Genomic in situ hybridization in Avena sativa.

Genomic fluorescent in situ hybridization was employed in the study of the genome organization and evolution of hexaploid oat (Avena sativa L. cv. Sun II, AACCDD, 2n = 6x = 42). Genomic DNAs from two diploid oat species, Avena strigosa (genomic constitution AsAs, 2n = 14) and Avena pilosa (genomic constitution CpCp, 2n = 14), were used as probes in the study. The DNA from A. strigosa labelled 28 of the 42 (2/3) chromosomes of the hexaploid oat, while 14 of the 42 (1/3) chromosomes were labelled with A. pilosa DNA, indicating a close relationship between the A and D genomes. Results also suggested that at least 18 chromosomes (9 pairs) were involved in intergenomic interchanges between the A and C genomes.     

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.     

Genetic relationships of the Japanese persimmon Diospyros kaki (Ebenaceae) and related species revealed by SSR analysis

Simple sequence repeat (SSR) molecular markers based on 18 primers were employed to study the genetic relationship of Japanese persimmon (Diospyros kaki) specimens. Two hundred and sixty-two bands were detected in 30 Japanese persimmon samples, including 14 Japanese and 10 Chinese genotypes of Japanese persimmon (Diospyros kaki) and six related species, D. lotus, D. glaucifolia, D. oleifera, D. rhombifolia, D. virginiana, and Jinzaoshi (unclassified - previously indicated to be D. kaki). All SSR primers developed from D. kaki were successfully employed to reveal the polymorphism in other species of Diospyros. Most of the primers were highly polymorphic, with a degree of polymorphism equal to or higher than 0.66. The results from the neighbor-joining dendrogram and the principal coordinate analysis diagram were the same; i.e., the Chinese and Japanese genotypes and related species were separated and the relationships revealed were consistent with the known pedigrees. We also concluded that 'Xiangxitianshi' from Xiangxi municipality, Hunan Province, China, is actually a sport or somaclonal variant of 'Maekawa-Jirou', and that 'Jinzaoshi' should be classified as a distinct species of Diospyros. We found that SSR markers are a valuable tool for the estimation of genetic diversity and divergence in Diospyros.     

The genomic composition of Tricepiro, a synthetic forage crop.

Chromosome in situ hybridization (FISH and GISH) is a powerful tool for determining the chromosomal location of specific sequences and for analysing genome organization and evolution. Tricepiro (2n = 6x = 42) is a synthetic cereal obtained by G. Covas in Argentina (1972), which crosses hexaploid triticale (2n = 6x = 42) and octoploid Trigopiro (2n = 8x = 56). Several years of breeding produced a forage crop with valuable characteristics from Secale, Triticum, and Thinopyrum. The aim of this work is to analyse the real genomic constitution of this important synthetic crop. In situ hybridization using total DNA of Secale, Triticum, and Thinopyrum as a probe (GISH) labelled with biotin and (or) digoxigenin showed that tricepiro is composed of 14 rye chromosomes and 28 wheat chromosomes. Small zones of introgression of Thinopyrum on wheat chromosomes were detected. The FISH using the rye repetitive DNA probe pSc 119.2 labelled with biotin let us characterize the seven pairs of rye chromosomes. Moreover, several wheat chromosomes belonging to A and B genomes were distinguished. Therefore, tricepiro is a synthetic hexaploid (2n = 6x = 42) being AABBRR in its genomic composition, with zones of introgression of Thinopyrum in the A genome of wheat.     

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids.

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.     

Differential organization of a LINE-1 family in Indian pygmy field mice

Southern blot hybridization analysis of genomic DNAs digested with restriction endonuclease EcoR I and Ava II from Mus musculus domesticus, Mus booduga and Mus terricolor with a cloned repetitive DNA fragment of Mus booduga as a probe showed difference in restriction pattern of this DNA in these three species. Further Southern analysis of the BamH I digested genomic DNAs from these species hybridized with cloned DNA fragment as a probe and sequencing of the cloned DNA revealed that this 252 bp cloned DNA fragment is a part of BamHI repeat element of genus Mus and is 87% homologous to the contiguous portion of the Mus musculus domesticus LINE-1 element. The species specific fragment pattern generated by different restriction endonucleases using this DNA as a probe revealed difference in the organization of LINE-1 repetitive element in the three species of genus Mus.     

