Detection of DNA sequence polymorphisms among wheat varieties

Summary A DNA marker detection strategy that allows the rapid, efficient resolution of high levels of polymorphism among closely related lines of common wheat (Triticum aestivum) has been developed to circumvent the apparent lack of restriction fragment length polymorphism in many important self-pollinated crop species. The technique of randomly amplified polymorphic DNA (RAPD) was combined with a denaturing gradient gel electrophoresis system (DGGE) to explore DNA sequence polymorphisms among different genotypes of wheat. Of the 65 primer combinations used for the polymerase chain reaction (PCR) amplifications, over 38% of them produced readily detectable and reproducible DNA polymorphisms between a spring wheat line, SO852, and a winter wheat variety, Clark. A high level of polymorphism was observed among a number of commercial varieties and breeding lines of wheat. This procedure was also used to detect polymorphisms in a recombinant inbred population to test the feasibility of its application in genome mapping. This DNA polymorphism detection system provides an opportunity for pedigree analysis and fingerprinting of developed wheat lines as well as construction of a high density genetic map of wheat. Without the need for ³²P and sophisticated DNA extraction procedures, this approach should make it feasible to utilize marker-based selection in a plant breeding program.     

Host range,mating type and population structure of Magnaporthe sp. of a single barley field in São Paulo state,Brazil

Brazil is blast disease hot spot because severe epidemics have occurred among wheat, triticale, rye, barley and oat crops. Although the first outbreak of barley blast appeared in 1998, little information is available. Therefore, this study aimed to examine host range, mating type composition and population structure of Magnaporthe sp. from a single barley field in São Paulo, Brazil. To examine pathogenicity, 25 Magnaporthe isolates were inoculated on five, three, two and two cultivars of barley, wheat, oat and rice, respectively, and one cultivar each of rye, corn, sorghum, triticale and certain weeds (Cenchrus echinatus, Setaria geniculata, Brachiaria plantaginea and Eleusine indica). Mating type distribution of 33 isolates was investigated by molecular tools. The genotypic divergence of 41 barley and five wheat isolates was investigated by 15 random amplified polymorphic DNA primers and unweighted pair group method with arithmetic mean. The host range of the barley blast pathogen included wheat, oat, rye and triticale but not rice and weeds. Sexual reproduction appeared to not be involved in the high genotypic diversity because only a single isolate, MAT1‐2, was identified. The majority of barley isolates clustered together with wheat blast, except for four, suggesting a different origin.     

Hydrolysis of RNA to 5′-nucleotide by seed sprouts,particularly malt sprouts

Conditions for the efficient conversion of commercial RNA to nucleoside 5′-monophosphate by means of a phosphodiesterase in malt sprouts have been determined. A comparison of the enzyme content of the rootlets, stems, and kernels of various plant seedlings, including barley, rye, oat, wheat, rice, and beans shows maximum amounts in the rootlets, and minimum quantities in the ungerminated kernels. Of all the seedlings tested, (mung bean, soy bean, oat, wheat, rice, barley) barley gave the highest conversion of RNA to 5′-nucleotides. Commercial malt sprouts prepared from 6 different malted barleys including 2-rowed and 6-rowed samples all showed about the same amount of phosphodiesterase content. Besides phosphodiesterase, other enzymes capable of hydrolyzing RNA and 5′-nucleotides were found in sprouts. These included 3′-phosphodiesterases, 5′-nucleotidases, and nucleosidases. By carefully pretreating both extracts and the solid sprouts at elevated temperatures for a limited time and by the addition of minimum amounts of Zn⁺², the action of these undesirable enzymes was either effectively destroyed or minimized so that the production of 5′-nucleotides was maximized. It was found that suspensions of appropriately washed and treated barley malt rootlets are substantially more effective than aqueous extracts for converting RNA to 5′-nucleotides.     

Development and molecular cytogenetic identification of new winter wheat--winter barley ('Martonvásári 9 kr1' - 'Igri') disomic addition lines.

