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WO1994001580A1 - Identification par genie genetique - Google Patents

Identification par genie genetique Download PDF

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Publication number
WO1994001580A1
WO1994001580A1 PCT/AU1993/000320 AU9300320W WO9401580A1 WO 1994001580 A1 WO1994001580 A1 WO 1994001580A1 AU 9300320 W AU9300320 W AU 9300320W WO 9401580 A1 WO9401580 A1 WO 9401580A1
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WO
WIPO (PCT)
Prior art keywords
rdna
nucleic acid
chromatin
sample
fragments
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PCT/AU1993/000320
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English (en)
Inventor
Nigel Steele Scott
Mark Raymond Thomas
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Commonwealth Scientific And Industrial Research Organisation
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Application filed by Commonwealth Scientific And Industrial Research Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to AU43007/93A priority Critical patent/AU4300793A/en
Publication of WO1994001580A1 publication Critical patent/WO1994001580A1/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
    • C12Q1/6876—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method of identifying vegetatively propagated crops and to probes for use in such a method.
  • Grapevines for example, are cultivated commercially on more than 10 million hectares throughout the world and the commercially important cultivars and clones of grape ⁇ vines are maintained by vegetative propagation.
  • the genus Vitis contains more than 70 species, and Vitis vinifera contains hundreds of different varieties and clones.
  • ampelography The science of ampelography, the identification of grapevines from the physical characteristics of the leaves, fruit, etc., is a highly specialised area of systematic botany.
  • ampelography still requires the ability of experts able to integrate subjectively a wide range of visual features and unerringly identify individual grapevines (Scott and Possingham, 1989) . It seems probable that younger scientists with such skills are unlikely to be available in the future. The loss of such experts is further compounded by an ever-increasing range of cultivars and clones which make reliable identification even more difficult.
  • ampelographic identification of grapevines requires leaf and fruit samples, together with observations at different stages of the season often over more than one year and the scoring of some 70 or 80 characteristics.
  • isoenzyme analysis depends on the observation that many plant enzymes differ marginally in protein structure (i.e. are polymorphic) from variety to variety. Often such a method can show that plants are different but it can never show that they are the same, because the presence or absence of polymorphic proteins in any sample can be a result of differential gene expression due to the stage of plant development and interactions with the environment. In other words, the limitation of isozyme analysis is that it is an observation of the genetic phenotype, rather than a direct observation of the plant genotype.
  • a ribosomal DNA (rDNA) probe sequence corresponding to a plant rDNA repeat unit or a part thereof.
  • the rDNA probe sequence may be isolated from Vitis.
  • the rDNA probe sequence may be isolated from V.vinifera or V.champini.
  • the rDNA probe sequence may be substantially homologous with the rDNA repeat unit of Vitis or a part thereof.
  • the rDNA probe sequences according to this aspect of the present invention may be utilized in a method of identifying grapevine cultivars from a small tissue fragment from any part of the plant. More particularly, the present invention relates to a method of identifying grapevine cultivars by examining polymorphisms in the IGS regions of rDNA repeat units.
  • ribosomal RNA genes in higher eukaryotes are organized in tandem arrays. In plants, the number of rDNA units per haploid genome is highly variable between species (from 570 in Arabidopsis to 31000 in Hvacinthus orientalis) and within species.
  • the rDNA is transcribed as a single RNA precursor molecule which is subsequently processed into the mature 18S, 5.8S and 25S rRNAs. Each unit contains a single transcription unit as well as long IGS sequence.
  • the IGS regions generally exhibit extensive intraspecific variability in both sequence and length. There may be some useful variability in the conserved part of these genes as well as the IGS region.
  • the rDNA probe sequence may be isolated from any suitable plant genus.
  • the rDNA probe sequence may be a homologous probe isolated from grapevine.
  • the rDNA probe sequence may be a heterologous probe.
