WO1990006375A1 - Procede d'identification de sequences d'adn dans des chromosomes de plantes - Google Patents

Procede d'identification de sequences d'adn dans des chromosomes de plantes Download PDF

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Publication number
WO1990006375A1
WO1990006375A1 PCT/GB1989/001440 GB8901440W WO9006375A1 WO 1990006375 A1 WO1990006375 A1 WO 1990006375A1 GB 8901440 W GB8901440 W GB 8901440W WO 9006375 A1 WO9006375 A1 WO 9006375A1
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WIPO (PCT)
Prior art keywords
dna
labelled
target plant
plant
source
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PCT/GB1989/001440
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English (en)
Inventor
Kesara Margrét ANAMTHAWAT-JÓNSSON
Michael David Bennett
John Seymour Heslop-Harrison
Andrew Rowland Leitch
Trude Schwarzacher
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The British Petroleum Company Plc
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Publication of WO1990006375A1 publication Critical patent/WO1990006375A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a method of identifying DNA sequences in the nuclear genome of a plant and to a method of plant breeding using this method.
  • Another method is to hybridize the chromosomes in a hybrid plant cell in situ with cloned DNA sequences as disclosed by N Lapitan e_t .al, (J Heredity 77, 415-419, 1986).
  • the probe DNA sequences will all be identical.
  • the sequences cloned are selected so as to be characteristic of one of the sources of the DNA in the target plant. However, the clones are difficult to isolate, and are unlikely to be generally applicable.
  • a method for identifying the origin of one or more sequences in the nuclear genome of a target eukaryotic plant containing genetic material from at least two different sources comprising hybridizing DNA from the target plant with labelled total genomic DNA fragments selected to hybridize to DNA from one of the sources, while preventing hybridization of the probe to sequences common to more than one source with unlabelled total genomic DNA fragments selected to block such common sequences by hybridizing with them in the target plant DNA and/or in the labelled DNA, and detecting the sites of hybridization of the labelled probe to the DNA of the target plant.
  • the scheme used for the purpose of the present specification is that given on page 33 of "Elementary Microbiology" by 0 Wyss, O B Williams and E W Gardener Jr., John Wiley & Sons Trie 1963.
  • the plant kingdom includes bacteria which are included in a phylum identified as the Protophvta (primitive plants without intracellular membranes). Eukaryotic plants therefore are all the members of the plant kingdom except the Protophyta.
  • the plant is preferably a vascular plant, more preferably one of the Spermatophvta (angiosperms, gymnosperms).
  • the plant is most preferably monocotyledonous, e.g. one of the Gramineae.
  • the target plant may contain DNA from Hordeum. Secale. Triticum. or Aegilops species.
  • Specific target plants to which the present inveniton may be applied are those containing a) DNA from a Hordeum and from a Secale source, b) DNA from a Triticum or a Aegilops source and a Secale source.
  • target plant is the plant whose DNA sequences are under investigation.
  • the target plant potentially contains DNA sequences originating from more than one distinct
  • target DNA sequences are some of the DNA sequences which may be genomes, chromosomes or chromosome segements potentially included in the nuclear genome of the target plant which are to be detected and followed through successive generations.
  • the "source" of the DNA ion the target plant may be the parents (e.g. in the first generation hybrid H. chilense x S. africanum) or be more distant ancestors of the target plant (e.g. S. cereale and T. durum Desf. in Triticale, or T. monococcum and others in T. aestivum).
  • the total genomic DNA used as labelled probe or blocking DNA can be isolates from the sources of DNA in the target plant.
  • the probe or blocking DNA may also be isolated from "remote sources” which are taxa related to the sources (e.g. of the same family, or the same genus).
  • At least one, and preferably all sources of the DNA sequences in the target plant should be distinct.
  • One source should preferably have some recognizable characteristic not present in the other sources which results from a difference in its genetic make-up (e.g. disease resistance, morphology and also including gene expression factors).
  • the fragments of nuclear genome used to block DNA sequences in common between the labelled probe and more than one of the sources in the target plant will be selected according to the degree of difference between the sources in the target plant.
  • the sources are closely related (e.g. H. vulgare and H. bulbosum) it will be preferably to use total genomic DNA from one of the sources as block.
