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WO2001027325A1 - Identification assistee par marqueur d'un gene associe a un trait phenotypique - Google Patents

Identification assistee par marqueur d'un gene associe a un trait phenotypique Download PDF

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Publication number
WO2001027325A1
WO2001027325A1 PCT/US2000/027719 US0027719W WO0127325A1 WO 2001027325 A1 WO2001027325 A1 WO 2001027325A1 US 0027719 W US0027719 W US 0027719W WO 0127325 A1 WO0127325 A1 WO 0127325A1
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Prior art keywords
members
expression
genetic marker
phenotypic trait
trait
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PCT/US2000/027719
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English (en)
Inventor
Steve Openshaw
Wesley B. Bruce
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Pioneer Hi-Bred International, Inc.
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Publication date
Application filed by Pioneer Hi-Bred International, Inc. filed Critical Pioneer Hi-Bred International, Inc.
Priority to AU78703/00A priority Critical patent/AU7870300A/en
Priority to EP00968844A priority patent/EP1230385A4/fr
Publication of WO2001027325A1 publication Critical patent/WO2001027325A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates generally to the field of plant breeding. More specifically, it relates to gene identification in plants.
  • SUMMARY OF THE INVENTION it is the object of the present invention to provide methods of selection of a gene associated with a phenotypic trait. It is an object of the present invention to provide a method of associating a gene with a phenotypic trait of interest and methods of associating an expression product with a phenotypic trait of interest.
  • the present invention relates to a method of associating a gene with a phenotypic trait of interest comprising (a) segregating members of a biological population by the presence or absence of one or more genetic markers statistically associated with a quantitatively inherited phenotypic trait; (b) expression profiling segregated members of (a); and, (c) determining from expression profiles of (b) the gene associated with said phenotypic trait.
  • the present invention relates to a method of associating an expression product with a phenotypic trait of interest comprising (a) segregating members of a population consisting of a biological population by the presence or absence of one or more genetic markers statistically associated with said phenotypic trait, wherein said phenotypic trait has a statistical association with more than one genetic locus; (b) expression profiling at least one segregated member of (a) possessing said genetic marker and at least one segregated member of (a) lacking said genetic marker; and, (c) determining from said expression profiles of (b) an expression product associated with said phenotypic trait.
  • the present invention relates to associating an expression product with a phenotypic trait of interest, comprising: (a) expression profiling a plurality of members of a biological population having one or more genetic markers statistically associated with a phenotypic trait of interest wherein said phenotypic trait exhibits statistical association with more than one genomic locus; (b) expression profiling a plurality of members from said population lacking said genetic marker; and, (c) determining from expression profiles of (a) and (b) an expression product associated with said phenotypic trait.
  • biological population includes reference to a group of individuals having the capacity to be genetically crossed, regardless of species.
  • a group of Glycine soja and Glycine max plants would be considered a "biological population” because they are capable of being crossed.
  • Individuals, as used herein, will refer to whole organisms, organism organs, cells, and progeny of same.
  • a plant biological population would include reference to whole plants, plant organs, plant cells, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • expression profiling includes reference to generating an expression profile.
  • expression profile is meant the quantitation of a plurality of DNA, RNA, or protein expression products from a cell, tissue or whole organism. Many RNA expression products of a cell or tissue can simultaneously be detected on a nucleic acid array, or by the technique of differential display or modification thereof, such as those described in WO
  • genetic locus is meant a location on a chromosome.
  • genomic locus is meant a location within the entire set of chromosomes of an organism.
  • linkage disequilibrium refers to a statistical association between two loci or between a trait and a marker.
  • marker includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome.
  • a genotype may be defined by use of one or a plurality of markers.
  • Phenotypic traits may be comprised of, but are not limited to, a combination of measurable traits reflected in, but not limited to, the following:
  • Barren plants The percent of plants per plot that were barren (lack ears).
  • Brittle Stalks This is a measure of the stalk breakage near the time of pollination, and is an indication of whether the stalk of a hybrid or inbred would snap or break near the time of flowering under severe winds.
  • Yield Yield of the grain at harvest in bushels per acre adjusted to 15.5% moisture.
  • Drydown The relative rate at which a hybrid will reach acceptable harvest moisture compared to other hybrids.
  • Dropped Ears A measure of the number of dropped ears per plot and represents the percentage of plants that dropped ears prior to harvest.
