WO1990005195A1 - Identification de varietes de tomates - Google Patents

Identification de varietes de tomates Download PDF

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Publication number
WO1990005195A1
WO1990005195A1 PCT/US1989/004849 US8904849W WO9005195A1 WO 1990005195 A1 WO1990005195 A1 WO 1990005195A1 US 8904849 W US8904849 W US 8904849W WO 9005195 A1 WO9005195 A1 WO 9005195A1
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pst
probes
probe
tomato
dna
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PCT/US1989/004849
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English (en)
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Willie H.-T. Loh
Lorin R. Debonte, Jr.
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Dna Plant Technology Corporation
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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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention is directed to an efficient and practical method of distinguishing tomato varieties by, detecting restriction fragment length polymorphisms that are characteristic of distinctive tomato varieties.
  • Previous attempts to obtain restriction fragment length polymorphisms in tomato have been frustrated by the lack of detected polymorphisms in cultivated tomato varieties.
  • the probes which generate such polymorphisms can be produced at high frequency using the methods to be disclosed.
  • Restriction fragment length polymorphisms is the term used to describe the identification of restriction fragments that are different in size although homologous in sequence when two DNA samples are compared (Botstein et al., Am. J. Hum. Genet. 32 : 314, 1980). RFLPs have an advantage as a genetic marker because of their lack of dominance, invariability, and absence of pleiotropic effect on other traits.
  • RFLPs The analysis of RFLPs requires digestion of DNA samples with a restriction enzyme, separation of the DNA fragments by size, transfer of the DNA fragments to a solid support (e.g., Southern blotting), hybridization to a probe which selectively recognizes fragments which vary, and detection of such fragments.
  • Modifications to the process can include modifying the DNA prior to digestion in order to alter restriction enzyme specificity, hybridization directly within the sizing material, and the use of different methods to detect the fragments.
  • RFLPs reflect minor differences found within or adjacent to very similar DNA sequences. If the sequences of the fragments were not substantially similar, the probe used to detect the RFLP would be unable to recognize the DNA fragment. If the fragments had no difference, there would be no size variation.
  • the size variation of the RFLP can reflect (1) microheterogeneity of the DNA sequence at the restriction site that defines the fragment resulting in the introduction or loss of a restriction enzyme site; (2) base additions within the fragment; (3) base deletions within the fragment; (4) prevention of cleavage of the restriction site by base modification; (5) sequence rearrangements and/or recombinations; or (6) combinations of these possibilities.
  • the probes known in the art which are used to detect the RFLP can be any one of various types. It can be anonymous: an unknown arbitrarily chosen nucleotide sequence that recognizes a useful RFLP.
  • An anonymous probe can include a coding sequence for a gene product, a non- coding portion or intron of a gene, a region that is unassociated with a particular gene, or a nucleotide
  • probes can consist of known gene sequences.
  • the number of times a probe's sequence is present in the DNA is usually irrelevant in detecting RFLPs: single copy probes which are present only once within the DNA, multicopy probes which are present in only a few copies or represent gene families, or repetitive sequence probes which are present in many copies have all been successfully used to detect RFLPs in DNA (Helentjaris and Gesteland, J. Molec. Appli. Genet. 2:237, 1983; Cooper and Schmidtke, Hum. Genet. 66:1, 1984; Jeffreys et al., Nature 314:67, 1985; Cullis and Cleary, Can. J. Genet. Cytol. 28:252-259, 1986).
  • Organisms differ in the amount and type of their methylated DNA bases. Bacteria have between 0.05-0.25 mol% of one or both of the methylated bases 5-methylcytosine or N6-methyladenine. However mammals have only one modified base, 5-methylcytosine, which is present at about 1 mol% of total bases (DNA Methylation, 1984, eds. Razin et al.,
  • methylation of internal cytosine residues include Hpa II, Msp I, Hha I, Eco RII, Bbv I, Pvu II, Xma I, Sma I, Nci I, Ava I, Hae II, Sal I, Xho I, Pst I and Pvu II.
  • methylation sites can abut or be included in restriction enzyme sites.
  • a methylase which methylates the 5' C of the DNA sequence CCGG adjacent to the
  • C of CCGG is methylated and the Bam HI enzyme is unable to cut within the GGATCCGG sequence.
