WO1989007647A1 - Liaisons genetiques entre des genes importants du point de vue agronomique et polymorphismes de longueurs de fragments a restriction - Google Patents

Liaisons genetiques entre des genes importants du point de vue agronomique et polymorphismes de longueurs de fragments a restriction Download PDF

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WO1989007647A1
WO1989007647A1 PCT/US1989/000709 US8900709W WO8907647A1 WO 1989007647 A1 WO1989007647 A1 WO 1989007647A1 US 8900709 W US8900709 W US 8900709W WO 8907647 A1 WO8907647 A1 WO 8907647A1
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corn plants
rflp
respect
profile
plants
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PCT/US1989/000709
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David Grant
Marc Christian Albertsen
William Dale Beavis
Robert Reid Fincher
William Edward Kuhn
John Stephen Charles Smith
Oscar Samuel Smith, Jr.
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Pioneer Hi-Bred International, Inc.
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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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • 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
    • 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

  • This invention is in the field of genetic engineering and corn breeding. More specifically, the invention concerns methods for finding correlations between agronomically important genes in corn and restriction fragment length polymorphisms through the use of DNA probes that are shown to reveal polymorphisms.
  • the goal of plant breeding is to combine in a single variety/hybrid various desirable traits of the parental lines and to exploit the heterosis exhibited by the cross of the parental lines.
  • these traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality.
  • uniformity of plant characteristies such as germination and stand establishment, growth rate, maturity and fruit size are important.
  • Field crops are bred through techniques that use the plant's method of pollination.
  • a plant is defined as self- pollinating if pollen from one flower is transferred to the same or another flower of the same plant.
  • a plant is cross- pollinated if the pollen comes from a flower on a different plant.
  • Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny.
  • a cross between two homozygous plants from differing backgrounds or two homozygous lines (inbred lines) produces a uniform population of hybrid plants that may be heterozygous for many gene loci.
  • a cross of two plants, each heterozygous at a number of gene loci, will produce a population of hybrid plants. Each of these plants differ genetically and the population will not be uniform.
  • Corn ( Zea mays L.) plants are bred by both self- pollination and cross-pollination techniques. Corn has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. Natural pollination occurs in corn when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears.
  • breeding programs are designed to combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desirable phenotypes.
  • breeding programs are designed to combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desirable phenotypes.
  • the new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential as F 1 hybrids.
  • the identification of desirable agronomic traits has traditionally been done by phenotypic selection.
  • the invention is based on the use of RFLPs to identify genetic linkages with agronomically important genes.
  • This invention consists of three major parts: (1) DNA probes shown to reveal polymorphisms between two parent inbred lines and having known chromosomal locations, (2) statistical techniques that can find correlations between the inheritance of one or more DNA probes and the phenotype of the plants under investigation, and (3) methods for using the identified genetic linkage between specific probes and genetic components of agronomically important traits in plant breeding.
  • this invention relates to a method for determining a particular trait in a maize plant which comprises analyzing each maize chromosome for DNA polymorphisms linked to a particular trait. Any of a variety of RFLPs, probes and restriction enzymes can be used, as illustrated herein.
  • the invention also relates to specific DNA probes that can be used in the method of this invention.
  • the invention further relates to use of newly-identified relationships between agronomic traits and genetic markers to enhance plant breeding.
  • restriction endonuclease or restriction enzyme is an enzyme that recognizes a specific base sequence in a double-stranded DNA molecule, and will cleave both strands of the DNA molecule at every place where this sequence appears.
  • restriction fragments The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome will be digested by a particular restriction endonuclease into a discrete set of restriction fragments. The DNA fragments that result from restriction enzyme cutting are separated and displayed by electrophoresis through agarose gels.
  • Restriction Fragment Length Polymorphism The genomic DNA of two individuals in a species, for example, will differ in sequence at many sites. When these differences occur in the recognition site for a restriction endonuclease, the enzyme will not cleave the DNA molecule at that point. Likewise, a variation may introduce a recognition site where none exists in the other individual, causing the DNA to be cut by the restriction enzyme at that point. Because of this, digestion of the two individuals' DNA will produce fragments having different lengths. A polymorphism in the length of restriction fragments produced by digestion of the DNA of the two individuals will result. Aoarose Gel Electrophoresis.
  • the DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure if the number of fragments included in the pattern is small.
  • most genomes, including the maize genome contain far too many DNA sequences to produce a simple pattern of restriction fragments.
  • a methodology referred to as the Southern hybridization procedure can be applied.