This paper describes a series of winter wheat - winter barley disomic addition lines developed from hybrids between winter wheat line Triticum aestivum L. 'Martonvásári 9 kr1' and the German 2-rowed winter barley cultivar Hordeum vulgare L. 'Igri'. The barley chromosomes in a wheat background were identified from the fluorescent in situ hybridization (FISH) patterns obtained with various combinations of repetitive DNA probes: GAA-HvT01 and pTa71-HvT01. The disomic addition lines 2H, 3H, and 4H and the 1HS isochromosome were identified on the basis of a 2-colour FISH with the DNA probe pairs GAA-pAs1, GAA-HvT01, and pTa71-HvT01. Genomic in situ hybridization was used to confirm the presence of the barley chromosomes in the wheat genome. The identification of the barley chromosomes in the addition lines was further confirmed with simple-sequence repeat markers. The addition lines were also characterized morphologically.     

Specific 5S ribosomal RNA primers for plant species i dentification in admixtures 3

The DNA sequences of the spacers between the 5S ribosomal RNA genes were determined for the cereals maize, barley, soghum, rye, rice, oat, and wheat. Species-specific primers were designed from the spacer region. PCR with these primers and a common primer from the conserved 5S ribosomal RNA gene sequence was investigated as a method for detection of the seven cereal species. DNA from these species could be specifically detected in mixtures. This technique could find application in the determination of the composition of admixtures or processed cereal products. The protocol described has potential for general application in the identification of plant species.     

Use of Polymerase Chain Reaction To Detect the Take-All Fungus, Gaeumannomyces graminis, in Infected Wheat Plants

Gaeumannomyces graminis, the causative agent of take-all disease of wheat, barley, and oats, was detected in infected wheat seedlings by using the polymerase chain reaction to amplify Gaeumannomyces-specific DNA fragments. Nested primers and two rounds of amplification were used to amplify two fragments, approximately 287 and 188 bp in size, from G. graminis-infected wheat seedlings. The use of nested primers greatly decreased the number of nonspecific amplification products. Polymerase chain reaction products were not obtained with DNA from seedlings infected with several other phytopathogenic fungi or with DNA from uninfected seedlings. Amplified products were visualized on agarose gels, and their identities were confirmed by DNA hybridization. This method did not require culturing the fungus and has potential for detecting G. graminis in infested wheat, barley, or oat fields.     

Assessment of genetic diversity in dent and popcorn (Zea mays L.) inbred lines using inter-simple sequence repeat (ISSR) amplification

Popcorn (Zea mays L.) hybrids grown in the United States are derived from narrow-based germplasm, and standard RFLP analysis detects relatively little polymorphism. Inter-simple sequence repeat (ISSR) amplification, a novel technique based on PCR amplification of inter-microsatellite sequences to target multiple loci in the genome, was employed to investigate its potential for detection of polymorphism among nineteen popcorn and eight dent corn inbred lines. ISSR yielded an average of 54 bands/primer/inbred line, with over 98% of the bands repeatable across DNA extractions and separate PCR runs. Ten primers based on di- and tri-nucleotide tandem repeats revealed 73% and 87% polymorphism among popcorn and dent corn lines, respectively, with an overall 95% polymorphism rate. Principal component and cluster analyses resulted in grouping of dent and popcorn lines corresponding to their heterotic breeding pools. ISSR amplification, in addition to being both simple and cost and time efficient, provides for rapid production of highly polymorphic markers which appear to correspond to known pedigree information. Therefore, the ISSR technique may have great potential for identifying polymorphism in species with narrow-based germplasm, and for use in DNA marker-assisted breeding approaches.     

Sequence haplotypes revealed by sequence-tagged site fine mapping of the Ror1 gene in the centromeric region of barley chromosome 1H