  • the sources are less closely related for example Hordeum vulgare and Secale africanum the total genomic DNA used as a blocking agent may be from a remote source if it is less closely related to the source of labelled DNA than to the other source or sources of DNA in the target plant.
  • hybridization as used for the artificial reannealing of single stranded forms of the DNA from plants with single stranded labelled DNA is understood in the art (as shown by Le et al and Lapitan et al (loc. cit. ) . for review see Henderson, Int.Rev.Cytol. 76, 1-46, 1982). It is, of course, distinct from the hybridization which takes place as a consequence of sexual reproduction in nature.
  • the single stranded DNA from the target plant may be in the form of chromosomes prepared for microscopy.
  • the present invention may be applied to DNA in the target plant which DNA is present as a metaphase or interphase chromosome preparation from the target plant.
  • the DNA may be extracted from the target plant and immobilized on a membrane, optionally on restricted areas of the membrane.
  • the DNA from the target plant may be digested with one or more restriction enzymes and size separated by electrophoresis before transfer to the membrane.
  • the DNA may be immobilized on the membrane by squashing target plant tissue and transferring the exudate on to the membrane.
  • Methods of producing single stranded DNA and of hybridizing it with labelled DNA probes are generally known and are for example disclosed by Lapitan et al and Henderson (loc. cit. ) .
  • the process of the present invention makes use of labelled total genomic DNA as a probe to hybridize to the target DNA sequences, blocked with unlabelled total genomic DNA from other sources.
  • the lengths of the labelled and unlabelled probe DNA sequences are generally less than that of the total length of the DNA sequence in the chromosome, 50 to 1000 base pairs typically.
  • the length reduction may be achieved by, for example, the DNA extraction process itself, autoclaving the DNA, sonication or mechanical shearing.
  • the DNA labelling procedure may also produce fragments of DNA sequences - oligo-labelling gives fragments which have a length of 80-120 base pairs.
  • Total genomic DNA from one of the sources present in the target plant is labelled and referred to as "labelled DNA”.
  • labelled DNA Preferably total genomic DNA from one or more of the sources present in the target plant, but not DNA from the source which supplied the labelled DNA, is used as "blocking DNA” and is unlabelled.
  • DNAs are made single-stranded ' before being placed under conditions which allow hybridization.
  • the blocking DNA which may be present in more than five times the concentration of the labelled DNA is brought in contact with (a) the target plant DNA, (b) the labelled DNA or (a) and (b) combined.
  • the labelled sequences can hybridize with those sequences of the target plant DNA which are still in single stranded form because they do not correspond to sequences in the blocking DNA.
  • many of those sequences of the labelled DNA which are not common to the blocking DNA remain single-stranded, and they are then the only labelled sequences remaining single-stranded and thus available to hybridize to the target plant DNA.
  • the blocking DNA hybridizes with the sequences of the target plant derived from the same source as the blocking DNA, and also sequences of the target plant DNA and/or labelled DNA which are in common with the blocking source DNA. In all cases, hybrid DNA between unlabelled and unlabelled, between labelled and unlabelled and between labelled and labelled will form. Because of the higher concentration of unlabelled DNA, the frequency of the latter will be lower, leaving labelled, generally low copy number sequences available to hybridize to corresponding sequences of the DNA from the target plant.
  • the labelled DNA is detected in some way as being different from the unlabelled DNA sequences, for example due to the presence of radioactive atoms or other atoms or groups, e.g. biotin or mercury.
  • Radioactive labelling may be used, which may be detected by autoradiography.
  • a preferred method is labelling with biotin, as described by, for example, Lapitan e_t t_l_ (loc. cit. ) . using nick translation.
  • the presence of biotin labelled material may be detected by fluorescence or colorimetric techniques.
  • An alternative method is to label the DNA with an enzyme and to detect the label by a reaction catalysed by the enzyme.
  • the marking of the potentially present target DNA may be positive or negative. Positive marking would mark the target DNA by the presence of e.g. radioactive atoms or biotin groups after the hybridization. Alternatively, negative marking may distinguish the target DNA by the absence of label.