  • the heterologous probe may be isolated from spinach or wheat. In these cases although the IGS regions are usually very heterologous the attachment of the probes to conserved regions of the rRNA genes makes heterologous probes almost as useful as homologous probes.
  • the rDNA probe sequence may include the entire grapevine rDNA repeat unit or a part thereof.
  • the rDNA probe sequence may span the entire IGS region or a part thereof.
  • the rDNA probe sequence may span a plurality of restriction sites within the IGS region.
  • the rDNA probe sequence may span a BamHI and EcoRI restriction site.
  • the rDNA probe sequence may span a BamHI and Hindlll restriction site.
  • the rDNA probe sequence may span a BamHI. EcoRI and Hindlll restriction site.
  • a rDNA probe sequence selected from pVc-A, pVvc-A and pVvc-B as hereinafter described.
  • the rDNA probe sequence may be labelled in any suitable manner. A radioactive or non-radioactive labelling may be used.
  • the rDNA probe sequence may be provided in the form of a cloned insert in a suitable plasmid cloning - universal vector.
  • a method for the analysis of a nucleic acid sample which method includes providing a genomic nucleic acid sample including a mixture of at least two fragments; and a rDNA probe sequence as hereinbefore described; subjecting the mixture of genomic nucleic acid fragments to a separation step; and hybridizing the separated fragments with the rDNA probe sequence.
  • the method for the analysis of a nucleic acid sample may include the preliminary step of providing a genomic nucleic acid sample; and at least one restriction enzyme; and digesting the nucleic acid sample with the restriction enzyme to form a mixture of at least two fragments.
  • the genomic nucleic acid sample may be DNA isolated from crops in which the whole plant or a scion is propagated vegetatively and may be applied to seed propagated crops.
  • the genomic DNA sample may be isolated from crops including fruits including citrus, pome fruits, stone fruits and nuts, including filberts and almonds, more specifically grapevine, citrus, macadamia, apples, pears, apricots, flowers including roses (for example Geraldton Wax), hops and vegetables.
  • propagated vegetatively includes a plant or scion thereof propagated clonally.
  • ribosomal RNA genes have been isolated from Vitus vinifera cv. Cabernet Sauvi ⁇ non and one from Vitis cha pini. Both length and restriction site variation is detected between these genes in the IGS region.
  • the rDNA copy number is about 1000 to 2500 per haploid genome.
  • the IGS regions are variable amongst Vitis species and between cultivars of V_-_ vinifera.
  • the separation step may be gel electrophoresis.
  • the electrophoresis, hybridization and restriction enzyme digestion may be performed using conventional techniques that would be familiar to a person skilled in the art.
  • the rDNA probe sequences as hereinbefore described may be utilized individually or in combination.
  • a method for the analysis of a nucleic acid sample which method includes providing at least two oligonucleotide primers complementary to substantially conserved regions of a genome; and a sample of genomic nucleic acids; performing an amplification step so as to amplify substantially the nucleic acid sequence flanked by the primers; and subjecting the amplified nucleic acid so obtained to an analytical step.
  • the 5S rRNA genes are arranged in long tandem arrays.
  • the repeating unit is composed of the 5S rRNA gene (about 120 bp in length) and a noncoding or spacer region that can range from 200 bp to greater than 1500 bp in plants.
  • the length and DNA sequence of the spacer region can be highly variable between and within a species. Length variation and DNA sequence differences of the spacer are also observed within individual plants. There is little DNA variation in the 5S rRNA gene of eukaryotes.
  • the oligonucleotide primers may be complementary to substantially conserved regions of the 5S rDNA sequence that flank the variable spacer region.
  • the primers may be of any suitable type so as to result in amplification of a sequence including the 5S rDNA variable spacer region.
  • the method for analysis of a nucleic acid sample may utilize two oligonucleotide primers complementary to substantially conserved regions of the Vitis genome, more preferably oligonucleotide primers having the sequences 5' G T A G T A C T A G G G A T G G G G T G A C C 3' and 5' C G G A G T T C T G A T G G G G A T C C G 3'.