  • Ear height is a measure from the ground to the highest placed developed ear node attachment and is measured in inches.
  • General Ear Mold This is based on overall rating for ear mold of mature ears without determining the specific mold organism, and may not be predictive for a specific ear mold.
  • European Corn Borer feeding resistance (Ostrinia nubilalis): Average inches of tunneling per plant in the stalk or post flowering degree of stalk breakage and other evidence of feeding by European Corn Borer.
  • GDU to shed The number of growing degree units (GDUs) or heat units required for an inbred line or hybrid to have approximately 50 percent of the plants shedding pollen and is measured from the time of planting. Growing degree units are calculated by the Barger Method, where the heat units for a 24-hour period are:
  • GDU (Max. temp. + Min. temp.) - 50
  • the highest maximum temperature used is 86° F. and the lowest minimum temperature used is 50° F.
  • the highest maximum temperature used is 86° F. and the lowest minimum temperature used is 50° F.
  • GDU to silk The number of growing degree units required for an inbred line or hybrid to have approximately 50 percent of the plants with silk emergence from time of planting.
  • Grain Appearance The general appearance of the shelled grain as it is harvested based on such factors as the color of harvested grain, any mold on the grain, and any cracked grain.
  • Harvest Moisture The moisture is the actual percentage moisture of the grain at harvest.
  • Moisture Advantage The moisture advantage of variety #1 over variety #2 as calculated by: Moisture of variety #2 - Moisture of variety #1 - Moisture Advantage of variety #1.
  • Grain Oil The amount of the kernel that is oil, expressed as a percentage on a dry weight basis.
  • Plant Height This is a measure of the height of the plant from the ground to the tip of the tassel in inches.
  • Pollen Score Rating indicating the amount of pollen shed.
  • Pollen Weight This is calculated by dry weight of tassels collected as shedding commences minus dry weight from similar tassels harvested after shedding is complete.
  • PRM Predicted Relative Maturity
  • PRM Shed Predicted relative maturity based on shed is based on the growing degree units
  • GDU required to reach 50% pollen shed. Relative values are predicted values from the linear regression of observed GDU's on relative maturity of commercial checks.
  • Protein Rating Comparison of relative amounts of protein in the grain compared to hybrids of similar maturity.
  • Root lodging The percentage of plants that root lodge; plants that lean from the vertical axis at an approximately 30° angle or greater would be counted as root lodged.
  • Scatter Grain lack of pollination or kernel abortion on the ear.
  • Seedling Vigor The amount of vegetative growth after emergence at the seedling stage
  • Stalk Count the final stand or number of plants per plot.
  • Stalk Lodge the percentage of plants that stalk lodged (stalk breakage) as measured by either natural lodging or pushing the stalks and determining the percentage of plants that break below the ear.
  • Tassel Blast the degree of blasting (necrosis due to heat stress) of the tassel at the time of flowering.
  • Tassel Size the relative size of the tassel.
  • Tassel Weight this is the average weight of a tassel (grams) just prior to pollen shed.
  • Ear Texture the relative hardness (smoothness of crown) of mature grain.
  • Number of tillers a count of the number of tillers per plot that could possibly shed pollen.
  • ASI the interval in GDU's between the GDU to shed and GDU to silk.
  • Grain composition amino acids The average amount and type of amino acids present in the kernel based on 25 kernels.
  • Grain composition carbohydrate The average amount and type of carbohydrate present in the kernel based on 25 kernels.
  • Ear length The length of the ear from the base to the tip of the cob.
  • Kernel Row Count The number of rows of kernels per ear.
  • Kernel per row The average number of kernels per row based on at least 4 rows.
  • Ear diameter The average diameter of the ear with intact kernels based on three measurements at different places on the ear.
  • Kernel row length The average distance from the first kernel at the base of the ear to the last kernel at the tip of the ear.
  • KWT100 The average mass of kernel in grams for 100 kernels either as fresh tissue or dried to moisture level of 15.5%.
  • KWT300 The average mass of kernel in grams for 300 kernels either as fresh tissue or dried to moisture level of 15.5%.
  • statically associated refers to the tendency of two events to occur together at a frequency greater than that attributable to chance, where the frequency attributable to chance is represented by a pre-determined level of significance.
  • Statistical association can be determined by any one of a number of significance tests well known to those in the art, for example, ANOVA or t-tests. See, e.g. Statistical Methods, Snedecor, G.W. and Cochran, W.G., Iowa State University Press, Ames, Iowa (1985).
