in several instances (Zischler et al. 1992; Arcot et al. 1995; Pederson et al. 1996; Heslop-Harrison and Schmidt, personal communications). Further amplification would involve unequal crossing over, gene conversion and transposition, as apparently happens with retrotransposons and tandem repeats, resulting in complex interactions at a number of levels (Dover 1993; Ohtsubo and Ohtsubo 1994; Schmidt and Heslop-Harrison 1998). The same basic evolutionary mechanisms of sequence
amplification are thus operational in the evolution of various repeated DNA families and types, but the timing and interaction of different mechanisms with each other might be different and specific for different repetitive classes and species, resulting in varying abundance and distribution patterns of sequence types. Acknowledgements. We thank Dr. P. Stephenson for help with the oligomer synthesis, and Professor J.S. Heslop-Harrison for many fruitful discussions. This work was supported by Univesidad de Alcala Project E009/97, a European Union Fellowship ERBB104CT965123 to A.C. and a BBSRC senior fellowship to T.S.
References Appels R, Driscoll C, Peacock WJ (1978) Heterochromatin and highly repeated DNA sequences in rye (Secale cereale). Chromosoma 70:67±89 Arcot SS, Wang Z, Weber JL, Deininger PL, Batzer MA (1995) Alu repeats: a source for the genesis of primate microsatellites. Genomics 29:136±144 Bedbrook JR, Jones J, O'Dell M, Thompson RD, Flavell RB (1980) A molecular description of telomeric heterochromatin in Secale species. Cell 19:545±560 Bryan GJ, Collins AJ, Stephenson P, Orry A, Smith JB, Gale MD (1997) Isolation and characterization of microsatellites from hexaploid bread wheat. Theor Appl Genet 94:557±563 Cuadrado A, Ceoloni C, Jouve N (1995) Variation in highly repetitive DNA composition of heterochromatin in rye studied by fluorescence in situ hybridization. Genome 38:1061±1069 Cuadrado A, Vitellozzi F, Jouve N, Ceoloni C (1997) Fluorescence in situ hybridization with multiple repeated DNA probe applied to the analysis of wheat-rye chromosome pairing. Theor Appl Genet 94:347±355 Dennis ES, Gerlach WL, Peacock WJ (1980) Identical polypyrimidine-polypurine satellite DNAs in wheat and barley. Heredity 44:349±366 Depeiges A, Goubely C, Lenoir A, Cocherel S, Picard G, Raynal M, Grellet F, Delseny M (1995) Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci. Theor Appl Genet 91:160±168 Dover GA (1993) Evolution of genetic redundancy for advanced players. Curr Opin Genet Dev 3:902±910 Fang DQ, Roose ML (1997) Identification of closely related citrus cultivars with inter-SSR markers. Theor Appl Genet 95:408±417 Gerlach WL, Peacock WJ (1980) Chromosomal locations of highly repeated DNA sequences in wheat. Heredity 44:269±276 Gill BS, Friebe B, Endo TR (1991) Standard karyotype and nomenclature system for description of chromosome bands and structural aberrations in wheat (Triticum aestivum). Genome 34:830±839 Gortner G, Nenno M, Weising K, Zink D, Nagl W, Kahl G (1998) Chromosomal localization and distribution of SSRs and the Arabidopsis-type telomere sequence in the genome of Cicer arietinum L. Chromosome Res 6:97±104 Hancock JM (1996) Simple sequences and the expanding genome. BioEssays 18:421±425 Heslop-Harrison JS, Schwarzacher T, Anamthawat-Jónsson K, Leitch AR, Shi M, Leitch IJ (1991) In situ hybridization with automated chromosome denaturation. Technique 3:109±115 Heslop-Harrison JS, Harrison GE, Leitch IJ (1992) Reprobing of DNA:DNA in situ hybridization preparations. Trends Genet 8:372±373 Lagercrantz U, Ellegren H, Andersson L (1993) The abundance of various polymorphic microsatellite motifs differs between plants and vertebrates. Nucleic Acids Res 21:1111±1115 Levinson G, Gutman GA (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol Biol Evol 4:203± 221