  • the advantages of analysing the 5S rRNA repeat locus in this way may include:
  • the primers appear to be universal. PCR products have been obtained from plants in different genera (Citrus. Vitis. Pisum. Lvcopersicum) . (3) The PCR product is predominantly the noncoding spacer region which is potentially a rich source of
  • eukaryotic genomes also contain simple sequences that are highly repeated.
  • eukaryotic genomes may contain repeated sequences that are composed of only one or two nucleotides, but the length of the repeat may be over one hundred nucleotides.
  • These serially repeated DNA or microsatellite sequences seem to have evolved unusually rapidly and there are usually marked differences between the serial repeat sequences of any two closely related species. This contrasts with the high degree of conservation of DNA sequence elsewhere in the genome.
  • the present invention provides a nucleic acid sequence including a serial repeat sequence as shown in Figure 5(a) to 5(t) or fragments thereof or sequences substantially homologous thereto or fragments thereof.
  • the oligonucleotide primers may be complementary to unique substantially conserved sequences flanking a serial repeat sequence.
  • the primers may be of any suitable type so as to result in amplification of a sequence including a serial repeat sequence.
  • the method for analysis of a nucleic acid sample for the sequence in Figure 5(a) may utilize two oligonucleotide primers having the sequences 5' A C A A T T G G A A A C C G C G T G G A G and 5' C T T C T C A A T G A T A T C T A A A A C C A T G
  • the method for analysis of a nucleic acid sample for the sequence in Figure 5(b) may utilize two oligonucleotide primers having the sequences 5' C A G C C C G T A A A T G T A T C C A T C and 5' A A A T T C A A A A T T C T A A T T C A A C T G G
  • nucleic acid sample for the sequence in Figures 5(c) to 5(t) may utilise two oligonucleotide primers that flank the serial repeat in each sequence.
  • the oligonucleotide primers shown in Figures 5(a) to 5(q) are particularly applicable to analysis of nucleic acid isolated from Vitis.
  • the oligonucleotide primers shown in Figures 5(r) to 5(t) are particularly applicable to analysis of nucleic acid isolated from hops.
  • the results obtained using more than one set of primers may be combined to complete an identification.
  • PCR the spacer region or serial repeat sequence is obtained in sufficient quantity for analysis. Length and base polymorphism of the amplified region may be investigated between and within a species or individual plants.
  • the method for analysis of a nucleic acid sample may include the step of subjecting the amplified nucleic acids to a separation step.
  • the separation step may be electrophoresis, for example agarose or acrylamide gel electrophoresis.
  • the amplified nucleic acid may be digested with one or more restriction enzymes to form a mixture of at least two fragments, prior to the separation step.
  • nucleotide sequence of the amplified nucleic acids may be determined by conventional techniques that would be familiar to a person skilled in the art.
  • the nucleic acids may be isolated from any of the plants described above. In a preferred aspect, the nucleic acids may be isolated from Citrus. Vitis. Pisum. Lycopersicum or hops.
  • nucleic acids may be isolated from eukaryotic tissue in a manner so as to minimise damage thereto.
  • the isolated nucleic acids may then be subjected to analysis as described above.
  • the present invention further provides a method for isolating nucleic acids from eukaryotic material which method includes providing a eukaryotic sample; and a chromatin isolation composition capable of maintaining chromatin in a substantially intact state; subjecting the eukaryotic sample to a crushing or grinding step; contacting the eukaryotic sample with the chromatin isolation composition; and isolating intact nuclei, fragmented nuclei and chromatin from other cell material prior to nucleic acid extraction.
  • the eukaryotic material may be a plant material or a non-plant material such as human, animal or fungal material.
  • the eukaryotic material may be a plant sample.
  • the plant sample may be of any suitable type as discussed above.
  • the plant sample may be a leaf sample.
  • the crushing or grinding step may be undertaken utilizing a grinding aid such as sand.
  • the crushing or grinding step may be conducted in the presence of liquid nitrogen.