We describe the development of polymerase chain reaction-based, sequence-tagged site (STS) markers for fine mapping of the barley (Hordeum vulgare) Ror1 gene required for broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. hordei). After locating Ror1 to the centromeric region of barley chromosome 1H using a combined amplified fragment length polymorphism/restriction fragment-length polymorphism (RFLP) approach, sequences of RFLP probes from this chromosome region of barley and corresponding genome regions from the related grass species oat (Avena spp.), wheat, and Triticum monococcum were used to develop STS markers. Primers based on the RFLP probe sequences were used to polymerase chain reaction-amplify and directly sequence homologous DNA stretches from each of four parents that were used for mapping. Over 28,000 bp from 22 markers were compared. In addition to one insertion/deletion of at least 2.0 kb, 79 small unique sequence polymorphisms were observed, including 65 single nucleotide substitutions, two dinucleotide substitutions, 11 insertion/deletions, and one 5-bp/10-bp exchange. The frequency of polymorphism between any two barley lines ranged from 0.9 to 3.0 kb, and was greatest for comparisons involving an Ethiopian landrace. Haplotype structure was observed in the marker sequences over distances of several hundred basepairs. Polymorphisms in 16 STSs were used to generate genetic markers, scored by restriction enzyme digestion or by direct sequencing. Over 2,300 segregants from three populations were used in Ror1 linkage analysis, mapping Ror1 to a 0.2- to 0.5-cM marker interval. We discuss the implications of sequence haplotypes and STS markers for the generation of high-density maps in cereals.     

Determination of variability in monosporidial lines of Tilletia indica by RAPD analysis

Genetic variability in 23 monosporidial lines developed from five isolates of Tilletia indica causing Karnal bunt of wheat isolated from four wheat growing states of India was determined by using 19 rapid amplified polymorphic DNA (RAPD) markers. Amplification profile generated with all the 19 primers produced 3–16 numbers of bands of 1.5–5 kb size. High level of polymorphism (95.2%) suggested wide range of variability. Maximum Jaccard's similarity coefficient (80%) was observed between KB2MsB and KB2MsC followed by KB5MsC and KB5MsE with 75% similarity, whereas it was minimum between KB3MsA and Kb4MsB (47%). The dendrogram derived from the fingerprint analysis with 19 RAPD primers by using UPGMA showed different levels of genetic similarity among monosporidial lines. At 35% genetic similarity, the monosporidial lines were grouped in two clusters. Some primers, viz., OPN-1, OPN-6, OPN-9, OPN-12, OPN-13, OPN-18, OPM-2, OPM-8, OPM-10, OPB-8, OPB-17 and OPB-20 showed 100% polymorphism. The RAPD fingerprint generated by OPN-1 and OPM-3 were analysed and showed high range of variation in genetic make-up of monosporidial lines.     

IRAP,REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (H^chH^chAABB; 2n?=?6x?=?42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (H^chH^ch; 2n?=?2x?=?14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n?=?4x?=?28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.     

RAPD-based analysis of introgression of barley genetic material into the genome of alloplasmic wheat lines (Hordeum geniculatum All./ Triticum aestivum L.)

Genomes of three alloplasmic wheat lines obtained on the basis of barley--wheat hybrid Horderum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42)(Pyrotrix 28) were examined using random amplified polymorphic DNA (RAPD) analysis. Line L-29 was obtained after first backcross of the initial hybrid with the wheat variety Pyrotrix 28 and ten subsequent self-pollinating generations. This line was represented by euploid plants with typical to the common wheat chromosome number (2n = 42), as well as by aneuploids, which contained an additional telocentric chromosome in the main karyotype (2n = 42 + t). Lines L-26 and L-27 were obtained by two backcrosses of one BC1 plant with the wheat variety Novosibirskaya 67 and one subsequent self-polination of one BC3 plant. Chromosome number in all these plants corresponded to 2n = 40 + 4t. RAPD analysis was carried out using seven primers, which were previously proved to be effective for identification of the barley genome fragments within hybrid genomes of alloplasmic lines. The presence of barley genome fragments in line L-29 was revealed by use of five primers, while in lines L-26 and L-27 these fragments were detected by use of one primer. The significant difference in the number of barley RAPD fragments in the genomes of alloplasmic lines obtained at different backcrossing stages suggests more intense displacement of barley genome during backcrossing compared to self-pollination in BC1 plants.     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

三系杂交稻亲本随机扩增多态性DNA(RAPD)分析

选用9个随机引物对31份杂交水稻亲本材料进行了RAPD分析, 共检测到60条多态性带。聚类分析结果表明,所有供试材料可以被明确地区分。在9个随机引物中,有8个具有较高的多态性检测能力。以这8个引物为基础,选用任两个引物即可在任一对材料中检测出多态性的频率在96.13%以上,而选用任3个引物则该频率在99.21%以上。这显示了运用RAPD鉴定稻种具有简便、灵敏、高效的优点,在鉴定杂交稻种的实践中有着良好的应用前景。 Abstract:Seven rice sterile lines,12 maintainer lines and 12 restorer lines were analyzed by RAPD with 9 primers.Altogether,118 fragments were generated,of which 60 detected polymorphisms among rice marker.Eight of nine primers can detect high polymorphism.The frequencies of polymorphism in any primers were used,the frequencies would be higher than 99.21%.The eight primers were therefore recommended as candidates for the identification of hybrid rice seeds.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

