Diversity and evolution of four dispersed repetitive DNA sequences in the genus Secale

We present the first characterization of 360 sequences in six species of the genus Secale of both cultivated and wild accessions. These include four distinct kinds of dispersed repetitive DNA sequences named pSc20H, pSc119.1, pSaO5(411), and pSaD15(940) belonging to the Revolver family. During the evolution of the genus Secale from wild to cultivated accessions, the pSaO5(411)-like sequences became shorter mainly because of the deletion of a trinucleotide tandem repeating unit, the pSc20H-like sequences displayed apparent homogenization in cultivated rye, and the second intron of Revolver became longer. In addition, the pSc20H-, pSc119.1-, and pSaO5(411)-like sequences cloned from wild rye and cultivated rye could be divided into two large clades. No single case of the four kinds of repetitive elements has been inherited by each Secale accession from a lone ancestor. It is reasonable to consider the vertical transmission of the four repetitive elements during the evolution of the genus Secale. The pSc20H- and pSaO5(411)-like sequences showed evolutionary elimination at specific chromosomal locations from wild species to cultivated species. These cases imply that different repetitive DNA sequences have played different roles in the chromosome development and genomic evolution of rye. The present study adds important information to the investigations dealing with characterization of dispersed repetitive elements in wild and cultivated rye.     

Painting the rye genome with genome-specific sequences

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.     

Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale

Eleven weedy or wild species or subspecies of the genus Secale L. were compared with a set of cultivated rye accessions, based on inter-simple sequence repeat (ISSR) markers to analyze their phylogenetic relationships. A total of 846 bands were amplified from reactions using 12 screening primers, including 79 loci with a mean of 10.1 alleles per locus. The number of amplified bands for each primer ranged from 12 to 134, with a mean of 70.5 amplified bands per primer. The presence and distribution of amplified bands in different accessions demonstrate that a rapid evolutionary trend of microsatellite repeats occurred during the speciation process from the perennial wild form to annual cultivated rye. In addition, variation, amplification, and deletion of microsatellites in genomes revealed phylogenetic relationships in the genus Secale. Analysis of the presence, number, and distribution of amplified bands in genomes, as well as the comparison with genetic similarity (GS) indices based on ISSR, indicate that Secale strictum subsp. africanum (Stapf) Hammer, Secale strictum anatolicum (Boiss.) Hammer, Secale sylvestre Host, and Secale strictum subsp. strictum (C. Presl) Hammer emerged in succession from a common ancestor of Secale following geographic separation and genetic differentiation. The annual weedy rye evolved from S. strictum subsp. strictum, which was domesticated as present-day cultivated rye. Data from ISSR analyses separated all investigated accessions of the genus Secale into three distinct groups. These results support the division of the genus Secale into three species: the annual wild species S. sylvestre; the perennial wild species S. strictum, including several differential subspecies forms such as strictum, africanum, and anatolicum; and S. cereale, including cultivated and weedy rye as subspecies forms.     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

Localisation of foldback DNA sequences in nuclei chromosomes of Scilla, Secale, and of mouse.

Foldback DNA, prepared from mouse and Scilla sibirica main band DNA, and from rye (Secale cereale) total DNA, was characterised by denaturation, renaturation, and electron microscopy. 3H-cRNA of this DNA was hybridised in situ to nuclei and chromosomes of the respective species. There is no universal labelling pattern among the three species. In mouse, highly repetitive foldback DNA is present in the whole chromatin including the satellite DNA-containing regions. In Scilla sibirica, on the contrary, the highly repetitive foldback sequences are excluded form the satellite DNA loci and are arranged in clusters in the remaining chromatin. In rye, there is a clear preferential labelling of the chromocenters in the interphase nuclei as well as metaphase chromosomes, indicating that highly repetitive foldback DNA is preferentially located among other highly repetitive sequences.     