  • the chromatin isolation composition may be of any suitable type.
  • the chromatin isolation composition may include a polyphenol-binding agent such as polyvinyl- pyrrolidone (PVP) .
  • PVP polyvinyl- pyrrolidone
  • a PVP sold under the trade name PVP 40,000 was found to be suitable.
  • the polyphenol binding agent may be present in amounts of from approximately 0.1% to 20% by weight, preferably 1% to 5% by weight, based on the total volume of the chromatin isolation composition.
  • the chromatin isolation composition may include a reducing agent such as mercaptoethanol to prevent oxidation of secondary metabolites.
  • the reducing agent may be present in amounts of from approximately 0.05% to 5% by weight, preferably 0.1% to 2.5% by weight, based on the total volume of the chromatin isolation composition.
  • the chromatin isolation composition may contain other components such as sodium chloride, tris(hydroxy- methyl)aminomethane (Tris), disodium ethylenediamine- tetraacetate (EDTA) , trisodium citrate; or mixtures thereof.
  • the components of the chromatin isolation composition may be present in concentrations such that the total ionic strength of the composition maintains the proteins and nucleic acids of the chromatin in a substantially associated state.
  • a buffer solution having a pH of approximately 6.5 to 8.5, preferably 7 to 8, may be used.
  • the contacting step may be undertaken in any suitable manner.
  • the ground plant sample may be placed in a container contacting the chromatin isolation composition.
  • the contacting step may be conducted at reduced temperature, for example from approximately 0°C to 10°C, to minimize damage to the nucleic acids present.
  • the container may be a centrifuge tube.
  • the chromatin isolation step may be any suitable process.
  • the chromatin isolation step may be a centrifugation process.
  • the sample may be centrifuged under reduced temperature for a period sufficient to substantially separate the intact nuclei, fragmented nuclei and chromatin from the other cell debris. Centrifugation may continue for approximately 5 to 25 minutes. In this way the nucleic acids are isolated from the majority of other eukaryotic components which impede nucleic acid purification while the nucleic acids are still protected by the proteins of the chromatin.
  • the nucleic acids may be extracted from the chromatin by conventional techniques that would be familiar to a person skilled in the art.
  • a nucleic acid extraction buffer Prior to extraction the intact nuclei, fragmented nuclei and chromatin may be suspended in a nucleic acid extraction buffer, which may be of any suitable type such that when the chromatin is contacted with said buffer the chromatin dissociates to form its constituent proteins and nucleic acids.
  • the nucleic acid extraction buffer may include a detergent such as N-lauryl sarcosine (sarkosyl) .
  • the detergent may be present in amounts of from approximately 0.5% to 20% by weight, preferably 1% to 5% by weight, based on the total volume of the nucleic acid extraction composition.
  • the nucleic acid extraction composition may include a primary alcohol such as ethanol.
  • the primary alcohol may be present in amounts from approximately 1% to 50% by weight, preferably 10% to 30% by weight, based on the total volume of the nucleic acid extraction composition.
  • the nucleic acid extraction composition may contain other components such as sodium chloride, Tris, a polyphenol-binding agent such as PVP 40,000, EDTA, a reducing agent such as mercaptoethanol mixtures thereof.
  • a buffer solution having a pH of approximately 6.5 to 8.5, preferably 7 to 8, may be used.
  • FIGURE 1 is a diagrammatic representation of a first figure.
  • FIGURE 2 Physical maps of rDNA repeat unit in V ⁇ . champini
  • FIGURE 4
  • FIGURE 5 Southern hybridization of Hindlll digests of genomic DNA from V___ vinifera cultivars with pVvc-A. Lane 1, Pinot Noir; Lane B, Sultana; Lane C, Tarran ⁇ o; Lane D, Cabernet Sauvi ⁇ non; Lane E, Touri ⁇ a; Lane F, Sauvi ⁇ non Rose.