  • Significance levels for ⁇ are preferably less than 0.01.
  • levels of significance for this invention could range between 0 and about 0.250, e.g. less than about 0.0001, 0.00050, 0.0010, 0.0050, 0.010, 0.025, 0.050, 0.100, or 0.250.
  • the present invention provides, among other things, methods of associating a gene or an expression product with a phenotypic trait.
  • the present invention provides utility in such exemplary applications as comparing gene expression between individuals or groups of individuals segregating for quantitatively inherited phenotypic traits.
  • this method can be used in breeding programs to produce "clean" bulks, so that differences in expression profiles between the bulks reflect allelic variation at the markers.
  • This method can also be used for the identification and isolation of genes associated with phenotypic traits.
  • the methods of the subject invention can be used with any biological population expressing a quantitative phenotypic trait.
  • Those of skill in the art will recognize that the methods of this invention can be applied to a biological populations of any organism such as bacteria, yeast, insect, mammalian, or preferably plant populations.
  • the present invention can be practiced over a broad range of plant types.
  • the invention can be used in species from the genera: Hordeum, Secale, Triticum, Sorghum (e.g., S. bicolor), Zea (e.g., Z.
  • Trifolium Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, and Avena.
  • the plant species is selected from the group consisting of: maize, soybean, wheat, canola, sunflower, alfalfa, sorghum, and rice.
  • the biological population used for the subject invention comprises at least 20 members.
  • a typical population includes between about 20 and 200 individuals but optionally may comprise at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1,000, 2500, or 5,000 individuals.
  • PHENOTYPIC TRAITS PHENOTYPIC TRAITS
  • the phenotypic trait selected for the current invention can be any quantitatively inherited phenotypic trait.
  • the phenotypic trait for a plant population is selected from the group consisting of: root lodging, stalk lodging, yield, insect resistance, and disease resistance.
  • the invention may be practiced using any phenotypic trait statistically associated with more than one genomic locus.
  • the trait used in the subject invention can be a QTL ("QTL” see, Edwards, et al, (1987) in Genetics 116:113).
  • QTL or quantitative trait loci, are regions of the genome containing one more markers statistically associated with a trait measured on a quantitative scale. As is known by those in the art, this association can be determined using a simple ANOVA or t-test. See, e.g., Asins and Campbell, (1988) 77zeor.
  • GENETIC MARKERS Members of the biological population are segregated on the basis of the presence or absence of at least one genetic marker statistically associated with the quantitatively inherited phenotypic trait of interest.
  • the genetic marker includes such markers as: RFLPs, RAPDs, AFLPs, SSRs, and SNPs.
  • RFLPs are the product of allelic differences between DNA restriction fragments caused by nucleotide sequence variability. As is well known to those of skill in the art, RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction endonuclease. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP (see, for example, Helentjaris et al., Plant Mol. Bio. 5:109-118 (1985), and U.S. 5,324,631).
  • random amplified polymorphic DNA are used as genetic markers.
  • the phrase "random amplified polymorphic DNA” or “RAPD” refers to the amplification product of the distance between DNA sequences homologous to a single oligonucleotide primer appearing on different sites on opposite strands of DNA. Mutations or rearrangements at or between binding sites will result in polymorphisms as detected by the presence or absence of amplification product (see, for example, Welsh and McClelland (1990), Nucleic Acids Res. 18:7213-7218; Hu and Quiros (1991) Plant Cell Rep. 10:505- 511).
  • amplified fragment length polymorphisms are used as a molecular marker.
  • AFLP amplified fragment length polymorphisms
  • SSR simple sequence repeats
  • the repeat region may vary in length between genotypes while the DNA flanking the repeat is conserved such that the same primers will work in a plurality of genotypes.
  • a polymorphism between two genotypes represents repeats of different lengths between the two flanking conserved DNA sequences (see, for example, Akagi et al (1996) Theor. Appl. Genet. 93:1071-1077; Bligh et al. (1995) Euphytica 86:83- 85; Struss et al. (1998) Theor. Appl. Genet. 97:308-315; Wu et al.
  • SSR single nucleotide polymorphism
  • SNP single nucleotide polymorphism
  • Expression profiling can be performed using essentially any cell or collection of cells from the organism, or the whole organism.