A second locus for the 5S multigene family in Secale L.: sequence divergence in two lineages of the family

The 5S RNA genes in Secale sp. are arranged as tandem arrays of a 460- and 480-bp repeating sequence. These size classes were initially discovered by restriction endonuclease analysis using BamHI and subsequently by DNA sequencing of cloned units. The length variation between short and long units originated from major deletion-insertion events in the noncoding spacer region of the 5S DNA repeat units. In situ hybridization with [3H]cRNA and biotin-labelled probes synthesized from both the short and long 5S DNA units of S. cereale localized the sites on chromosome 1R and a new site on a chromosome identified as 5R. We propose that the chromosome 1R locus, which has been mapped previously, be named 5SDna-R1 and the second locus, reported in the present paper, be referred to as 5SDna-R2. A preferential hybridization of a probe from the long unit to the 5SDna-R2 locus and of a probe from the short unit to the 5SDna-R1 locus is reported. The clustering of long units in the 5SDna-R2 locus was confirmed by restriction endonuclease digestion of DNA from rye chromosome 5R additions to wheat. Nucleotide sequence alignment of 5S DNA repeat units from a number of Secale species, using both phenetic and cladistic computer programmes, demonstrated that two clear lineages corresponding to the long and short units existed in this genus. The different Secale species could not be unambiguously differentiated using the 5S DNA sequences.     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Polyploidization-induced genome variation in triticale.

Polyploidization-induced genome variation in triticale (x Triticosecale Wittmack) was investigated using both AFLP and RFLP analyses. The AFLP analyses were implemented with both EcoRI-MseI (E-M) and PstI-MseI (P-M) primer combinations, which, because of their relative differences in sensitivity to cytosine methylation, primarily amplify repetitive and low-copy sequences, respectively. The results showed that the genomic sequences in triticale involved a great degree of variation including both repetitive and low-copy sequences. The frequency of losing parental bands was much higher than the frequency of gaining novel bands, suggesting that sequence elimination might be a major force causing genome variation in triticale. In all cases, variation in E-M primer-amplified parental bands was more frequent in triticale than that using P-M primers, suggesting that repetitive sequences were more involved in variation than low-copy sequences. The data also showed that the wheat (Triticum spp.) genomes were relatively highly conserved in triticales, especially in octoploid triticales, whereas the rye (Secale cereale L.) genome consistently demonstrated a very high level of genomic sequence variation (68%-72%) regardless of the triticale ploidy levels or primers used. In addition, when a parental AFLP band was present in both wheat and rye, the tendency of the AFLP band to be present in triticale was much higher than when it was present in only one of the progenitors. Furthermore, the cDNA-probed RFLP analyses showed that over 97% of the wheat coding sequences were maintained in triticale, whereas only about 61.6% of the rye coding sequences were maintained, suggesting that the rye genome variation in triticale also involved a high degree of rye coding sequence changes. The data also suggested that concerted evolution might occur in the genomic sequences of triticale. In addition, the observed genome variation in wheat-rye addition lines was similar to that in triticale, suggesting that wheat-rye addition lines can be used to thoroughly study the genome evolution of polyploid triticale.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.     

The molecular-cytogenetic analysis of grasses and its application to studying relationships among species of the Triticeae

An analysis of four species from the genus Secale, including the study of different accessions, has shown that the properties of DNA clones of monomer units from three repeated sequence loci, namely, Ter, Nor, and 5S DNA, proved to be representative of the entire loci from which they were isolated. This finding in Secale species, including the discovery of a new locus for 5S DNA on chromosome 5R, has been used to interpret information on the Ter, Nor, and 5S DNA loci from 15 species in the Triticeae complex. The evolutionary relationship among species suggested by the DNA sequence data has shown many consistencies with a number of other characters such as those used in classical systematics, as well as geographical distribution data and isozyme and chromosome-pairing studies. Apparent inconsistencies such as a close relationship between the R and P genomes at the Ter loci are interpreted in terms of amplification-deletion phenomena known to occur at repetitive sequence loci. In addition, this study included species endemic to Australia and thus provided a broad time span in which to consider some features of repeated sequence family evolution, such as the conservation of certain parts of 5S DNA spacer regions.     

