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WO1999004621A1 - Marqueur moleculaire pour le gene determinant le rapport fructose/glucose dans une tomate mure - Google Patents

Marqueur moleculaire pour le gene determinant le rapport fructose/glucose dans une tomate mure Download PDF

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Publication number
WO1999004621A1
WO1999004621A1 PCT/IL1998/000336 IL9800336W WO9904621A1 WO 1999004621 A1 WO1999004621 A1 WO 1999004621A1 IL 9800336 W IL9800336 W IL 9800336W WO 9904621 A1 WO9904621 A1 WO 9904621A1
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WIPO (PCT)
Prior art keywords
plants
tomato
fructose
primer
marker
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Ceased
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PCT/IL1998/000336
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English (en)
Inventor
Ilan Levin
Arthur A. Shaffer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
STATE OF ISRAEL - MINISTRY OF AGRICULTURE
State of Israel
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STATE OF ISRAEL - MINISTRY OF AGRICULTURE
State of Israel
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by STATE OF ISRAEL - MINISTRY OF AGRICULTURE, State of Israel filed Critical STATE OF ISRAEL - MINISTRY OF AGRICULTURE
Priority to KR1020007000715A priority Critical patent/KR20010052053A/ko
Priority to EP98933866A priority patent/EP0998191A1/fr
Priority to AU83548/98A priority patent/AU8354898A/en
Priority to JP2000503701A priority patent/JP2001510685A/ja
Publication of WO1999004621A1 publication Critical patent/WO1999004621A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • 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
    • C12Q2600/13—Plant traits
    • 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 generally to a method of breeding tomatoes having superior taste characteristics and to tomatoes having superior taste characteristics, and particularly to a molecular marker for the gene determining the fructose to glucose ratio in mature tomato fruit.
  • TSS total soluble solids
  • Tomatoes with high fructose to glucose ratios have been developed, using a method of selection described in applicant/assignee's Israel patent application 105243, PCT patent application PCT/US94/03522 and US patent application 08/530,216, the disclosures of which are incorporated herein by reference.
  • this method consists of hybridizing a tomato plant of the L. esculentum species with a plant of the L.
  • the analysis of mature fruit sugars in the described method is via direct chemical analysis of the fruit sugars, for example by chromatographic separation of individual sugars
  • RFLP markers require a cloned probe, endonuclease digestion of genomic DNA and time consuming DNA transfer, labeling and hybridization steps At the end of this laborious process only a single locus of a very limited polymorphic content is usually revealed More efficient polymorphism assays can be obtained from multilocus DNA probes yielding DNA fingerprints (DFP, see Jeffreys A J , Wilson V and Thein S L 1985 a
  • SSR analysis is low cost and easy to perform because no prior target DNA sequence information in polymorphic DNA regions is required for its implementation.
  • AFLP is more expensive to produce but has the capacity to detect a much greater number of polymorphic loci in a single assay than other currently available PCR-based techniques.
  • Microsatellites are expensive to produce since they require allele specific primers and detect only a single polymorphic locus in a single assay.
  • a molecular marker In the case of selection for sugar content of mature fruit, a molecular marker has the advantage of allowing for selection at the young seedling stage , in contrast to selection only at the mature fruit stage. Furthermore, selection using a molecular marker eliminates the confounding effects of environmental influences on the plant phenotype which can limit the effectiveness of selection for a phenotypic trait such as mature fruit sugar content.
  • the present invention seeks to provide a molecular marker for a gene determining fructose to glucose ratio in mature tomato fruit.