  • a variety of profiling methods are available, including hybridization of expressed or amplified nucleic acids to a nucleic acid array, hybridization of expressed polypeptides to a protein array, hybridization of peptides or nucleic acids to an antibody array, subtractive hybridization, differential display and, hybridization of either proteins or nucleic acids to an array of nucleic acids or proteins, respectively.
  • the expression profile is an RNA profile.
  • the expression products which are detected in the methods of the invention are RNAs, e.g., mRNAs expressed from genes within a cell of the plant or tissue profiled. RNAs can be detected using any of several techniques available. For example, northern blot hybridization is widely used for RNA detection, and is generally taught in a variety of standard texts on molecular biology, including Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press, Inc., San Diego, CA ("Berger”); Sambrook et al., Molecular Cloning - A Laboratory Manual (2 nd Ed.), Vol.
  • RNA can be converted into a double stranded DNA using a reverse transcriptase enzyme and a polymerase, see Ausubel, supra.
  • detection of mRNAs can be performed by converting, e.g., mRNAs into DNAs, which are subsequently detected in, e.g., a standard Southern blot technique.
  • These general methods can be used for expression profiling. For example, arrays of probes can be spotted onto a surface and expression products (or in vitro amplified nucleic acids corresponding to expression products) can be labeled and hybridized with the array. For convenience, it may be helpful to use several arrays simultaneously. It is expected that one of skill is familiar with nucleic acid hybridization.
  • nucleic acid probe is chemically linked to a solid support and a target nucleic acid (e.g., an RNA or corresponding amplified DNA) is hybridized to the probe.
  • target nucleic acid e.g., an RNA or corresponding amplified DNA
  • Either the probe, or the target, or both, can be labeled, typically with a fluorophore.
  • hybridization is detected by detecting bound fluorescence.
  • hybridization is typically detected by quenching of the label by the bound nucleic acid.
  • detection of hybridization is typically performed by monitoring a signal shift such as a change of color, fluorescent quenching, or the like, resulting from proximity of the two bound labels.
  • the probe is a mass label
  • the mass of the label can be detected quantitatively by mass spectrometer.
  • an array of probes are synthesized on a solid support.
  • chip masking technologies and photoreceptive chemistry it is possible to generate ordered arrays of nucleic acid probes with large numbers of probes.
  • These arrays which are known, e.g., as "DNA chips," or as very large scale immobilized polymer arrays can include millions of defined probe regions on a substrate having an area of about 1 cm 2 to several cm 2 .
  • arrays of chemicals, nucleic acids, proteins, or the like can also be printed on a solid substrate using printing technologies. The construction and use of solid phase nucleic acids arrays to detect target nucleic acids is well described in the literature. See, Fodor, et al.
  • a combinatorial strategy allows for the synthesis of arrays containing a large number of probes using a minimal number of synthetic steps. For instance, it is possible to synthesize and attach all possible DNA 8- mer oligonucleotides (4 8 , or 65,536 possible combinations) using only 32 chemical synthetic steps. In general, these procedures provide a method of producing 4 n different oligonucleotide probes on an array using only 4 n synthetic steps.
  • probe design is influenced by the intended application. For example, where several allele-specific probe-target interactions are to be detected in a single assay, e.g. on a single nucleic acid chip, it is desirable to have similar melting temperatures for all the probes.
  • the length of the probes are adjusted so that the melting temperatures for all the probes on the array are closely similar (it will be appreciated that different lengths for different probes may be needed to achieve a particular T m where different probes have different GC contents).
  • melting temperature is a primary consideration in probe design, other factors are also optionally used to further adjust probe construction, such as elimination of self-complementarity in the probe (which can inhibit hybridization of a target nucleotide).
  • restriction site or amplification template for a second primer is incorporated, the primers are optionally longer than those described above by the length of the restriction site, or amplification template site.
  • Standard restriction enzyme sites include 4 base sites, 5 base sites, 6 base sites, 7 base sites, and 8 base sites.
  • An amplification template site for a second primer can be of essentially any length, for example, the site can be about 15-25 nucleotides in length.
  • the amplified products are optionally labeled and are typically resolved by electrophoresis on a polyacrylamide gel; the location(s) where label is present are excised and the labeled product species is/are recovered from the gel portion, typically by elution.