  • FIGURE 5 Southern hybridization of Hindlll digests of genomic DNA from V___ vinifera cultivars with pVvc-A. Lane 1, Pinot Noir; Lane B, Sultana; Lane C, Tarran ⁇ o; Lane D, Cabernet Sauvi ⁇ non; Lane E, Touri ⁇ a; Lane F, Sauvi ⁇ non Rose.
  • FIGURE 5 Southern hybridization of Hindlll digests of genomic DNA from V___ vinifera cultivars with pVvc-A. Lane 1, Pinot Noir; Lane B, Sultana; Lane C, Tarran ⁇ o; Lane D, Cabernet Sauvi ⁇ non; Lane E, Touri ⁇ a; Lane F, Sauvi ⁇ non Rose.
  • Grapevine sequences designated (a) G7, (b) 17GA, (c) A21GA, (d) INSKA4, (e) INSKGA28, (f) M13A3, (g) SKA19, (h) IN36GT, (i) SKT30, (j) 213GT and (k)IN18GA. Positions of oligonucleotide primers are shown by underlining. In each case the simple repeat sequence is flanked by the primers.
  • DNA digested with restriction enzymes was electro- phoresed through 0.7% or 2% agarose gels, then transferred to a nylon membrane (Gene Screen Plus, New England Nuclear) by alkaline blotting.
  • the filter was hybridized with random-primed fragments in 6xSSC, 1%SDS and following hybridization the filter was washed with 2xSSC, 1%SDS at 65°C.
  • the rDNA repeat unit identified from most plants so far contains single sites for EcoRV and Xbal, and it was likely that the bands on the Xbal blots and the ca 10.5-12.21 kbp bands on the Hindlll blot of V.vinifera cv. Cabernet Sauvi ⁇ non were the full length Vitis rDNA repeat units.
  • rRNA gene copy number varies widely in many plants. From inspection of Southern blots of Vitis species and V.vinifera cultivars, it appears that rRNA gene copy number is relatively constant and dot blot hybridizations gave copy numbers of 2500 per diploid genome for V.vinifera cv. Cabernet Sauvi ⁇ non, and 1000 for Sultana. These two cultivars contained the highest and lowest copy numbers, respectively. The average copy number was about 1500 per diploid genome (e.g. Pinot cultivars) and V.champini also contained about 1500 copies per diploid genome. Ribosomal DNA repeat unit polymorphism in Vitis
  • the rDNA polymorphisms in 29 different V.vinifera cultivars were also examined using pVvc-A as a probe.
  • the simplest hybridization patterns were obtained with Hindlll digested fragments. These patterns in relation to the rDNA genes contain both complete rDNA unit and some IGS fragments.
  • Six different Hindlll digestion patterns were found amongst these varieties ( Figure 4) and all 29 cultivars could be matched to one of these patterns (Table 1) . More complicated patterns were found when the genomic DNA was digested with BamHI or EcoRI (results not shown). These patterns could also be grouped by their similarities and allowed further subdivision of the groups in Table 1.
  • the development of repetitive nuclear DNA sequences as markers for grapevine (Vitis) identification offers an objective and rapid technique as an alternative to ampelography. These DNA markers would also prove useful in studies of genome mapping and organisation.
  • the nuclear genome of grapevine is small with a 2C value of 0.7 pg, but is composed of a variety of repeated sequences.
  • the microsatellite squences or simple sequence repeats are present with copy number dependent on the particular repeated motif.
  • Minisatellite sequences similar to the tandem repeat in M13 phage are also present.
  • Clones containing simple repeat sequences were identified in a genomic libraries of grapevine DNA using simple di-, tri- or tetra- nucleotide repeats such as (AT) 8 ' (GT) ⁇ o' (CGT >io and the like P r obes. The clones were then sequenced. Nineteen such sequences are shown in Figures 5(a) to (t) inclusive. The first sixteen sequences are suitable for Vitis. The remaining three sequences are suitable for hops. TABLE 1 Classification of V.vinifera cultivars according to the rDNA polymorphism.