Repetitive DNA and chromosome evolution in plants

Most higher plant genomes contain a high proportion of repeated sequences. Thus repetitive DNA is a major contributor to plant chromosome structure. The variation in total DNA content between species is due mostly to variation in repeated DNA content. Some repeats of the same family are arranged in tandem arrays, at the sites of heterochromatin. Examples from the Secale genus are described. Arrays of the same sequence are often present at many chromosomal sites. Heterochromatin often contains arrays of several unrelated sequences. The evolution of such arrays in populations is discussed. Other repeats are dispersed at many locations in the chromosomes. Many are likely to be or have evolved from transposable elements. The structures of some plant transposable elements, in particular the sequences of the terminal inverted repeats, are described. Some elements in soybean, antirrhinum and maize have the same inverted terminal repeat sequences. Other elements of maize and wheat share terminal homology with elements from yeast, Drosophila, man and mouse. The evolution of transposable elements in plant populations is discussed. The amplification, deletion and transposition of different repeated DNA sequences and the spread of the mutations in populations produces a turnover of repetitive DNA during evolution. This turnover process and the molecular mechanisms involved are discussed and shown to be responsible for divergence of chromosome structure between species. Turnover of repeated genes also occurs. The molecular processes affecting repeats imply that the older a repetitive DNA family the more likely it is to exist in different forms and in many locations within a species. Examples to support this hypothesis are provided from the Secale genus.     

Isoenzymes of aspartate aminotransferase in perennial and annual rye and their hybrids.

Aspartate aminotransferase patterns were screened in a collection of rye genotypes that included 24 accessions of wild perennial rye (Secale montanum Guss.), 6 accessions of cultivated perennial Derzhavin and Tsitsin rye (Secale cereale x S. montanum), 15 accessions of winter and spring rye cultivars (S. cereale L.), and 9 accessions of perennial and annual rye genotypes bred from S. montanum ssp. kuprijanovii, Derzhavin rye, and winter rye for their resistance to fungal diseases. Aspartate aminotransferase is coded for by four loci. The data fit the model where AAT 1/4 is coded by Aat 1 and Aat 4, two duplicate loci, with null and two active alleles for each locus, alleles 1 and 3 for locus Aat 1 and alleles 2 and 4 for locus Aat 4; dimeric AAT 1/4 enzyme molecules are the products of both intralocus and interloci complementation. Allele 1 of Aat 1 was the most prominent in the isoenzyme patterns of the rye species. Alleles null and 2 of Aat 4 were twice as frequent in the perennial rye accessions, including Derzhavin and Tsitsin rye, than in winter and spring rye. In contrast, allele 4 of Aat 4 was characteristic of S. cereale. Within the screened collection, locus Aat 2 was monomorphic. Among three alleles of Aat 3, allele 2 dominated isoenzyme profiles of both rye species, whereas the other two alleles were species-specific: allele 1 was characteristic of S. montanum and allele 3 was found only in S. cereale. Key words : rye, Secale cereale, Secale derzhavinii, Secale montanum, aspartate aminotransferase, isoenzymes, perennial habit, polymorphism.     

Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.     

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences

The rye-specific R173 family of repeated DNA sequences consists of ca. 15 000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins >20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheatGlu-D1 gene.     

Amplification and dispersion of repeated DNA sequences in theTriticeae

Four representatives of a family of dispersed repetitive sequences which were prominent and dispersed in the E genome ofThinopyrum elongatum but poorly represented in wheat, were studied in detail. The 1.4kb sequences were present both as part of tandem and more complex arrays and appeared to have resulted from repeated amplification of the sequence and their dispersion throughout the genome. Subcloning of sections of the 1.4 kb sequences resulted in probes which improved the resolution of the E genome from the genomes in wheat and enabled identification of single E genome chromosomes introduced into wheat. The generality of these types of sequences in the tribeTriticeae was confirmed by isolating analogous sequences from the R (rye,Secale cereale), V (Dasypyrum villosum), and N (Psathyrostachys juncea) genomes. — The cloned repetitive sequences from the R, V, and N genomes each showed characteristic fluctuations in amount within the grasses examined in addition to being virtually absent from wheat. It is thus possible that these sequences may provide useful taxonomic indicators for establishing relationships within theTriticeae, as well as valuable probes for tracing alien chromatin introduced into wheat.     