  • the resultant recovered product species can be subcloned into a replicable vector with or without attachment of linkers, amplified further, and/or detected, or even sequenced directly. Sequencing methods are described in Berger, Sambrook and Ausubel, supra.
  • Expression profiling methods are used to determine a gene or an expression product statistically associated with the phenotypic trait of interest. For genes or expression products associated with the marker, one group will have alleles favorable for the trait while the other group will have unfavorable alleles. By comparing differences in expression between the groups segregated on the basis of the presence or absence of at least one genetic marker, genes or expression products associated with the trait can be identified.
  • members possessing the gene or expression product exhibit at least a 2-fold variation relative to members lacking said genetic marker.
  • the gene or expression product could exhibit variation in expression between members possessing the marker and relatives lacking the marker of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Abstract

L'invention concerne un procédé permettant d'associer un gène ou un produit d'expression à un trait phénotypique hérité de manière complexe recherché dans une plante. Les plantes sont triées selon qu'elles contiennent ou non un marqueur génétique. Un ou plusieurs des groupes triés sont classés par profil d'expression afin d'obtenir le gène associé au trait phénotypique recherché. Le gène associé au trait phénotypique recherché peut être identifié et/ou isolé.
PCT/US2000/027719 1999-10-08 2000-10-06 Identification assistee par marqueur d'un gene associe a un trait phenotypique WO2001027325A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU78703/00A AU7870300A (en) 1999-10-08 2000-10-06 Marker assisted identification of a gene associated with a phenotypic trait
EP00968844A EP1230385A4 (fr) 1999-10-08 2000-10-06 Identification assistee par marqueur d'un gene associe a un trait phenotypique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15853799P 1999-10-08 1999-10-08
US60/158,537 1999-10-08

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WO2001027325A1 true WO2001027325A1 (fr) 2001-04-19

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WO2008018790A2 (fr) * 2006-08-07 2008-02-14 Nsure Holding B.V. Diagnostics de qualité basés sur la génomique pour des produits agricoles frais
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CN105567857A (zh) * 2016-03-09 2016-05-11 中国农业科学院作物科学研究所 384个snp位点及其在大豆品种资源鉴定中的应用
CN106676176A (zh) * 2017-01-18 2017-05-17 天津农学院 一种利用多重pcr对四倍体紫花苜蓿进行ssr分析的方法
CN107354232A (zh) * 2017-09-18 2017-11-17 江苏省农业科学院 一种开发与小麦特定染色体区段连锁分子标记的方法
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CN108060262B (zh) * 2018-02-08 2020-07-14 中国农业科学院作物科学研究所 与小麦根系性状相关的kasp标记及其应用

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Cited By (15)

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Publication number Priority date Publication date Assignee Title
US8669056B2 (en) 2002-12-31 2014-03-11 Cargill Incorporated Compositions, methods, and systems for inferring bovine breed
US11053547B2 (en) 2002-12-31 2021-07-06 Branhaven LLC Methods and systems for inferring bovine traits
US10190167B2 (en) 2002-12-31 2019-01-29 Branhaven LLC Methods and systems for inferring bovine traits
US9982311B2 (en) 2002-12-31 2018-05-29 Branhaven LLC Compositions, methods, and systems for inferring bovine breed
US9206478B2 (en) 2002-12-31 2015-12-08 Branhaven LLC Methods and systems for inferring bovine traits
EP1626621A4 (fr) * 2003-05-28 2009-10-21 Pioneer Hi Bred Int Methode de selection vegetale
EP1626621A2 (fr) * 2003-05-28 2006-02-22 Pioneer Hi-Bred International, Inc. Methode de selection vegetale
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WO2008018790A3 (fr) * 2006-08-07 2008-04-03 Nsure Holding B V Diagnostics de qualité basés sur la génomique pour des produits agricoles frais
WO2008018790A2 (fr) * 2006-08-07 2008-02-14 Nsure Holding B.V. Diagnostics de qualité basés sur la génomique pour des produits agricoles frais
US10695562B2 (en) 2009-02-26 2020-06-30 The University Of North Carolina At Chapel Hill Interventional drug delivery system and associated methods
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CN106676176A (zh) * 2017-01-18 2017-05-17 天津农学院 一种利用多重pcr对四倍体紫花苜蓿进行ssr分析的方法
CN107354232A (zh) * 2017-09-18 2017-11-17 江苏省农业科学院 一种开发与小麦特定染色体区段连锁分子标记的方法

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