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Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS

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Abstract

We have applied a refined microdissection procedure to create a plasmid library of the barley (Hordeum vulgare L.) chromosome arm 1HS. The technical improvements involved include synchronization of meristematic root tissue, a metaphase drop-spread technique, paraffin protection of the collection drop to avoid evaporation, and a motorized and programmable microscope stage. Thirteen readily-discernible telocentric chromosomes have been excised from metaphases of synchronized root-tip mitoses. After lysis in a collection drop (2 nl), the DNA was purified, restricted withRsaI, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. Finally, the amplified DNA was cloned into a standard plasmid vector. The size of the library was estimated to be approximately 44,000 recombinant plasmids, of which approximately 13% can be utilized for RFLP analysis. Tandem repetitive probes could be rapidly excluded from further analysis after colony hybridization with labelled total barley DNA. Analysis of 552 recombinant plasmids established that: (1) the insert sizes ranged between 70 and 1150 bp with a mean of 250 bp, (2) approximately 60% of the clones contained highly repetitive sequences, and (3) all single- or low-copy probes tested originate from chromosome 1HS. Four probes were genetically mapped, using an interspecificH. vulgare xH. spontaneum F2 population. One of these probes was found to be closely linked to theMla locus conferring mildew resistance.

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References

  • Ainsworth CC, Miller TE, Gale MD (1986) The genetic control of grain esterases in hexaploid wheat. II. Homoeologous loci in related species. Theor Appl Genet 72:219–225

    Google Scholar 

  • Anderson KL, Stack SM, Mitchel JB (1982) An investigation of the basis of a current hypothesis for the lack of G-banding in plant chromosomes. Exp Cell Res 138:433–436

    Google Scholar 

  • Antequera F, Bird AP (1988) Unmethylated CpG islands associated with genes in higher plant DNA. EMBO J 7:2295–2299

    Google Scholar 

  • Bonierbale MW, Plaisted RL, Tanksley SD (1988) RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics 120:1095–1103

    Google Scholar 

  • Coe EH, Neuffer MG, Hoisington DA (1988) The genetics of corn. In: Sprague GF, Dudley JW (eds) Corn and corn improvement, vol 3. American Society of Agronomy Madison Wisconsin, pp 81–258

    Google Scholar 

  • Conia J, Bergounioux C, Perennes C, Muller P, Brown S, Gadal P (1987) Flow-cytometric analysis and sorting of plant chromosomes fromPetunia hybrida protoplasts. Cytometry 8:500–508

    Google Scholar 

  • Davies KE, Young BD, Elles RG, Hill ME, Williamson R (1981) Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry. Nature 293:374–376

    Google Scholar 

  • Doležel J, Čihaliková J, Lucretti S (1992) A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L. Planta 188:93–98

    Google Scholar 

  • Edström J-E, Kaiser R, Röhme D (1987) Microcloning of mammalian metaphase chromosomes. Methods Enzymol 151:503–516

    Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Annal Biochem 132:6–13

    Google Scholar 

  • Gebhardt C, Ritter E, Debener T, Schachtschabel U, Walkemeier B, Uhrig H, Salamini F (1989) RFLP-analysis and linkage mapping inSolanum tuberosum. Theor Appl Genet 78:65–77

    Google Scholar 

  • Golds TJ, Babczinsky J, Rauscher G, Koop H-U (1992) Computer-controlled tracking of single cell development inNicotiana tabacum L. andHordeum vulgare L. protoplasts embedded in agarose/alginate films. J Plant Physiol 140:582–587

    Google Scholar 

  • Graner A, Siedler H, Jahoor A, Herrmann RG, Wenzel G (1990) Assessment of the degree and the type of restriction fragment length polymorphism in barley (Hordeum vulgare). Theor Appl Genet 80:826–832

    Google Scholar 

  • Graner A, Jahoor A, Schondelmaier J, Siedler H, Pillen K, Fischbeck G, Wenzel G, Herrmann RG (1991) Construction of an RFLP map of barley. Theor Appl Genet 83:250–256

    Google Scholar 

  • Greilhuber J (1977) Why plant chromosomes do not show Gbands. Theor Appl Genet 50:121–124

    Google Scholar 

  • Hadano S, Watanabe M, Yokoi H, Kogi M, Kondo I, Tsuchiya H, Kanazawa I, Wakasa K, Ikeda J-E (1991) Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome. Genomics 11:364–373

    Google Scholar 

  • Heun M, Kennedy AE, Anderson JA, Sorrels ME, Tanksley SD (1991) Construction of a restriction-fragment-length-polymorphism map for barley (Hordeum vulgare). Genome 34:437–447

    Google Scholar 

  • Hinze K, Thompson RD, Ritter E, Salamini F, Schulze-Lefert P (1991) RFLP-mediated targeting of theml-o resistance locus in barley (Hordeum vulgare). Proc Natl Acad Sci USA 88:3691–3695

    Google Scholar 

  • Islam AKMR (1983) Ditelosomic additions of barley chromosomes to wheat. Proc 6th Int Wheat Genet Symp Kyoto, Japan, pp 233–238

  • Jahoor A, Fischbeck G (1987) Source of resistance to powdery mildew in barley lines derived fromHordeum spontaneum collected in Israel. Plant Breed 99:274–281