  • a methylase may modify a base within only one of the possible
  • the Hinc II restriction enzyme digests DNA containing any of the sequences GTCGAC, GTCAAC, GTTGAC and GTTAAC, because of its degenerate recognition of the sequence GTPyPuAC.
  • the methylation of the A base within the TCGA methylation site eliminates the digestion of the GTCGAC sequences by Hinc II resulting in a novel restriction pattern. This A-methylation does not occur in plants, however. 2.4. DNA-BASED PLANT GENETIC MARKERS
  • pea DNA presents a high level of restriction fragment polymorphism, in that eleven experimental lines could be easily distinguished using two probes that encoded either the ribulose-1, 5-biphosphate carboxylase (rbcS) or the chlorophyll a/b binding protein (cab) (Polans et al., Proc. Natl. Acad. Sci. USA 82:5083, 1985). Only five restriction enzyme digests were necessary to distinguish the eleven lines (Eco RI, Bam HI, Hind III, Bel I, and Bgl II).
  • Genomic actin probes from maize and soybean were used to identify homologous sequences in tomato by analyzing the hybridization of the probes to specific restriction
  • LA1563 is a breeding line developed from an
  • chmielewskii The study used four restriction enzymes and sixty random cDNA clones to detect a RFLP that was the same between L. chmielewskii and the breeding line LA1563, but was different in VF36. One cDNA clone was associated with the soluble solids gene and could be used to select the gene in a tomato breeding program. In a subsequent study
  • Random cDNA clones complementary to the poly- adenylated mRNA of VF36 tomato leaves were characterized by copy number (Bernatzky and Tanksley, Mol. Gen. Genet. 203:8, 1986). The authors estimated that fifty-three percent of the clones represented single copy DNA and thirty-two percent corresponded to two genetically independent loci. The remaining fifteen percent of the clones represented loci for multi-gene families that were present in three to five places in the tomato genome.
  • Chromosomal assignment of random genomic clones, produced by cloning Pst I fragments of VF36 was accomplished by quantitative densitometry following hybridization to DNA isolated from tomato lines that were trisomic for each of the twelve tomato chromosomes (Young et al., Nucl. Acids Res. 15:9339, 1987). After selection to eliminate
  • organellar clones ninety-two percent of the clones were single copy. Twenty-three of the assigned single copy clones were used for linkage analysis with known RFLPs from the tomato map containing two hundred loci. Twenty-one of the twenty-three assignments were correct and increased the known loci of the tomato map of L. esculentum x L.
  • a linkage map of the tomato genome was developed that was based on isozyme markers (Tanksley , Isozyme Bull . 18 : 43,
  • the RFLPs were detected as differences between the cultivated parental species L. esculentum and the wild parental species L. pennellii and forty-six of their F2 progeny.
  • Helentjaris et al. also developed a linkage map of tomato based on the RFLPs detected between L. esculentum var. Manapal and L. hirsutum using tomato leaf cDNA library and an Hind III genomic library.
  • the map produced following linkage analysis of a L. esculentum x L. hirsutum hybrid line consists of 104 RFLP loci organized into twenty linkage groups with four loci not linked to any others.
  • RFLPs can be detected that distinguish one individual within a species from most other individuals within the species, or that can distinguish one variety from another, or that can distinguish one species from another species or one family from another family. Each type has its uses although the frequencies of detecting any RFLP within its own characteristic group can vary. Most human RFLPs distinguish one individual from another and are reflective of the genetic diversity resulting from unplanned random breeding patterns. Most domesticated plants are the result of planned breeding programs, but still contain enough diversity between species that it is relatively simple to detect species specific RFLPs. However, only some plants like pea and maize have enough diversity to
  • Lycopersicon varieties UC204B and VFNT cherry.
  • a cDNA probe library produced from mRNA isolated from leaves, a large number of restriction enzymes (Bgell, Hindlll, BamHI, BstEII, EcoRI, EcoRV, Kpnl, Sstl, Bstnl and Taql), a large number of tomat varieties (12), and a large number of probes (22) had to be used as compared to the low numbers of enzymes, probes and varieties that were needed in maize.