  • Southern Hybridization Procedure The purpose of the Southern hybridization procedure, also referred to as Southern blotting, is to transfer physically DNA fractionated by agarose gel electrophoresis onto a support such as nylon membrane or nitrocellulose filter paper while retaining the relative positions of DNA fragments resulting from the fractionation procedure.
  • the methodology used to accomplish the transfer from agarose gel to the support is to draw the DNA from the gel into the support by capillary action.
  • Nucleic Acid Hybridization is used to detected related DNA sequences by hybridization of single-stranded DNA on supports such as nylon membrane or nitrocellulose filter papers. Nucleic acid molecules that have complementary base sequences will reform the double- stranded structure if mixed in solution under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a support. In the Southern hybridization procedure, the latter situation occurs.
  • the maize genomic DNA is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single stranded form, and transferred to the support, making it available for reannealing to the hybridization probe.
  • Hybridization Probe To detect a particular DNA sequence in the Southern hybridization procedure, a labeled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to a support such as nylon membrane or nitrocellulose filter paper. The areas on the filter that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the reannealing reaction. The areas of the filter that exhibit such labeling can then be detected according to the type of label used.
  • the hybridization probe is generally produced by molecular cloning of a specific DNA sequence from the maize genome.
  • This invention is based on the use of restriction fragment length polymorphism (RFLP) to identify genetic linkages to agronomically important genes.
  • RFLP restriction fragment length polymorphism
  • This invention consists of methods for locating agronomically important genes based on RFLPs and of novel DNA probes for use in the methods.
  • this invention is based on the identification of restriction fragments from the ten chromosomes of maize that define genetic linkages between specific chromosomes and agronomically important traits using analysis techniques that can find correlations between the inheritance of one or more DNA sequences and the phenotype of the plant under investigation.
  • Identifying RFLPs involves the use of restriction endonucleases, DNA mapping, and cloned DNA probes. Restriction endonucleases cleave the genomic DNA molecules at specific sites. Cloned RFLPs are detected as the differences in the size of restriction fragments observed in Southern blotting experiments using cloned DNA probes free of repetitive sequences. Certain polymorphisms can thus be genetic markers that are associated with a specific agronomic trait. Establishment of such an association permits the monitoring of heritable sequences of genomic DNA. Probes that can detect sequences associated with specific traits can therefore be derived from known gene loci or from anonymous DNA segments. RFLPs appear to be present in all regions of the maize genome, thus making it feasible to construct a detailed maize genetic linkage map and thereby localize agronomically important genes.
  • RFLPs that identify genetic characteristics are particularly useful in breeding programs in order to select for certain traits.
  • Other uses of RFLPs may include varietal identification, identification and mapping of quantitative trait loci (QTL), quantification of genetic diversity in a crop population, screening genetic resource strains, or populations for useful quantitative trait alleles and their marker-assisted introgression from the resource strain to a commercial variety, and marker-assisted early selection of recombinant inbred lines in plant pedigree breeding programs.
  • QTL quantitative trait loci
  • RFLPs are used to identify genetic linkage to agronomically important genes.
  • This invention consists of three major parts: (1) DNA probes shown to reveal polymorphisms between two parent inbred lines and having known chromosomal locations, (2) statistical techniques that can find correlations between the inheritance of one or more DNA probes and the phenotype of the plants under investigation, and (3) use of the identified genetic linkages between specific probes and genetic components of agronomically important traits as an aid in selecting plants and populations in "classical" plant breeding based on Mendelian genetics.
  • the invention comprises determining a particular trait in a maize plant by analyzing maize chromosomes for DNA polymorphisms and linkage to that trait.
  • Specific traits determined include adjusted yield, plant yield, plant height, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, and stay green.
  • a definition of each of these traits is as fol1 ows:
  • Plant Yield The plant yield is the field weight divided by the number of plants per plot.
  • Plant Height This is a measure of the height of the hybrid from the ground to the tip of the tassel.
  • the ear height is a measure from the ground to the top ear node attachment.
  • the GDU is the number of growing degree units (GDU) required for an inbred line or hybrid to shed pollen from the time of planting. Growing degree units are calculated by the Barger Method, where the heat units for a 24-hour period are: The highest maximum used is 86°F and the lowest minimum used is 50°F.
  • GDUs are a way of measuring plant maturity.
  • the grain moisture is the percentage moisture of the grain at harvest.
  • Root Lodging The root lodging is the percentage of plants that do not root lodge; i.e., those that lean from the vertical axis at an approximately 30° angle or greater would be counted as root lodged.
  • Stalk Lodging This is the percentage of plants that do not stalk lodge, i.e., stalk breakage, as measured by either natural lodging or pushing the stalks and determining the percentage of plants that break off below the ear.