Identification of mitotic chromosomes of tuberous and non-tuberous solanum species (Solanum tuberosum and Solanum brevidens) by GISH in their interspecific hybrids.

GISH (genomic in situ hybridization) was applied for the analysis of mitotic chromosome constitutions of somatic hybrids and their derivatives between dihaploid clones of cultivated potato (Solanum tuberosum L.) (2n = 2x = 24, AA genome) and the diploid, non-tuberous, wild species Solanum brevidens Phil. (2n = 2x = 24, EE genome). Of the primary somatic hybrids, both tetraploid (2n = 4x) and hexaploid (2n = 6x) plants were found with the genomic constitutions of AAEE and AAEEEE, respectively. Androgenic haploids (somatohaploids) derived from the tetraploid somatic hybrids had the genomic constitutions of AE (2n = 2x = 24) and haploids originating from the hexaploid hybrids were triploid AEE (2n = 3x = 33 and 2n = 3x = 36). As a result of subsequent somatic hybridization from a fusion between dihaploid S. tuberosum (2n = 2x = 24, genome AA) and a triploid somatohaploid (2n = 3x = 33, genome AEE), second-generation somatic hybrids were obtained. These somatic hybrids were pentaploids (2n = 5x, genome AAAEE), but had variable chromosome numbers. GISH analysis revealed that both primary and second-generation somatic hybrids had lost more chromosomes of S. brevidens than of S. tuberosum.     

基于叶绿体DNA ndhA和nrDNA ITS序列变异分析栽培柿及其近缘种亲缘关系

以柿属植物（Diospyros spp.）中与柿近缘的8种共30个基因型为试材,进行核糖体DNA(nrDNA)内转录间隔区（ITS）和叶绿体DNA ndhA序列变异分析,并通过软件计算两个序列及合并后的进化模型,依据进化模型采用ML法(maximum likelihood method)分析进化关系。为进一步弄清柿属植物种间亲缘关系和供试柿（Diospyros kaki Thunb.）种内分子差异提供了理论依据。结果表明：（1）ndhA序列长度变异范围在1 492~1 511,14个信息位点;ITS序列长度变异范围在660~761,56个信息位点。ITS、ndhA和ndhA+ITS（ndhA和ITS合并）最适碱基进化模型分别为（TrN+I+G）、（F81+I）和（GTR+I+G）。综合ITS和ndhA序列分析表明：柿与油柿和云南野毛柿亲缘关系最近,与美洲柿和乌柿最远。（2）21份柿品种材料的ITS长度均为730,包括4个变异位点,据此4个变异位点对供试柿种内21个品种进行聚类分析。研究认为,ndhA和ITS能较清楚解释了柿与其近缘种间的亲缘关系,并通过柿品种ITS的差异位点分析鉴别出栽培柿种内的差异。     

Genomic relationships between Dasypyrum villosum (L.) Candargy and D. hordeaceum (Cosson et Durieu) Candargy.

The origin and genomic constitution of the tetraploid perennial species Dasypyrum hordeaceum (2n = 4x = 28) and its phylogenetic relationships with the annual diploid Dasypyrum villosum (2n = 2x = 14) have been investigated by comparing the two genomes using different methods. There is no apparent homology between the conventional or Giemsa C-banded karyotypes of the two Dasypyrum species, nor can the karyotype of D. hordeaceum be split up into two similar sets. Polymorphism within several chromosome pairs was observed in both karyotypes. Cytophotometric determinations of the Feulgen-DNA absorptions showed that the genome size of D. hordeaceum was twice as large as that of D. villosum. Both the cross D. villosum x D. hordeaceum (crossability rate 12.1%) and the reciprocal cross (crossability rate 50.7%) produced plump seeds. Only those from the former cross germinated, producing sterile plants with a phenotype that was intermediate between those of the parents. In these hybrids (2n = 21), an average of 13.77 chromosomes per cell paired at meiotic metaphase I. Trivalents were only rarely observed. Through dot-blot hybridizations, a highly repeated DNA sequence of D. villosum was found not to be represented in the genome of D. hordeaceum. By contrast, very similar restriction patterns were observed when a low-repeated DNA sequence or different single-copy sequences of D. villosum or two sequences in the plastidial DNA of rice were hybridized to Southern blots of the genomic DNAs of the two Dasypyrum species digested with different restriction endonucleases. By analyzing glutamic-oxaloacetic-transaminase, superoxide dismutase, alcohol dehydrogenase, and esterase isozyme systems, it was shown that both Dasypyrum species shared the same phenotypes, which differed from those found in hexaploid wheat. In situ hybridizations using DNA sequences encoding gliadins showed that these genes were located close to the centromere of three pairs of D. villosum chromosomes and that they had the same locations in six pairs of D. hordeaceum chromosomes. We conclude that the autoploid origin of D. hordeaceum from D. villosum, which cannot be defended on the basis of chromosomal traits, is suggested by the other findings obtained by comparing the two genomes. Key words : Dasypyrum hordeaceum, Dasypyrum villosum, phylogenetic relationships.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