  • Plant DNAs Total genomic DNA was isolated from leaves of Hordeum vulgare. H. ehilense Roem & Schult, Secale cereale, S africanum Stapf and the hybrids H. vulgare x S. africanum and H. ehilense x S. africanum. Following a standard dot blotting protocol, the DNA was denatured and transferred to a hybridization transfer membrane (GeneScreenPlus membrane, E I du Pont de Nemours & Co Inc, Boston, USA), applying approximately 0.08, 0.2, 0.8 and 2.0 ⁇ g DNA of each species to different dots of the dot blotter, before alkaline denaturation.
  • a hybridization transfer membrane GeneScreenPlus membrane, E I du Pont de Nemours & Co Inc, Boston, USA
  • Blocking DNA Autoclaved total genomic DNA from H. vulgare.
  • the transfer membrane was pre-hybridized with 200 ⁇ g of denatured blocking DNA in 2 ml of a standard hybridization solution (Sharp PJ, Kreis M, Shewry PR, Gale MD. Theor. Appl. Genet. 75:289-290, 1988).
  • a standard hybridization solution Sharp PJ, Kreis M, Shewry PR, Gale MD. Theor. Appl. Genet. 75:289-290, 1988.
  • 0.5 ⁇ g denatured labelled DNA and 1 g denatured herring carrier DNA were added to the plastic bag and incubated overnight at 70*C.
  • Post-hybridization washes were performed with O.l ⁇ xSSC (20xSSC: 3M sodium chloride, 0.3M sodium citrate, pH7) at 60*C. Hybridization under such conditions should allow sequences with 80% homology to form hybrids.
  • Hybridized biotinylated DNA was visualised (made visible) using the streptavidin/alkaline phosphatase colorimetric detection system by Bethesda Research Laboratories (Maryland, USA).
  • Hybridization to both S. africanum and S. cereale was strongly visible in the dots containing 2.0 and 0.8 ⁇ g of DNA and only weakly to 0.2 ⁇ g DNA.
  • the hybridization to the DNA of the two Hordeum species was only detected in the 2.0 ⁇ g dot. Differentiation between S. africanum and the Hordeum species and detection of the S. africanum DNA in the hybrids was possible (Example 1), but impossible between S. africanum and S. cereale (Test A).
  • Example 3 and Comparative Test B A hybridization membrane was prepared and hybridized as described in Example 1. Total genomic DNA from H. vulgare (0.5 ⁇ g) was labelled with biotin by nick-translation, and autoclaved total genomic DNA (100 ⁇ g) from H. ehilense was used as blocking DNA.
  • Example 2 The dots containing H. vulgare and H. vulgare x S. africanum DNA (Example 2) showed more hybridization signal than the dots containing H. ehilense and H. ehilense x S. africanum DNA (Test B).
  • Example 4 and Comparative Tests C and D The dots containing H. vulgare and H. vulgare x S. africanum DNA (Example 2) showed more hybridization signal than the dots containing H. ehilense and H. ehilense x S. africanum DNA (Test B).
  • Plant DNAst Total genomic DNA from H. vulgare and H. bulbosum Nevski was digested with the restriction enzymes EcoRI and Dral, size separated by agarose gel electrophoresis and transferred to Hybond N + (Amersham International pic, Amersham, U.K.) support membrane using alkaline transfer methodology. Labelled DNA; Total genomic DNA from H. bulbosum. Blocking DNA; Autoclaved total genomic DNA from H. vulgare. For probe labelling, hybridization and detection of hybridization sites, the chemiluminescence method ⁇ CL (Amersham) was used following the manufacturer's protocol. Briefly, the transfer membrane was pre-incubated for 30 min in the hybridization solution and denatured blocking DNA (6 ⁇ g/ml).
  • Examples 5 and 6 showed that the DNA from a remote source can be used to block the common sequences in hybridization experiments.
  • T. aestivum cv. Chines'e Spring showed only weak hybridization. Stronger hybridization was detected to DNA of S. cereale. triticale, T. aestivum cv. Beaver and cv. Glennson.
  • rye DNA can be discriminated in triticale (a hybrid between wheat and rye) and in wheat varieties containing a rye chromosome segment (cv. Beaver and Glennson). Signal quantification showed that the hybridization was approximately proportional to the amount of rye material present.
  • Chromosome preparations were made from fixed root tips from
  • H. ehilense x S. africanum by placing them in a mixture of 4% cellulase and 40% liquid pectinase in 0.01M citric acid/sodium citrate buffer (pH 4.6) for l-2h at 37 ⁇ C. They were subsequently squashed in 45% acetic acid following standard cytological procedures.
  • Hybridized labelled probe was detected using fluoresceinated avidin, and amplified with biotinylated anti-avidin. Chromatin was counterstained with propidium iodide (1-2 ⁇ g/ml in phosphate buffered saline).
  • Example 9 Prosphases, telophases and interphases which were treated as described in Example 8 showed distinct domains belonging to either yellow labelled chromosomes from S. africanum or red unlabelled chromosomes from H. ehilense.
  • Example 10 Chromosome preparations from root tips of H. vulgare x S.