  • the marker can be used to find this gene and produce tomato seeds, plants and/or fruit with the desirable characteristic of increased fructose to glucose ratio.
  • the marker includes a first amplification product generated by a primer called an MS6 primer, the MS6 primer including a nucleotide sequence TCTCTCTCTCTCTCCC. Further in accordance with a preferred embodiment of the present invention the marker includes a fragment having a nucleotide sequence as follows:
  • the first amplification product is allelic to a second amplification product obtained by a primer called an MS8 primer, the MS8 primer including a nucleotide sequence TCTCTCTCTCTCTCCG.
  • a method for breeding tomato plants that produce tomatoes having superior taste characteristics including the steps of crossing at least one Lycopersicon esculentum plant with a Lycopersicon spp. to produce hybrid seeds, collecting the hybrid (Fi) seeds, growing plants from the Fi seeds, pollinating the Fi plants, collecting the hybrid seeds produced by the Fj plants, growing plants from the seeds produced by the F t plants, measuring glucose and fructose content of ripe fruit produced from the plants grown from the seeds of the Fi plants, providing at least one marker for a gene which provides increased fructose to glucose ratio in a tomato plant, and using the at least one marker to select a tomato plant with tomato fruit having desired characteristics including a fructose to glucose ratio greater than a ratio of standard Lycopersicon esculentum .
  • a method for finding a gene that produce tomatoes having superior taste characteristics including the steps of providing at least one marker for a gene whicn provides increased fructose to glucose ratio in tomato plants, and using the at least one marker to find the gene.
  • the method further includes cloning the gene.
  • the method includes the step of propagating the plants with tomato fruits having the desired characteristics.
  • the plants may be propagated by vegetative propagation or by seed.
  • a tomato plant, tomato fruit and/or tomato seed may be produced in accordance with any of the methods of the present invention.
  • the high fructose to glucose ratio breeding line was derived from the introgression of the trait of high fructose to glucose ratio from the wild species Lycopersicon hirsutum (LAI 777), as described in PCT patent application PCT US94/03522. The following procedure was carried out for soluble sugar determination. Fruit portions of about 500 mg fresh weight were placed in 80% ethanol and soluble sugars were extracted from the tissue by heating to 70°C, as described in Miron and Schaffer (1991).
  • Genomic DNA was extracted from the 2 parental lines with divergent fructose to glucose ratio in the mature fruit and from individual plants of the Fi and F populations generated by crossing the two parental lines.
  • the individual plants from the F 2 population segregated for the trait of fructose to glucose ratio, the range being 1-3.75. Individual plants from the F 2 population could therefore be easily ranked for the trait of fructose to glucose ratio.
  • the genomic DNA was extracted as in Fulton, T.M., Chunwongse, J. and Tanksley,
  • a collection of 18 base pairs single strand DNA primers containing di- and tri - nucleotide repeats were synthesized (Pharmacia Biotech, Inc., Austria) and used in the presence of template DNA to screen by a polymerase amplification reaction DNA sequences linked to the gene encoding high fructose to glucose ratio. Initially, DNA samples extracted from individual plants from the two parental lines were used to identify, by an amplification reaction, polymorphic DNA patterns. Amplification reactions (25 ml final volume) contained 10 ng template DNA, 25 mM
  • TAPS (pH 9.3 at 25°C), 50 mM KC1, 2mM MgC12, 1 mM ⁇ -mercaptoethanol, 0.2 mM of each of the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP and dTTP), 20 ng of a single primer (or 10 ng of each of two primers when using a combination of two primers), and 1 unit of thermostable Taq DNA polymerase (SuperNova Taq Polymerase, MADI LTD., Israel). Rmixtures were overlaid by 15 ml of light mineral oil, and reactions were carried out in an automated thermocycler (MJ Research Inc., Watertown, Massachusetts, USA).