  • the present invention is directed to detection, characterization and use of restriction fragment length polymorphisms (RFLPs) which specifically identify one variety from another variety within a tomato species .
  • RFLPs restriction fragment length polymorphisms
  • the variety differences that are specifically illustrated in the invention are the RFLP differences between the cultivated varieties within the L. esculentum domestic tomato species.
  • the probes described herein are also capable of
  • RFLPs described in this invention result from the hybridization of a specific probe with target variety DNA after digestion with a methylation sensitive restriction enzyme.
  • probes are cloned from genomic fragments of tomato DNA cleaved with methylation sensitive restriction enzymes that have been characterized as
  • the restriction fragments are sized and ligated to a suitable cloning vector.
  • the ligated vector is transformed into cells and preferably selected for inserts which are present in tomato in low copy number.
  • the cloned low copy fragments are radioactively labelled and hybridized to blots containing lanes of digested DNA from different varieties of cultivated tomato. Probes which illuminate a RFLP are identified and saved.
  • a catalog of RFLPs characteristic of a given variety of tomato, a restriction enzyme digest of the DNA from that tomato variety, and a given probe are developed. RFLPs are then identified in samples of unknown tomato variety DNA by digesting with any of the characterized restriction enzymes which produce RFLPs in known DNA samples and by using the identified probes to hybridize the digested DNA. Comparison of the RFLP pattern in the unknown sample of DNA with the catalog of variety specific RFLPs allows identification of the unknown DNA.
  • a Tomato Cultivar Specific RFLP is
  • Rutgers, Walter and Floradade were digested to completion with either Bst NI, Bam HI, Eco RI or Eco RV restriction enzyme. After gel electrophoresis, the DNA fragments were transferred to a nylon membrane using the protocol of
  • FIG. 1 is a diagram representing RFLP patterns for several species of tomato. Each letter denotes a different
  • Figure 3 is a diagram representing RFLP patterns for several distantly related cultivars of L. esculentum.
  • Figure 4 is a diagram representing RFLP patterns for several closely related cultivars of L. esculentum.
  • DNA used for digestion by methylation sensitive restriction enzymes can be prepared from isolated nuclei of tomato cultivar leaves, or other convenient sources.
  • Contaminating organelles are removed by centrifugation of the nuclei through a 60% sucrose step gradient.
  • the DNA used to produce a library of genomic clones was prepared from Pstl digested nuclear DNA of cultivar E6203.
  • any tomato line may be used as the source of genomic DNA.
  • the DNA is digested to completion and separated by gel electrophoresis in 1% agarose. Fragments between 0.3-2.0 kB and 2.0-4.0 kB were excised and extracted by electroelution and then
  • Clones which contained a fragment of the tomato genome were screened by dot blot analysis to identify low copy clones.
  • the selected low copy number plasmids were nick translated to produce a radioactive probe. Probes were hybridized to digests of genomic DNA to identify those that detected RFLP's when the DNA of two varieties was compared.
  • Any restriction enzyme which will cut plant DNA is suitable to digest cultivar DNA, for probing, including Eco RI, Eco RV, Bam HI, Bst NI, Xba I, Hind III, Rsa I, Taq I, Msp I, Hind I, and the like.
  • Methylation sensitive enzymes which are inhibited by methylation of an internal or end cytosine are employed to generate the genomic clones used to produce labelled probes. These methylation sensitive restriction enzymes have been identified above. The enzymes are employed under conditions well known to those skilled in the art, i.e., according to manufacturer directions,
  • DNA fragments were separated in a size dependent method.
  • gel electrophoresis in agarose is the method chosen in the specific examples, any sizing method including methods based on charge and size is sufficient and is encompassed in the invention, since sizing methods for DNA fragments are well known in the art (e.g., Southern,
  • Hybridization of the probe to the size separated DNA is a procedure known in the art (Denhart, Biochem. Biophys.
  • Fragments can be hybridized while still in the gel matrix (Purello et al., Anal. Biochem. 128:393, 1983) or following transfer to a solid support (e.g.,
  • the solid support can be either nitrocellulose membranes, activated paper, nylon membranes br other types of supports known in the art.