三例小麦细胞质雄性不育系线粒体DNA的扩增片段长度多态性分析

细胞质雄性不育是小麦杂种优势利用的重要途径,为了鉴定3例小麦雄性不育系的细胞质类型,对其线粒体DNA(mtDNA)进行扩增片段长度多态性(Amplified fragment length polymorphism,AFLP)分析。文中利用差速离心法和不连续蔗糖密度梯度超速离心法提取纯化小麦线粒体。结果表明:通过该提取方法获得的mtDNA,其质量和纯度能够满足PCR反应和遗传学分析。在64对选扩引物中,筛选到了4对特异性引物,其中引物E1/M7在ms(Kots)-90-110不育系扩增出3条特异条带;引物E4/M2在ms(Ven)-90-110不育系扩增出2条特异条带;引物E7/M6在ms(S)-90-110不育系中扩增出2条特异条带;引物E6/M4在ms(Kots)-90-110不育系中扩增出2条特异条带。这些特异引物可以用来作为鉴定具有粘果山羊草Aegilops kotschyi、偏凸山羊草Ae.ventricosa、斯卑尔脱小麦Triticum spelta 3类不育细胞质型小麦雄性不育系的细胞质分子标记,为研究小麦细胞质雄性不育机理奠定了分子基础。     

Identification of a RAPD marker linked to progressive rod-cone degeneration in dogs

Random amplified polymorphic DNA (RAPD) analysis has been used widely in plant and fungi for identification of markers linked to genetic traits and mapping, but its use is limited to identification of intra- and inter-species difference in domestic mammals. We report here identification of a RAPD-derived marker linked to progressive rod-cone degeneration (prcd), an inherited autosomal recessive retinal disease of dogs. A total of 400 standard 10-mer primers were used for amplification by use of DNA samples from normal (+/+) and affected (prcd/prcd) dogs. A single primer was identified which amplified a 1.5-kb DNA fragment only from normal dogs. PCR with longer primers designed from the sequence-characterized amplified region of the 1.5-kb DNA fragment identified a co-dominant multi-allelic polymorphism in the prcd-informative pedigree. Three recombinants were identified among 34 informative offsprings, yielding a LOD score of 5.568 at θ= 0.091. This marker was mapped to two canine–rodent hybrid cell lines in which two genes (canine homologues of human breast cancer 1 susceptibility gene, and cGMP phosphodiesterase γ-subunit gene), and three anonymous microsatellites have been identified. This is the first reported identification of a RAPD-derived marker with multiple alleles linked to a mammalian disease locus. Received: 20 February 1998 / Accepted: 24 April 1998     

Maize centromeres: organization and functional adaptation in the genetic background of oat

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from approximately 300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize centromeres.     

Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution

In plant species with large genomes such as wheat or barley, genome organization at the level of DNA sequence is largely unknown. The largest sequences that are publicly accessible so far from Triticeae genomes are two 60 kb and 66 kb intervals from barley. Here, we report on the analysis of a 211 kb contiguous DNA sequence from diploid wheat (Triticum monococcum L.). Five putative genes were identified, two of which show similarity to disease resistance genes. Three of the five genes are clustered in a 31 kb gene-enriched island while the two others are separated from the cluster and from each other by large stretches of repetitive DNA. About 70% of the contig is comprised of several classes of transposable elements. Ten different types of retrotransposons were identified, most of them forming a pattern of nested insertions similar to those found in maize and barley. Evidence was found for major deletion, insertion and duplication events within the analysed region, suggesting multiple mechanisms of genome evolution in addition to retrotransposon amplification. Seven types of foldback transposons, an element class previously not described for wheat genomes, were characterized. One such element was found to be closely associated with genes in several Triticeae species and may therefore be of use for the identification of gene-rich regions in these species.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.