  • MS6 TCTCTCTCTCTCTCCC
  • MS8 TCTCTCTCTCTCTCTCTCCG
  • MS6 and MS8 primers were further used to analyze individual DNA samples extracted from the entire F 2 population.
  • the analysis of the amplification products obtained from DNA samples extracted from individual F 2 plants revealed that the product obtained by the MS6 primer was allelic to the amplification product obtained by the MS 8 primer. Therefore, individual plants from the entire F 2 population could be characterized as homozygous to the marker allele generated by MS6 that is associated with the trait of high fructose to glucose ratio, homozygous to the marker allele generated by MS8 that is associated with low fructose to glucose ratio and heterozygous.
  • Genotype-phenotype relation Analyses of variance were carried out using results obtained from the F 2 population to determine the effect of association between each of the marker bands or their combination (zygosity) and the trait of fructose to glucose ratio and the percentage of fructose to glucose variation explained by these variation components.
  • the DNA markers obtained were found highly and significantly associated with the trait of fructose to glucose ratio (Table 1).
  • the association between the zygosity status and the trait of fructose to glucose ratio was highly significant at a high log-of-differences (LOD) score explaining 40.5% of the total variation in fructose to glucose ratios observed in the F 2 population (Table 1 ).
  • LOD log-of-differences
  • a nested analysis of variance was also carried out to determine the total variation explained by the zygosity status of F 2 plants, plants within zygosity status and fruits within plants (Table 2). The zygosity status appeared to be highly significant in this analysis as well
  • fructose to glucose ratio i.e. fructose and glucose levels
  • the gene identified can directly modulate fructose to glucose ratios without an apparent effect on the components of this ratio i.e., fructose and glucose levels.
  • the genetic effect of the gene encoding fructose to glucose ratio could be estimated based on the DNA markers amplified by the MS6 and MS8 primers.
  • the allele encoding high fructose to glucose ratio appeared to be partially dominant.
  • the DNA fragment amplified by the MS6 primer was extracted from the agarose gel using the Geneclean II kit (BIO 101, Vista CA, USA) and cloned using the p-GEM easy vector cloning system (Promega corporation, Madison WI, USA).
  • the nucleotide sequence of the cloned fragment was determined using an ABI PRISM 377 DNA sequencer and is as follows:
  • the underlined are annealing sites of the MS6 primer used to identify the marker linked to fructose to glucose ratio. It is possible that the amplification product generated by the MS6 primer may be part of, in the area of, or lead to finding the gene that determines the increased fructose to glucose ratio.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention concerne un marqueur moléculaire pour un gène déterminant le rapport fructose/glucose dans une tomate mûre. Dans une forme de réalisation préférée de cette invention le marqueur comprend un premier produit d'amplification généré par une amorce appelée amorce MS6, cette amorce MS6 comprenant une séquence nucléotidique TCTCTCTCTCTCTCTCCC.
PCT/IL1998/000336 1997-07-23 1998-07-16 Marqueur moleculaire pour le gene determinant le rapport fructose/glucose dans une tomate mure Ceased WO1999004621A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
KR1020007000715A KR20010052053A (ko) 1997-07-23 1998-07-16 성숙된 토마토 열매 중의 프럭토스 대 글루코스 비를결정하는 유전자에 대한 분자 마커
EP98933866A EP0998191A1 (fr) 1997-07-23 1998-07-16 Marqueur moleculaire pour le gene determinant le rapport fructose/glucose dans une tomate mure
AU83548/98A AU8354898A (en) 1997-07-23 1998-07-16 A molecular marker for the gene determining the fructose to glucose ratio in mature tomato fruit
JP2000503701A JP2001510685A (ja) 1997-07-23 1998-07-16 成熟トマト果物におけるフルクトース対グルコース比を決定する遺伝子のための分子マーカー