  • RFLPs can be visualized by hybridizing with a
  • radioactive probe prepared by nick translation of DNA, random prime hexanucleotide end labeling of DNA fragments, in vivo labeling of plasmid or M13 phage clones,
  • Radioactive probes are washed from the blot and the hybridized fragments are detected by autoradiography.
  • Plasmid pT7T3 was the plasmid of choice for cloning tomato DNA digested with methylation sensitive restriction enzymes because it contains a pBR322 origin of replication and the lacZ gene with a multi-linker cloning sequence. Insertion of a DNA fragment into pT7T3 produces a color detectable difference between bacterial colonies that contain or do not contain an insert because of the
  • Ligations were performed between the cloning vector pT7T3 that had been linearized with Pst I, and the two sized fractions of digested nuclear DNA at various molar ratios. Ligation ratios of 1:1, 1:2 and 1:5 and 1:12.5 (plasmid to insert DNA) were tested. Recombinant clones were visualized by the growth of white colonies in the presence of x-gal. The optimal ligation ratio for cloning the small fragments was determined to be 1 : 12 .5. A total of 439 potential transformants were selected and glycerol stocks were made of each clone strain for permanent storage.
  • the Pst I clones were dot-blotted and screened to select single and low copy number sequences. 154 of 180 Pst I clones (85%) were shown to contain low copy number
  • the pT7T3 contains an Ace I site within the B- galactosidase gene. Recombination of Hpa II inserts in the Ace I site can be visualized by the production of white colonies in the presence of x-gal because of the
  • the pT7T3 plasmid was linearized with Ace I to create a two-base compatible cloning site for Hpa II. Following digestion, the plasmid preparation was electrophoresed to purify digested plasmid DNA from undigested circular forms. The linearized fraction was excised, electroeluted and concentrated by ethanol precipitation.
  • L. esculentum E6203 nuclear DNA was digested with Hpa II and the digest was electrophoresed. Hpa II fragments between 0.5 and 4 kb were eluted from an anion exchange resin with 0.64 M NaCl.
  • the Ace I digested pT7T3 plasmid was ligated to the Hpa II fragments. The optimal ligation ratio was determined to be 1:10 (plasmid: insert DNA).
  • the ligation mixture was transformed into E. coli DH5 cells , which were plated on 10.0 mg/l ampicillin and 50 ug/ml X-gal. 478 white colonies were picked. 80 recombinant plasmids were recovered per ng of ligated DNA. DNA was isolated from 180 recombinant plasmids using a small scale boiling lysis method. 54 clones (56%) were found to contain inserts.
  • Glycerol stocks were made of each clone for permanent storage.
  • RFLP identification can be carried out as follows.
  • Each clone is hybridized to Southern blots containing four digests of selected tomato species and accessions. Each blot can usually only be probed 5-7 times because the stripping process needed for reuse removes DNA. Following each hybridization, the probe is stripped off with 0.1 N NaOH to allow reprobing.
  • Clones of dispersed repeat sequences which give discrete bands on hybridization can be used to identify RFLPs. Each hybridizing band represents a potential RFLP.
  • Such repetitive sequences can be used to fingerprint an individual within a homogeneous population.
  • conserved repeats which appear as a single conserved band, are far more useful for constructing the fingerprinting catalog of RFLPs.
  • Probes were produced and hybridized to Southern blots of genomic digests of tomato species to generate RFLP patterns that were distinguished by autoradiography of the washed blot. All of the 39 Pst I clones tested in 156 hybridizations were able to generate RFLPs which can distinguish tomato species or groups of species using tomato DNA digested with four restriction endonucleases. 144 RFLP patterns were generated of which 22 were unique.
  • Probes were produced from the Pst I clones to test the genomic digests of fifteen tomato varieties spanning a range of modern and old, processing and fresh market varieties. Total DNA from each variety was digested with restriction enzymes, electrophoresed, and transferred to blots. The blots were hybridized with the probes and RFLPs were
  • the probes which identified RFLPs were tested further to identify RFLPs in more closely related tomato varieties. Genomic DNA from each variety was digested with Eco RI, Eco RV, Bst NI, Xba I and Hind III (double digest), Hinf I, Rsa I, Msp I, and Taq I. Sixteen individual probes generated a total of 21 RFLP patterns. 6 of the 12 closely related varieties could be uniquely fingerprinted on the basis of their RFLP patterns.
