Abstract
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.
Similar content being viewed by others
References
Devos KM, Gale MD (1992) The use of random amplified polymorphic DNA in wheat. Theor Appl Genet 84:567–572
Devos KM, Atkinson MD, Chinoy CN, Liu CJ, Gale MD (1992) RFLP-based genetic map of the homoeologous group 3 chromosomes of wheat and rye. Theor Appl Genet 83:931–939
Driscoll CJ, Sears ER (1971) Individual additions of the chromosomes of ‘Imperial’ rye to wheat. Agron Abstr 1971:6
Flavell RB, Dean C, Moore G (1993) Exploiting plant genomes, small and large
Flavell RB, Heslop-Harrison JS (eds) The chromosome. Bios Scientific Publ., London, pp 249–264
Flavell RB, Rimpau J, Smith DB (1977) Repeated sequence DNA relationships in four cereal genomes. Chromosoma 63:205–222
Gosden J, Hanratty D (1991) Comparison of sensitivity of three haptens in the PRINS (oligonucleotide primed in situ DNA synthesis) reaction. Technique 3:159–165
Heslop-Harrison JS, Leitch AR, Schwarzacher T, Anamthawat-Jonsson K (1990) Detection and characterisation of 1B/1R translocaions in common wheat. Heredity 65:385–392
Islam AKMR, Shepherd KW, Sparrow DB (1978) Isolation and characterisation of euplasmic wheat-barley chromosome addition lines. Heredity 46:161–174
Javornik B, Sinkovic T, Vapa L, Koebner RMD, Rogers WJ (1991) A comparison of methods for identifying and surveying the presence of 1BL.1RS translocations in bread wheat. Euphytica 54:45–53
King IP, Purdie KA, Rezanoor HN, Koebner RMD, Miller TE, Reader SM, Nicholson P (1993) Characterization of Thinopyrum besarabicum chromosome segments in wheat using random amplified polymorphic DNAs and in situ hybridization. Theor Appl Genet 86:895–900
Koebner RMD (1990) Subtilisin inhibitor — polymorphic protein produced by a gene on the short arms of wheat homoeologous group 1 chromosomes. J Genet Breed 44:49–52
Koebner RMD (1994a) Generation of PCR-based markers for the detection of rye chromatin in a wheat background. Theor Appl Genet (in press)
Koebner RMD (1994b) Predigestion of DNA template improves the level of polymorphism of RAPDs in wheat. Genet Anal Tech Appl (in press)
Leitch IJ, Heslop-Harrison JS (1993) Physical mapping of four sites of 5S rDNA sequences and one site of the α-amylase-2 gene in barley (Hordeum vulgare). Genome 36:517–523
Leitch IJ, Leitch AR, Heslop-Harrison JS (1991a) Physical mapping of plant DNA sequences by simultaneous in situ hybridization of two differently labelled fluorescent probes. Genome 34:329–333
Leitch AR, Schwarzacher T, Mosgöller W, Bennett MD, Heslop-Harrison JS (1991b) Parental genomes are separated throughout the cell cycle in a plant hybrid. Chromosoma 101:206–213
Leitch AR, Schwarzacher T, Wang ML, Leitch IJ, Kurlan-Momirovich G, Moore G, Heslop-Harrison JS (1993) Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture. Plant Cell Tiss Org Cult 33:287–296
Martin GB, Williams JGK, Tanksley SD (1991) Rapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near isogenic lines. Proc Natl Acad Sci USA 88:2336–2340
Meinkoth J, Wahl G (1984) Hybridization of nucleic acids immobilized on solid supports. Anal Biochem 138:267–284
Moore G, Cheung W, Schwarzacher T, Flavell R (1991) BIS 1, a major component of the cereal genome and a tool for studying geomic organization. Genomics 10:469–476
Moore G, Abbo S, Cheung W, Foote T, Gale M, Koebner R, Leitch A, Leitch I, Money T, Stancombe P, Yano M, Flavell R (1993) Key features of cereal genome organization as revealed by the use of cytosine methylation-sensitive restriction endonucleases. Genomics 15:472–482
Paran I, Kesseli R, Michelmore R (1991) Identification of RFLP and RAPD markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations. Proc Natl Acad Sci USA 88:9828–9838
Penner GA, Bush A, Wise R, Kim W, Domier L, Kasha K, Laroche A, Scoles G, Molnar SJ, Fedak G (1993) Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Methods Applic 2:341–345
Rajaram S, Mann CHE, Ortiz-Ferrara G, Mujeeb-Kazi A (1983) Adaptation, stability and high yield potential of certain 1B/1R CIMMYT wheats. In: Sakamoto S (ed) Proc 6th Int Wheat Genet Symp. Maruzen, Kyoto, Japan, pp 613–621
Reiter RS, Williams JGK, Feldmann KA, Rafalski JA, Tingey SV, Scolnik PA (1992) Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs. Proc Natl Acad Sci USA 89:1477–1481
Rogowsky PM, Guidet FLY, Langridge P, Shepherd KW, Koebner RMD (1991) Isolation and characterisation of wheat-rye recombinants involving chromosome arm 1DS of wheat. Theor Appl Genet 82:537–544
Schwarzacher TS, Leitch AR (1994) Enzymatic treatment of plant material to spread chromosomes for in situ hybridization. In: Isaac P (ed) Methods in molecular biology vol. 28: protocols for nucleic acid analysis by non-radioactive probes. Humana Press, Totowa, N J, pp 153–160
Sears ER (1966) Nullisomic-tetrasomic combinations in hexaploid wheat. In: Riley R, Lewis KR (eds) Chromosome manipulations and plant genetics. Oliver and Boyd, London, pp 29–45
Sharp PJ, Kreis M, Shewry PR, Gale MD (1988) Location of β-amylase sequences in wheat and its relatives. Theor Appl Genet 75:286–290
Shepherd KW (1973) Homoeology of wheat and alien chromosomes controlling endosperm protein phenotypes. In: Sears ER, Sears LMS (eds) Proc 4th Int Wheat Genet Symp. University of Missouri, Columbia, Mo., pp 745–760
Singh NK, Shepherd KW (1985) The structure and genetic control of a new class of disulphide-linked proteins in wheat endosperm. Theor Appl Genet 71:79–92
Author information
Authors and Affiliations
Additional information
Communicated by K. Tsunewaki
Rights and permissions
About this article
Cite this article
Francis, H.A., Leitch, A.R. & Koebner, R.M.D. 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. Theoret. Appl. Genetics 90, 636–642 (1995). https://doi.org/10.1007/BF00222127
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00222127