Abstract

On identifie l'origine d'une séquence d'ADN dans le génome nucléaire d'une plante eucaryotique contenant du matériau génétique provenant d'au moins deux sources distinctes, en hybridant les ADNs provenant de la plante avec tous les fragments marqués du génome sélectionnés de façon à s'hybrider sur les ADNs provenant d'une des sources, tout en bloquant les séquences communes à plus d'une source avec tous les fragments non marqués du génome sélectionnés de façon à bloquer ces séquences communes.
PCT/GB1989/001440 1988-12-02 1989-12-01 Procede d'identification de sequences d'adn dans des chromosomes de plantes WO1990006375A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB888828130A GB8828130D0 (en) 1988-12-02 1988-12-02 Method of identifying dna sequences in chromosomes of plants
GB8828130.8 1988-12-02

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WO1990006375A1 true WO1990006375A1 (fr) 1990-06-14

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EP (1) EP0446292A1 (fr)
JP (1) JPH04502253A (fr)
AU (1) AU4660289A (fr)
CA (1) CA2004408A1 (fr)
GB (1) GB8828130D0 (fr)
WO (1) WO1990006375A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0444649A2 (fr) * 1990-02-27 1991-09-04 Agrilab Biotechnology Ltd. Procédé pour le dépistage des agents pathogènes des plantes, pouvant être utilisés sur le terrain et trousse diagnostique pour sa rÀ©alisation
FR2711143A1 (fr) * 1993-10-13 1995-04-21 United Kingdom Government Procédé d'identification d'un genre, d'une espèce ou d'une variété de fruits; kit de test, oligonucléotide et sonde d'hybridation pour cette identification.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0242991A2 (fr) * 1986-03-20 1987-10-28 Lubrizol Genetics Inc. Carte chromosomique élargie de la tomate ainsi que banque d'ADN complémentaire

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JPH07828B2 (ja) * 1984-10-26 1995-01-11 大同特殊鋼株式会社 浸炭部品
JPS61253346A (ja) * 1985-04-30 1986-11-11 Kobe Steel Ltd 高強度歯車用鋼
JPS624465A (ja) * 1985-07-01 1987-01-10 Nippon Kokan Kk <Nkk> プライマ塗布装置
JPS62274052A (ja) * 1986-05-21 1987-11-28 Daido Steel Co Ltd 軸受用肌焼鋼

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0242991A2 (fr) * 1986-03-20 1987-10-28 Lubrizol Genetics Inc. Carte chromosomique élargie de la tomate ainsi que banque d'ADN complémentaire

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Cancer Research, Vol. 48, 1988 Peter Devilee et al: "Detection of Chromosome Aneuploidy in Interphase Nuclei from Human Primary Breast Tumors Using Chromosome-specific Repetitive DNA Probes ", see page 5825 - page 5830, see especially p 5825 col 2 lines 11-33. *
Dialog Information Services, File 55, BIOSIS 1980-90BIOSIS number 83075427, Lapitan N L V et al, "Wheat-rye translocations detection of chromosome breakpoints by in-situ hybridization with a biotin- labeled DNA probe", J HERED 77 (6), 1986 (RECD.1987)415-419 *
Dialog Information Services, File 55, BIOSIS 1980-90BIOSIS number 88119452, Schwarzacher T et al: "In situ localization of parental genomes in a wide hybrid", ANN BOT (LOND) 64 (3), 1989, 315-324 *
Hum Genet, Vol. 80, 1988 T. Cremer et al: "Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes ", see page 235 - page 246, see especially the summery and p 236 col 1 lines 26 - col 2 line 4. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0444649A2 (fr) * 1990-02-27 1991-09-04 Agrilab Biotechnology Ltd. Procédé pour le dépistage des agents pathogènes des plantes, pouvant être utilisés sur le terrain et trousse diagnostique pour sa rÀ©alisation
EP0444649A3 (en) * 1990-02-27 1991-12-27 Agrilab Biotechnology Ltd. Procedure for the detection of plant pathogens under field conditions and a diagnostic kit for its application
FR2711143A1 (fr) * 1993-10-13 1995-04-21 United Kingdom Government Procédé d'identification d'un genre, d'une espèce ou d'une variété de fruits; kit de test, oligonucléotide et sonde d'hybridation pour cette identification.

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EP0446292A1 (fr) 1991-09-18
JPH04502253A (ja) 1992-04-23
GB8828130D0 (en) 1989-01-05
AU4660289A (en) 1990-06-26
CA2004408A1 (fr) 1990-06-02

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