  • the labelled probes are denatured with 2 mg of sonicated herring sperm DNA at 95oC for 10 minutes, then quick cooled on ice.
  • To the probe DNA is added 2 mis of 42o C hybridization solution (50% formamide, 1% sodium dodecyl sulfate (SDS), 1 M NaCl, 7% dextran sulfate, 50 MM Tris, pH 7.5.
  • the probe mixture is added to the genomic digest membrance (10x14 cm) which has been prehybridized at 42°C for 18 hours. The membrance is then immersed in a low stringency wash (0.3 M sodium chlordie, 0.03 M sodium citrate, 0.1% SDS) at 20°C twice for 10 minutes each.
  • the membrance is then washed in a high stringency wash (0.03 M sodium chloride, 0.03 M sodium citrate, 0.1% SDS) at 65°C for 30 minutes.
  • the filters are exposed to x-ray film through intensifying screens for 3-6 days at '70oC to visualize hybridization.
  • the labelled probe is stripped off to prepare the nylon membrane for the next round of screening.
  • the number of times a blot can be reprobed is directly related to the efficiency of the probe screening process. Resolution of single copy
  • the hybridized probles are removed from the nylon membrances by agitating in 0.1 sodium hydroxide at 20oC for 30 minutes.
  • the membranes are neutralized in 0.015 M sodium chloride, 0.0015 sodium citrate, 0.1% SDS, 0.2 M Tris-HCl, pH 7.5 at 20oC for 30 minutes.
  • probes of the present invention can be made and used. It is understood, for example, that the source of the DNA employed for probe preparation may be any tomato variety, and the methylation sensitive enzyme employed may also be varied, although Pst I and Hpa II are preferred.
  • the present invention encompasses not only the specific probes described herein, but also all DNA sequences hybridizable to the clones described herein and thus functionally equivalent to the described probes in their utility for detecting RFLPs.
  • One skilled in the art can readily determine if a given DNA sequence, whether produced by enzymatic digestion of genomic
  • DNA is equivalent to the instant claimed clones by the hybridcation procedure outlined below.
  • the DNA sequence or clone to be tested is purified by electrophoresis in low melting-point agar.
  • the purified fragment is then slot blotted onto a DNA binding nylon membrane (according to manufacturer's instructions).
  • the membrane is then probed with radioactively labelled ( ⁇ 5 X 10 7 cpm/ug DNA) probes of the present invention, as follows:
  • the labelled probes are denatured with 2 mg of
  • the probe is added to the slot blotted membrane which has been prehybridized at 32oC in 8 ml of hybridization solution; and incubated at 32oC for 24 hours under
  • RFLPs RFLPs have been identified for each. of the varieties and species tested. The methods have been useful in distinguishing among a number of wild species as well as among cultivated L. esculentum varieties of relatively distant and close backgrounds.
  • the first experiments were designed to determine species specific RFLPs.
  • Pst I sequences cloned in pT7T3 were subsequently tested as RFLP probes.
  • the probes were hybridized to genomic digests of wild tomato species to generate RFLP patterns.
  • Each clone was hybridized to four filters
  • Each filter contains genomic DNA froma the following tomato species and accession: L. cheesmariii LA483 and LA1449, L. esculentum UC204C and 2-327-5, L.
  • L. chmielewskii accessions LA1028 and LA1306, have distinguishable RFLP patterns indicating that the extent of genetic diversity may be variable between different tomato accessions. Every species and accession tested can be identified by probing Eco RV digests with Pst-20 and Pst-7.
  • Hpa-67 uniquely distinguished Hunt 100 from all other varieties tested in Eco RI, Eco RV, Dra I and Bst NI genomic digests.
  • VFNT Cherry was also uniquely distinguished by Hpa-67 in the Eco RV digest.
  • Figure 3 The probe/enzyme combinations which can distinguish tomato cultivars of the Wide Germplasm set are summarized in Figure 3.
  • the Narrow Germplasm set includes MX144, F749-E2, CXN114, X2974-WE52B, Acc. 29,
  • Genomic DNA from these plants were digested with Eco RI, Eco RV, Bst NI, Xba I and Hind III (double digest), Hinf I, Rsa I, Msp I, and Tac I. Sixteen Pst I probes were labelled and tested singly or in groups of 2 or 3 against genomic digests of the 12 Narrow Germplasm lines (see Figure 4).