  • DNA spacer region its structure and variation in populations and among species. Theor. Appl. Genet. 63;337-348.

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Abstract

Une séquence constituant une sonde à ADN ribosomique correspond à l'unité répétitive de l'ADN ribosomique d'une plante ou à une de ses parties. On décrit aussi des procédés permettant d'analyser et d'isoler des échantillons d'acide nucléique.
PCT/AU1993/000320 1992-07-03 1993-06-30 Identification par genie genetique WO1994001580A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0785281A4 (fr) * 1995-07-28 1999-11-24 Sapporo Breweries Methode de differentiation genetique des varietes du houblon
DE19856064A1 (de) * 1998-12-04 2000-06-29 Invitek Gmbh Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008117A1 (fr) * 1990-10-17 1992-05-14 Applied Biosystems, Inc. Procede de determination d'un genotype par comparaison de la sequence de nucleotides de membres d'une famille de genes et materiel a cet effet
AU3107493A (en) * 1992-01-17 1993-07-22 Pioneer Hi-Bred International, Inc. Detection of nucleotide sequences using simple sequence repeats and oligonucleotide ligation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008117A1 (fr) * 1990-10-17 1992-05-14 Applied Biosystems, Inc. Procede de determination d'un genotype par comparaison de la sequence de nucleotides de membres d'une famille de genes et materiel a cet effet
AU3107493A (en) * 1992-01-17 1993-07-22 Pioneer Hi-Bred International, Inc. Detection of nucleotide sequences using simple sequence repeats and oligonucleotide ligation

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BIOCHIM. BIOPHYS. ACTA, Vol. 825, issued 1985, (Elsevier, Holland), KATO A. et al., "Repeated DNA Sequences Found in the Large Spacer of Vicia Faba rDNA", pages 411-415. *
CAN. J. GENET. CYTOL., Vol. 28(5), issued 1986, by the Genetics Society of Canada (Ottawa), APPELS R. et al., "Alien Chromatin in Wheat: Ribosomal DNA Spacer Probes for Detecting Specific Nucleolar Organizer Region Loci Introduced Into Wheat", pages 665-672. *
CHROMOSOMA, (BERL.), Vol. 86, issued 1982, (Springer-Verlag, Berlin), HADLACZKY G. et al., "Structure of Isolated Protein-Depleted Chromosomes of Plants", pages 643-659. *
GENE, Vol. 99, issued 1991, (Elsevier Holland), UNFRIED K. et al., "Subrepeats of rDNA Intergenic Spacer Present as Prominent Independent Satellite DNA in Vigna Radiata but not Vigna Angularis", pages 63-68. *
GENOME, Vol. 32, issued 1989, MOLNAR S.J. and FEDAK G., "Polymorphism in Ribosomal DNA Repeat Units of 12 Hordeum Species", pages 1124-1127. *
MOL. CELL. BIOCHEM., Vol. 89, issued 1989, (Kluwer Academic Publishers, Holland), KNOSP O. et al., "Biochemical Characterization of Chromatin Fractions Isolated from Induced and Uninduced Friend Erythroleukemia Cells", pages 37-46. *
PLANT MOLECULAR BIOLOGY, Vol. 15, issued 1990, (Kluwer Academic Publishers, Belgium), PROCUNIER J.D. and KASHA K.J., "The rDNA Intergenic Spacer Region Nucleotide Sequence of Hordeum Bulbosum L", pages 661-662. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0785281A4 (fr) * 1995-07-28 1999-11-24 Sapporo Breweries Methode de differentiation genetique des varietes du houblon
CZ296930B6 (cs) * 1995-07-28 2006-07-12 Sapporo Breweries Ltd. Zpusob identifikace genetických druhu chmelu
DE19856064A1 (de) * 1998-12-04 2000-06-29 Invitek Gmbh Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien
DE19856064C2 (de) * 1998-12-04 2000-11-30 Invitek Gmbh Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien

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