Applications Claiming Priority (2)

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IL121373 1997-07-23
IL12137397A IL121373A0 (en) 1997-07-23 1997-07-23 A molecular marker for the gene determining the fructose to glucose ratio in mature tomato fruit

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EP (1) EP0998191A1 (fr)
JP (1) JP2001510685A (fr)
KR (1) KR20010052053A (fr)
AU (1) AU8354898A (fr)
IL (1) IL121373A0 (fr)
WO (1) WO1999004621A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075277A3 (fr) * 1999-06-09 2004-02-19 Israel State Marqueur moleculaire base sur le gene fructokinase 2 (frk2)
WO2012095841A1 (fr) * 2011-01-10 2012-07-19 State Of Israel, Ministry Of Agriculture And Rural Development, A.R.O. - Volcani Center Plants de tomate améliorés
US10745710B2 (en) 2015-08-06 2020-08-18 University Of Tsukuba Plant having mutant cyclin F-box gene
CN120099213A (zh) * 2025-03-21 2025-06-06 上海市农业科学院 一种检测与番茄果实糖度相关的snp位点的引物组及其应用
CN120330378A (zh) * 2025-06-17 2025-07-18 潍坊科技学院 一种番茄果实含糖量相关的分子标记及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022289A1 (fr) 1993-03-31 1994-10-13 Peri Development Applications (1985) Ltd. Procede pour obtenir des tomates presentant des caracteristiques de gout ameliorees et produit obtenu par ce procede
US5434344A (en) * 1990-04-16 1995-07-18 The Regents Of The University Of California Sucrose accumulating tomato technology
US5750869A (en) * 1995-01-15 1998-05-12 Calgene, Inc. Soluble solids modification using sucrose phosphate synthase encoding sequences
US5817913A (en) 1993-03-31 1998-10-06 Peri Devlopment Applications, Ltd. Method for breeding tomatoes with superior taste characteristics and product of the method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5434344A (en) * 1990-04-16 1995-07-18 The Regents Of The University Of California Sucrose accumulating tomato technology
WO1994022289A1 (fr) 1993-03-31 1994-10-13 Peri Development Applications (1985) Ltd. Procede pour obtenir des tomates presentant des caracteristiques de gout ameliorees et produit obtenu par ce procede
US5817913A (en) 1993-03-31 1998-10-06 Peri Devlopment Applications, Ltd. Method for breeding tomatoes with superior taste characteristics and product of the method
US5750869A (en) * 1995-01-15 1998-05-12 Calgene, Inc. Soluble solids modification using sucrose phosphate synthase encoding sequences

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHETELAT R. T., ET AL.: "INHERITANCE AND GENETIC MAPPING OF FRUIT SUCROSE ACCUMULATION IN LYCOPERSICON CHMIELEWSKII.", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, vol. 04., no. 04., 1 January 1993 (1993-01-01), GB, pages 643 - 650., XP002913666, ISSN: 0960-7412, DOI: 10.1046/j.1365-313X.1993.04040643.x *
STOMMEL J. R., ET AL.: "GENETIC CONTROL OF FRUIT SUGAR ACCUMULATION IN A LUCOPERSICON ESCULENTUM C L. HIRSUTUM CROSS.", JOURNAL OF THE AMERICAN SOCIETY FOR HORTICULTURAL SCIENCE., AMERICAN SOCIETY FOR HORTICULTURAL SCIENCE., ALEXANDRIA, VA, US., vol. 118., no. 06., 1 January 1993 (1993-01-01), ALEXANDRIA, VA, US., pages 859 - 863., XP002913665, ISSN: 0003-1062 *
TANKSLEY S. D., ET AL.: "USE OF MOLECULAR MARKERS IN BREEDING FOR SOLUBLE SOLIDS CONTENT IN TOMATO - A RE-EXAMINATION.", THEORETICAL AND APPLIED GENETICS., SPRINGER, BERLIN, DE, vol. 75., 1 January 1988 (1988-01-01), DE, pages 811 - 823., XP002913667, ISSN: 0040-5752, DOI: 10.1007/BF00265610 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075277A3 (fr) * 1999-06-09 2004-02-19 Israel State Marqueur moleculaire base sur le gene fructokinase 2 (frk2)
WO2012095841A1 (fr) * 2011-01-10 2012-07-19 State Of Israel, Ministry Of Agriculture And Rural Development, A.R.O. - Volcani Center Plants de tomate améliorés
US10745710B2 (en) 2015-08-06 2020-08-18 University Of Tsukuba Plant having mutant cyclin F-box gene
CN120099213A (zh) * 2025-03-21 2025-06-06 上海市农业科学院 一种检测与番茄果实糖度相关的snp位点的引物组及其应用
CN120330378A (zh) * 2025-06-17 2025-07-18 潍坊科技学院 一种番茄果实含糖量相关的分子标记及其应用

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AU8354898A (en) 1999-02-16
IL121373A0 (en) 1998-01-04
KR20010052053A (ko) 2001-06-25
EP0998191A1 (fr) 2000-05-10
JP2001510685A (ja) 2001-08-07

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