  • DNA is extracted from the plant cell and digested with a given restriction endonuclease. After the digest is obtained, and the same is separated by a standard technique such, as, for example, agarose gel electrophoresis, the separated bands are probed with a DNA fragment coding for the RFLP sequence.
  • methods for generating additional new DNA fragments also linked with the gene for a particular trait are as follows:
  • a first method is to test randomly chosen maize DNA fragments (either genomic or c-DNA clones) that map to the appropriate region of the maize genomic map. Such mapping can be achieved by two techniques:
  • the new fragment need not be polymorphic, but for (b), polymorphisms must first be identified by comparing the restriction pattern of the genomic DNA at the new site in unrelated plants.
  • the mapped fragment must still be shown to detect a polymorphism in maize DNA.
  • the polymorphism which represents a genetic marker can then be tested for genetic linkage with genes affecting agronomic traits or can be tested for linkage to other DNA probes, such as those described below.
  • Another method for obtaining DNA clones is to construct a library from maize DNA isolated from metaphase chromosomes that have been sorted on a fluorescence activated sorter. This method can sometimes yield purified chromosomes of 95% or greater purity.
  • a final method of obtaining new DNA probes from the region of the chromosome containing the agronomically important gene is to use any probes already mapped to the region in order to "fish out” adjacent overlapping pieces of DNA from genomic libraries (commonly called “chromosome walking”).
  • a probe must ultimately be found to detect a polymorphism if it is to be useful for testing for the desired trait.
  • the polymorphism must be found to be linked to genes affecting traits or to other useful markers in studies, or to be immediately adjacent to preexisting markers.
  • the particular probe can be of any desired sequence length as long as it is capable of identifying the polymorphism in the involved DNA region or locus. It can be a DNA fragment by itself, or be present in longer genetic sequences or fragments, or even in a plasmid or other appropriate vehicle. Labelling and hybridization conditions can be readily determined by those of skill in the art. Usually, the stringency is standard for unique sequence DNA from within the species.
  • a genetic linkage map was constructed from the data presented in the Examples by utilizing the algorithms described by E. Lander et al., Genomics, 1:174-181 (1987), herein incorporated by reference. These genetic linkage groups were assigned to chromosome arms by (1) using as probes DNA sequences whose location was known, and/or (2) using as markers isozyme loci whose location was known.
  • this invention also provides methods for augmenting conventional plant breeding by identification of individual plants which have the desired genotype at a genetic marker locus associated with one of the foregoing traits, comprising the steps of - constructing a preferred RFLP profile for genetic markers identified herein as being associated with the trait or traits in question; - determining the RFLP profiles of individuals in a segregating population with respect to the associated genetic markers; and - selecting individuals from the population having RFLP profiles which most closely match the preferred profile.
  • the construction of the preferred RFLP profile is a matter within the skill of the typical plant breeder. For example, the breeder will select a particular allele which provides a desirable contribution in terms of ear circumference. The contribution viewed as "desirable" will vary according to the objectives of the breeding program. In one instance, a large ear circumference be be desirable, while in another, a small ear circumference may be preferred. In yet another instance, the ear circumference per se may be unimportant, but the breeder may be working to develop a plant which is homozygous, i.e., has the same contribution from both parents, with respect to that trait. In any event, it will be a simple matter for the breeder to prepare a list of desired traits and to construct a preferred RFLP profile for the ideal plant from that list and the genetic marker linkages identified herein.
  • a segregating population of plants is easy to obtain, and is typically found, for example, in the progeny of selfed F 1 hybrids of two different inbred lines. Once the population has been identified, RFLP profiles of each plant are performed using the well-known techniques described above.
  • Another individual will offer the desired allele for ear circumference and stay green, but will lack the desired allele for kernel row length. Selection between the two will simply involve a decision by the breeder on whether it is preferable to have the allele for kernel row length or for stay green, and in fact such decisions will be a relatively trivial exercise compared to the judgements made in conventional plant breeding, which are based on less clear-cut information about the genetic makeup of the individuals. The breeder also has the option of proceeding to the next stage of breeding with both individuals.
  • Markers identified by the prefix PIO- are available commercially from Pioneer Hi-Bred International, Des Moines, IA 50309. Markers identified by the prefixes BNL- and UMC- are publicly available markers which can be obtained from Brookhaven National Laboratory and the University of Missouri, respectively. The remainder of the markers are either published isozyme markers of known genetic location, such as AMP1, MDH2 and GLU1, or are probes for specific mutant genes which are well known and whose location has been identified, as mentioned above.
  • Grain quality This is a subjective score based on visual grading of the shelled corn in terms of moldy and cracked kernels.
  • Cold test germination This is a percentage germination test performed in a cold chamber, and evaluates germination performance under adverse conditions.