  • RFLP patterns were generated. Three probes (Pst-83, Pst-154 and Pst-157) were responsible for all of these RFLP patterns. Six unique RFLP patterns were identified by two probes (Pst-83 and Pst-154) and three enzymes (Eco RI, Eco RV, and Msp I). Unique RFLP patterns for CXN114 were produced by probing Eco RI and Eco RV digests with Pst-154. Similarly, unique RFLPs were
  • Table 1 below provides a list of plasmids containing probes of the present invention which have been deposited with the American Type Culture Collection, Rockville,
  • Each vial contains several probes of the present invention.
  • the probes in each vial can be identified from each other by the size of the Pstl tomato DNA insert (Table 1).
  • the cloning vector, pT7T3, to which they have ligated is 2.88 Kb in length.
  • the Pst I inserts can be isolated or visualized by digesting 300 ng of the vial contents with the restriction enzyme Pstl following the suppliers procedure.
  • the released inserts can be separated from each other and the cloning vector by electrophoresis through a 1.0% agarose gel.
  • the inserts can be visualized by staining with
  • the present invention is not limited in scope by the deposited probes, however, which are intended only as illustrations of the present invention.
  • a complete library of Pst I and Hpa II clones are kept at DNA Plant Technology, 2611 Branch Pike, Cinnaminson, NJ, and samples are available upon request.

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Abstract

La présente invention se rapporte à des sondes permettant d'identifier dans la tomate des fragments de restriction par analyse des polymorphismes de longueur. Les sondes sont des fragments obtenus par digestion de l'ADN de la tomate par une enzyme sensible à la méthylation. L'invention concerne également un procédé d'identification d'espèces sauvages de tomate ainsi que de variétés de tomate de culture.
PCT/US1989/004849 1988-10-31 1989-10-30 Identification de varietes de tomates WO1990005195A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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WO1995011991A1 (fr) * 1993-10-27 1995-05-04 Universite Claude Bernard Lyon I SONDE ET PROCEDE DE DETECTION DES LEVURES DE L'ESPECE $i(CANDIDA KRUSEI)
GB2283568A (en) * 1993-10-13 1995-05-10 Mini Agriculture & Fisheries Identification of the origin of fruit or fruit juice
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn
WO2003023065A1 (fr) * 2001-09-06 2003-03-20 Syngenta Participations Ag Types de methylation de l'adn
CN112695031A (zh) * 2021-01-26 2021-04-23 北京市农林科学院 一种鉴定番茄品种dna指纹的方法及其使用的snp引物组合

Citations (1)

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Publication number Priority date Publication date Assignee Title
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GB2283568A (en) * 1993-10-13 1995-05-10 Mini Agriculture & Fisheries Identification of the origin of fruit or fruit juice
GB2283568B (en) * 1993-10-13 1997-12-24 Mini Agriculture & Fisheries Identification of fruit juice components
WO1995011991A1 (fr) * 1993-10-27 1995-05-04 Universite Claude Bernard Lyon I SONDE ET PROCEDE DE DETECTION DES LEVURES DE L'ESPECE $i(CANDIDA KRUSEI)
FR2711669A1 (fr) * 1993-10-27 1995-05-05 Bernard Lyon I Universite Clau Sonde et procédé de détection des levures de l'espèce Candida krusei.
US5955267A (en) * 1993-10-27 1999-09-21 Bio Merieux S.A. Probe and method for detecting yeast of species Candida krusei
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn
US6514698B1 (en) 1997-08-29 2003-02-04 Osvaldo J. Lopez DNA methyltransferase genotyping
WO2003023065A1 (fr) * 2001-09-06 2003-03-20 Syngenta Participations Ag Types de methylation de l'adn
CN112695031A (zh) * 2021-01-26 2021-04-23 北京市农林科学院 一种鉴定番茄品种dna指纹的方法及其使用的snp引物组合
CN112695031B (zh) * 2021-01-26 2023-03-14 北京市农林科学院 一种鉴定番茄品种dna指纹的方法及其使用的snp引物组合

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