594 McMurray CT (1995) Mechanisms of DNA expansion. Chromosoma 104:2±13 Morgante M, Olivieri AM (1993) PCR-amplified microsatellites as markers in plant genetics. Plant J 3:175±182 Mukai Y, Nakahara Y, Yamamoto M (1993) Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome 36:489±494 Nanda I, Zischler H, Epplen C, Guttenbach M, Schmid M (1991) Chromosomal organization of simple repeated DNA sequences used for DNA fingerprinting. Electrophoresis 12:193±203 Ohtsubo H, Ohtsubo E (1994) Involvement of transposition in dispersion of tandem repeat sequences (TrsA) in rice genomes. Mol Gen Genet 245:449±455 Pedersen C, Rasmussen SK, Linde-Laursen I (1996) Genome and chromosome identification in cultivated barley and related species of the Triticeae (Poaceae) by in situ hybridization with the GAA-satellite sequence. Genome 39:93±104 Powell W, Machray GC, Provan J (1996) Polymorphism revealed by SSRs. Trends Plant Sci 1:215±222 Röder MS, Korzun V, Gill BS, Ganal MW (1998) The physical mapping of microsatellite markers in wheat. Genome 41:278±283 Schlegel R, Gill BS (1984) N-banding analysis of rye chromosomes and the relationship between N-banded and C-banded heterochromatin. Can J Genet Cytol 26:765±769 Schlötterer C, Tautz D (1992) Slippage synthesis of simple sequence DNA. Nucleic Acids Res 20:211±215 Schmidt T, Heslop-Harrison JS (1996) The physical and genomic organization of microsatellites in sugar beet. Proc Natl Acad Sci USA 93:8761±8765 Schmidt T, Heslop-Harrison JS (1998) Genomes, genes and junk: the large-scale organization of plant chromosomes. Trends Plant Sci 3:195±199 Schmidt T, Boblenz K, Metzlaff M, Kaemmer D, Weising K, Kahl G (1993) DNA fingerprinting in sugar beet (Beta vulgaris) identification of double-haploid breeding lines. Theor Appl Genet 85:653±657
Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS (1989) In situ localization of parental genomes in a wide hybrid. Ann Bot 64:315±324 Strand M, Prolla TA, Liskay RM, Petes TD (1993) Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365:274±276 Tautz D, Renz M (1984) Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res 12:4127± 4138 Traut W (1991) Chromosomen: Klassische und Molekulare Cytogenetik. Springer, Berlin Heidelberg New York, 390 pp Vershinin A, Schwarzacher T, Heslop-Harrison JS (1995) The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes. Plant Cell 7:1823±1833 Wallace RB, Johnson MJ, Hirose T, Miyake T, Kawashima EH, Itakura K (1981) The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit û-globin DNA. Nucleic Acids Res 9:879±894 Wang Z, Weber JL, Zhong G, Tanksley SD (1994) Survey of plant short tandem DNA repeats. Theor Appl Genet 88:1±6 Weber JL, May PE (1989) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388±396 Weising K, Weigand F, Driesel AJ, Kahl G, Zischler H, Epplen JT (1989) Polymorphic simple GATA/GACA repeats in plant genomes. Nucleic Acids Res 17:10128 Weissenbach J, Gyapay G, Dib C, Vignal A, Morissette J, Millasseau P, Vaysseix G, Lathrop M (1992) A second-generation linkage map of the human genome. Nature 359:794±801 Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by SSR (SSR)-anchored polymerase chain reaction amplification. Genomics 20:176±183 Zischler H, Kammerbauer C, Studer R, Grzeschik KH, Epplen JT (1992) Dissecting (CAC)5/(GTG)5 multilocus fingerprints from man into individual locus-specific, hypervariable components. Genomics 13: 983±990