    Google Scholar 

  • Jahoor A, Fischbeck G (1993) Identification of new genes for mildew resistance of barley at theMla locus inHordeum spontaneum-derived lines. Plant Breed (in press)

  • Jahoor A, Backes G, Graner A, Herrmann RG, Fischbeck G (1991) Development of RFLP markers for barley. Plant Breed 107:73–76

    Google Scholar 

  • Jung C, Fischer F, Claussen U, Horsthemke B, Herrmann RG (1992) A DNA library from an individualBeta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes. Plant Mol Biol 20:503–511

    Google Scholar 

  • Kao F-T, Yu J-W (1991) Chromosome microdissection and cloning in human genome and genetic disease analysis. Proc Natl Acad Sci USA 88:1844–1848

    Google Scholar 

  • Kosambi DD (1944) The estimation of map distances from recombination values. Ann Eugen 12:172–175

    Google Scholar 

  • Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181

    Google Scholar 

  • Landry BS, Kesseli RV, Farrara B, Michelmore RW (1987) A genetic map of lettuce (Lactuca sativa L.) with restriction fragment length polymorphism, isozyme, disease resistance and morphological markers. Genetics 116:331–337

    Google Scholar 

  • Lüdecke H-J, Senger G, Claussen U, Horsthemke B (1989) Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348–350

    Google Scholar 

  • Lüdecke H-J, Senger G, Claussen U, Horsthemke B (1990) Construction and characterization of band-specific DNA libraries. Hum Genet 84:512–516

    Google Scholar 

  • Pan WH, Houben A, Schlegel R (1993) Highly effective cell synchronization in plant roots by hydroxyurea, amiprophosmethyl and colchicine. Genome (in press)

  • Pillen K, Steinrücken G, Wricke G, Herrmann RG, Jung C (1992) A linkage map of sugar beet (Beta vulgaris L.). Theor Appl Genet 84:129–135

    Google Scholar 

  • Rimpau I, Smith DB, Flavell RB (1980) Sequence organization in barley and oat chromosomes revealed by interspecies DNA/DNA hybridization. Heredity 44:131–149

    Google Scholar 

  • Sandery MJ, Forster JW, Macadam SR, Blunden R, Jones RN, Brown SDM (1991) Isolation of a sequence common to Aand B-chromosomes of rye (Secale cereale) by microcloning. Plant Mol Biol Rep 9:21–30

    Google Scholar 

  • Schondelmaier J, Jacobi A, Fischbeck G, Jahoor A (1992) Genetical studies on mode of inheritance and localization of theamo1 (high amylose) gene in barley. Plant Breeding 109:274–280

    Google Scholar 

  • Schüller C, Backes G, Fischbeck G, Jahoor A (1992) RFLP markers to identify the alleles on theMla locus conferring powdery mildew resistance in barley. Theor Appl Genet 84:330–338

    Google Scholar 

  • Senger G, Lüdecke H-J, Horsthemke B, Claussen U (1990) Microdissection of banded human chromosomes. Hum Genet 84:507–511

    Google Scholar 

  • Siedler H, Graner A (1991) Construction of physical maps at theHor1 locus of two barley cultivars by pulsed-field gel electrophoresis. Mol Gen Genet 226:177–181

    Google Scholar 

  • Spangenberg G, Koop H-U (1992) Low-density cultures: microdroplets and single-cell nurse cultures. In: Lindsey K (ed) Plant tissue culture manual, supplement 1, section A. Kluwer Academic Publishers Dordrecht, Boston London, pp 1–28

    Google Scholar 

  • Tanksley SD (1988) Molecular mapping of plant chromosomes. In: Gustafson P, Appels R (eds) Chromosome structure and function. Plenum Publ Corp, New York, pp 157–173

    Google Scholar 

  • Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS (1992) Construction of a chromosome-enrichedHpaII library from flow-sorted wheat chromosomes. Nucleic Acids Res 20:1897–1901

    Google Scholar 

Download references

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Authors and Affiliations

  1. Institute of Botany, Ludwig-Maximilians-University Munich, Menzinger Straße 67, W-8000, Munich 19, Germany

    R. Martin, H. -U. Koop, R. G. Herrmann & C. Jung

  2. Institute for Plant Breeding, Technical University Munich, W-8050, Freising-Weihenstephan, Germany

    J. Schondelmaier & A. Jahoor

  3. Institute for Resistance Genetics, Federal Biological Research Center for Agriculture and Forestry, W-8059, Grünbach, Germany

    A. Graner

  4. Institute of Plant Genetics and Crop Plant Research, Corrensstraße 3, O-4325, Gatersleben, Germany

    A. Houben

Authors
  1. J. Schondelmaier
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  2. R. Martin
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  3. A. Jahoor
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  4. A. Houben
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  5. A. Graner
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  6. H. -U. Koop
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  7. R. G. Herrmann
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  8. C. Jung
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Communicated by F. Salamini

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Schondelmaier, J., Martin, R., Jahoor, A. et al. Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS. Theoret. Appl. Genetics 86, 629–636 (1993). https://doi.org/10.1007/BF00838719

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  • DOI: https://doi.org/10.1007/BF00838719

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