The evolutionary history of Drosophila buzzatii. XXIII. High content of nonsatellite repetitive DNA in D. buzzatii and in its sibling D. koepferae.

The frequency and types of repetitive nonsatellite DNA of two sibling species of the repleta group of Drosophila, D. buzzatii, and D. koepferae have been determined. For each species, the analysis is based on a sample of more than 100 clones (400 kb) obtained from genomic DNA. A theoretical model has been developed to correct for the presence of a mixture of repetitive and unique DNA in these clones. After correction, a high content of repetitive DNA has been demonstrated for both species (D. buzzatii, 19-26%; D. koepferae, 27-32%). The repetitive sequences have been classified according to their hybridization pattern when used as probes against genomic DNA and by their in situ hybridization signals on polytene chromosomes. Data suggest that the main nonsatellite component of these species is simpler and more repetitive than that of D. melanogaster, pointing to a wide variability in content and class size distribution of repetitive DNA among Drosophila species.     

Identification of a pentanucleotide telomeric sequence, (TTAGG)n, in the silkworm Bombyx mori and in other insects.

A pentanucleotide repetitive sequence, (TTAGG)n, has been isolated from a silkworm genomic library, using cross-hybridization with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and Bal 31 exonuclease experiments revealed major clusters of (TTAGG)n at the telomeres of all Bombyx chromosomes. To determine the evolutionary origin of this sequence, two types of telomeric sequence, (TTAGG)5 and a hexanucleotide repetitive sequence, (TTAGGG)4, which is conserved mainly among vertebrate and several invertebrate telomeres so far examined, were hybridized to DNAs from a wide variety of eukaryotic species under highly stringent hybridization conditions. The (TTAGGG)5 oligonucleotide hybridized to genomic DNAs from vertebrates and several nonvertebrate species, as has been reported so far, but not to any DNAs from insects. On the other hand, the Bombyx type of telomere sequence, (TTAGG)n, hybridized to DNAs from 8 of 11 orders of insect species tested but not to vertebrate DNAs, suggesting that this TTAGG repetitive sequence is conserved widely among insects.     

Biosystematic study of hexaploids Elymus tschimganicus and E. glaucissimus. II. Interspecific hybridization and genomic relationship.

To investigate genomic relationships of Elymus tschimganicus (Drobov) Tzvelev (2n = 6x = 42, S1S2Y genomes) and E. glaucissimus (M. Pop.) Tzvelev (2n = 6x = 42, S1S2Y genomes), interspecific hybridizations of the two target species were carried out with 27 other Elymus species containing the SH, SY, SYH, SYP, SYW, and SH1H2 genomes, respectively, collected from different geographic regions. Chromosome pairing behavior was analyzed at metaphase I in 27 hybrids representing 23 hybrid combinations, and overall genomic relationships of the two target species with the other Elymus taxa were estimated. The study concluded that (i) interspecific hybridization was principally easy to perform between the Elymus species, but no general pattern of crossability was obtained, and all hybrids were completely sterile, (ii) the two species have a similar meiotic pattern in their hybrids with the other Elymus species, and (iii) species containing the SY, SYP, and SYH genomes have a generally higher level of genomic homology to the target species than those possessing the SH genomes, and the South American hexaploid with the SH1H2 genomes has the lowest level of genomic homology to the two target taxa.     

