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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
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
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
Antequera F, Bird AP (1988) Unmethylated CpG islands associated with genes in higher plant DNA. EMBO J 7:2295–2299
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
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
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
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
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
Edström J-E, Kaiser R, Röhme D (1987) Microcloning of mammalian metaphase chromosomes. Methods Enzymol 151:503–516
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Annal Biochem 132:6–13
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
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
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
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
Greilhuber J (1977) Why plant chromosomes do not show Gbands. Theor Appl Genet 50:121–124
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
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
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
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
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
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
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
Kosambi DD (1944) The estimation of map distances from recombination values. Ann Eugen 12:172–175
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
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
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
Lüdecke H-J, Senger G, Claussen U, Horsthemke B (1990) Construction and characterization of band-specific DNA libraries. Hum Genet 84:512–516
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
Rimpau I, Smith DB, Flavell RB (1980) Sequence organization in barley and oat chromosomes revealed by interspecies DNA/DNA hybridization. Heredity 44:131–149
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
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
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
Senger G, Lüdecke H-J, Horsthemke B, Claussen U (1990) Microdissection of banded human chromosomes. Hum Genet 84:507–511
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
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
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
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
Author information
Authors and Affiliations
Additional information
Communicated by F. Salamini
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00838719