Characterization of ω-secalin genes from rye, triticale, and a wheat 1BL/1RS translocation line

Sixty-two DNA sequences for the coding regions of omega-secalin (ω-secalin) genes have been characterized from rye (Secale cereale L.), hexaploid and octoploid triticale (× Triticosecale Wittmack), and wheat (Triticum aestivum L.) 1BL/1RS translocation line. Only 19 out of the 62 ω-secalin gene sequences were full-length open reading frames (ORFs), which can be expressed into functional proteins. The other 43 DNA sequences were pseudogenes, as their ORFs were interrupted by one or a few stop codons or frameshift mutations. The 19 ω-secalin genes have a typical primary structure, which is different from wheat gliadins. There was no cysteine residue in ω-secalin proteins, and the potential celiac disease (CD) toxic epitope (PQQP) was identified to appear frequently in the repetitive domains. The ω-secalin genes from various cereal species shared high homology in their gene sequences. The ω-secalin gene family has involved fewer variations after the integration of the rye R chromosome or whole genome into the wheat or triticale genome. The higher Ka/Ks ratio (i.e. non-synonymous to synonymous substitutions per site) in ω-secalin pseudogenes than in ω-secalin ORFs indicate that the pseudogenes may be subject to a reduced selection pressure. Based on the conserved sequences of ω-secalin genes, it will be possible to manipulate the expression of this gene family in rye, triticale, or wheat 1BL/1RS translocation lines, to reduce its negative effects on grain quality.     

Detection and mapping of SSRs in rye ESTs from aluminium-stressed roots

Aluminium toxicity is a major problem for crop production on acid soils. Rye (Secale cereale L.) has one of the most efficient group of genes for aluminium tolerance, at least, four independent and dominant loci, Alt1, Alt2, Alt3 and Alt4, located on chromosome arms 6RS, 3RS, 4RL and 7RS, have been described. The increasing availability of expressed sequence tags in rye and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In order to obtain simple sequence repeat (SSR) markers related with Al tolerance more than 1,199 public accessible rye cDNA sequences from Al-stressed roots were exploited as a resource for SSR markers development. From a total of 21 S. cereale microsatellite (SCM) loci analysed, 12 were located on chromosomes 1R, 2R, 3R, 4R and 5R, using wheat–rye addition lines or mapped using a F₂ population segregating for Al tolerance. Seven SCM loci were included in a rye map with other SCIM and RAPD markers. Moreover, 14 SCM loci could be associated to proteins with known or unknown function. The possible implications of these sequences in aluminium tolerance mechanisms are discussed.     

The structure,amount and chromosomal localisation of defined repeated DNA sequences in species of the genus Secale

The structure, copy number and chromosomal location of arrays of four families of highly repeated sequences have been investigated in representative species of the genus Secale. The four unrelated families, previously characterised in Secale cereale, have repeating units of 480, 610, 630 and 120 base pairs respectively. The following general conclusions can be drawn in addition to detailed knowledge of the sequence content of heterochromatin in each accession studied: (1) Every species is unique in its complement or chromosomal distribution or both of the four highly repeated sequence families. S. montanum and S. cereale accessions studied here show the same complement of repeated sequences, but they differ substantially in the amounts they contain of the 610 and 630 base pair (bp) families, and in the distribution over the chromosomes of the 480 bp family. The structure of the repeating unit is also different in many members of the 480 bp family in S. montanum. — (2) The substantial differences between species in the amounts of the most highly repeated DNA sequences exist in the absence of any such conspicuous differences in most other repeated sequences which were detected as fluorescent bands after restriction enzyme digestion and gel electrophoresis. — (3) Each of the different highly repeated families can exist independently of the other families, though all the families have telomeric sites. Also, in the outbreeding species, heteromorphisms are frequent, and are particularly conspicuous in hybridisation detecting the 480 bp sequence family. — (4) The association of the highly repeated sequences with heterochromatin, discussed in the accompanying paper is generally true for other species in the genus, and the lower amounts of heterochromatin in other Secale species compared to S. cereale are associated with lower amounts of specific families of highly repeated DNA sequences. — (5) Analysis of highly repeated sequence families is likely to provide an easy method of identification of new accessions of Secale.