柿树炭疽菌侵染不同柿树种、品种和部位的细胞学特征

用柿树炭疽病菌Colletotrichumgloeosporioides的分生孢子制备孢子悬浮液,接种无核柿、野柿、冬柿和浙江柿的新梢、叶柄和叶片,并观察致病性、附着胞形成和侵染特性。柿树炭疽菌可以侵染无核柿枝条和叶柄以及野柿枝条,但不侵染无核柿叶片、野柿叶柄和叶片,也不侵染冬柿和浙江柿枝条、叶柄和叶片。室内接种试验与田间自然发病结果一致。柿树炭疽菌在不同柿树表面均能形成附着胞,附着胞产生在寄主表皮背斜细胞壁间结合处(JACWs)或近结合处的百分率达81%~93%。接种12h后,不同柿树表面都有附着胞形成;36h后,无核柿枝条、叶柄中有侵染菌丝存在;48h后,无核柿枝条、叶柄中观察到膨大初生菌丝和较细次生菌丝,初生菌丝可扩展到相邻细胞中,而野柿枝条中仅观察到侵染菌丝;60h后,野柿枝条中也观察到膨大的初生菌丝和较细的次生菌丝,但初生菌丝仅局限在最初侵染的细胞中,无核柿枝条和叶柄以及野柿枝条中都有分枝的次生菌丝在细胞内、细胞间或相邻的细胞中扩展;直到接种90h后,在冬柿和浙江柿上都未观察到侵染菌丝的形成。结果表明,柿树炭疽菌在不同柿树种和品种上侵染菌丝的形成和扩展方式可能是其寄主专化性(或致病性)差异的重要机制之一。     

Decaploidy in Fragaria iturupensis (Rosaceae)

The strawberry genus, Fragaria (Rosaceae), has a base chromosome number of x = 7. Cultivated strawberries (F. ×ananassa nothosubsp. ananassa) are octoploid (2n = 8x = 56) and first hybridized from F. chiloensis subsp. chiloensis forma chiloensis × F. virginiana subsp. virginiana. Europe has no known native octoploid species, and only one Asian octoploid species has been reported: F. iturupensis, from Iturup Island. Our objective was to examine the chromosomes of F. iturupensis. Ploidy levels of wild strawberry species, include diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), pentaploid (2n = 5x = 35), hexaploid (2n = 6x = 42), octoploid (2n = 8x = 56), and nonaploid (2n = 9x = 63). Artificial triploid (2n = 3x = 21), tetraploid, pentaploid, octoploid, decaploid (2n = 10x = 70), 16-ploid, and 32-ploid plants have been constructed and cultivated. Surprisingly, chromosome counts and flow cytometry revealed that F. iturupensis includes natural decaploid genotypes with 2n = 10x = 70 chromosomes. This report is the first of a naturally occurring decaploid strawberry species. Further research on F. iturupensis and exploration on northern Pacific islands is warranted to ascertain the phylogeny and development of American octoploid species.     

Acaricidal activity and function of mite indicator using plumbagin and its derivatives isolated from Diospyros kaki Thunb. roots (Ebenaceae)

Acaricidal effects of materials derived from Diospyros kaki roots against Dermatophagoides farinae and D. pteronyssinus were assessed using impregnated fabric disk bioassay and compared with that of the commercial benzyl benzoate. The observed responses varied according to dosage and mite species. The LD50 values of the chloroform extract of Diospyros kaki roots were 1.66 and 0.96 microg/cm2 against D. farinae and D. pteronyssinus. The chloroform extract of Diospyros kaki roots was approximately 15.2 more toxic than benzyl benzoate against D. farinae, and 7.6 times more toxic against D. pteronyssinus. Purification of the biologically active constituent from D. kaki roots was done by using silica gel chromatography and high-performance liquid chromatography. The structure of the acaricidal component was analyzed by GCMS, 1H-NMR, 13C-NMR, 1H-13C COSY-NMR, and DEPTNMR spectra, and identified as plumbagin. The acaricidal activity of plumbagin and its derivatives (naphthazarin, dichlon, 2,3-dibromo-1,4-naphthoquinone, and 2-bromo-1,4- naphthoquinone) was examined. On the basis of LD50 values, the most toxic compound against D. farinae was naphthazarin (0.011 microg/cm2) followed by plumbagin (0.019 microg/cm2), 2- bromo-1,4-naphthoquinone (0.079 microg/cm2), dichlon (0.422 microg/ cm2), and benzyl benzoate (9.14 microg/cm2). Additionally, the skin color of the dust mites was changed from colorless-transparent to dark brown-black by the treatment of plumbagin. Similar results have been exhibited in its derivatives (naphthazarin, dichlon, and 2-bromo-1,4-naphthoquinone). In contrast, little or no discoloration was observed for benzyl benzoate. From this point of view, plumbagin and its derivatives can be very useful for the potential control agents, lead compounds, and indicator of house dust mites.     

Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

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.