  • Soak test germination This is a test of percentage germination under ideal germination conditions, but after the seed has been soaked prior to planting.
  • European corn borer second brood tolerance This is a visual evaluation of plant damage caused by infestation by the second annual brood of the European corn borer caterpillar.
  • Plant yield This is the yield of a test plot divided by the number of plants in the test plot.
  • TRAIT GDU Shed (GDUSHD)
  • TRAIT Dropped Ears, Statistically transformed (FOGDE) P1020690
  • TRAIT Grain quality (GRNOUL)
  • TRAIT European Corn Borer Second Brood Tolerance (ECB2SC)
  • TRAIT Root lodging, statistically transformed (FOGRTL)
  • TRAIT Stalk lodging (STKLDG)
  • TRAIT Stalk lodging, statistically transformed (FOGSTL)
  • TRAIT Yield, Bushels/Acre (BU_ACR) BNL5.59 BNL6.20 UMC042 PI020608
  • TRAIT Weight of 100 kernels (CKWT)
  • TRAIT Dropped ears, statistically transformed (FOGDE) PI0205 PI02017 PI020558 PI020508
  • TRAIT Cold test germination (CTST) UMC034 BNL8.33 PI01017 PI067
  • TRAIT Soak test germination (SKTST) PI020518 PI020511 PI020509 BNL8.33 PI020569 PI020708 PI020746 BNL9.11 PI02052 GLU1
  • TRAIT Early stand count (ESTCNT) UMC061 PI020713 PI020725 PI020597
  • TRAIT Ear circumference (EARCIR)
  • TRAIT Cob circumference (COBCIR)
  • PI065 GLU1 PI020646 TRAIT Bare tip (NOSBAC)
  • TRAIT Kernel row length (ROWLEN)
  • TRAIT Kernel Depth (KDEPTH)
  • TRAIT Kernels per kernel row (KERPRO)
  • PI020626 TRAIT Numberer of kernel rows per ear (KERROW)
  • TRAIT Kernel width (KWIDTH)
  • TRAIT GDU Shed (GDUSHD)
  • TRAIT GDU Silk (GDUSLK)
  • TRAIT Plant heioht (PLTHT)
  • TRAIT European Corn Borer Second Brood Tolerance (ECB2SC)
  • TRAIT Root lodging (RT_LDG)
  • TRAIT Root lodging, statistically transformed (FOGRTL)
  • TRAIT Stalk lodging, statistically transformed (FOGSTL)
  • TRAIT Grain quality (GRNOUL)
  • the inbreds B73 and Mo17 were crossed to produce the F 1 hybrid designated B73/Mol7. Hybrid seed was planted, several plants selfed, and the seed bulked to produce the F2 generation designated B73/Mo17)X. 175 seeds from the F2 generation were planted in peat pots in a greenhouse. Seedlings were transplanted to the field at normal planting time. Each plant was self-pollinated using the usual procedures for pollination of corn to produce the F3 ears designated B73/Mo17)Xn where n ranged from 1 to 175 and represents the specific F3 ear. Ears were harvested, identified to plant number, and kept separate. Each ear therefore contained seed that would generate an F3 family.
  • a winter nursery was used to produce seed for field testing from the F3 ears. Twenty-four kernels from each of 112 F3 ears were planted in an isolated crossing block. Interplanted around those 112 rows were rows of Pioneer inbred V78. Tassels were removed by hand from every plant of the 112 F3 families. Thus, the ear of each F3 plant from the B73/Mo17 cross was pollinated by the inbred V78. For each of the 112 F3 families , each ear from the 24 plants were harvested, dried, shelled, and bulked together. These 112 entities of seed were considered as 112 F3 topcrosses to inbred V78.
  • each F3 ear was planted in 112 rows, one for each F3 family. Within each row, ten plants were self-pollinated to derive F4 ears. For each of the 112 F3 families, each of the self-pollinated ears were harvested, dried, shelled, and bulked together. These 112 entities of seed were considered as 112 F4 bulks.
  • the 112 F3 topcrosses and 112 F4 bulks were evaluated in field performance tests (yield tests).
  • the tests were conducted according to standard yield test procedures as used in the profession of agronomy and crop breeding.
  • Each of the 112 F3 topcrosses comprised an entry in a randomized complete block design. Check entries were added making a total entry list of 125. The experiment was grown in two replications at each of four locations in central Iowa. Each replication of an entry was planted in a two-row plot. Plots were 5.3 meters long with .76 meters between rows. Plots were overplanted and thinned to even and uniform stand of 50 plants per plot. These plants were allowed to grow to maturity and data were collected for various traits throughout the season.
  • the 112 F4 bulks were tested identically except there were three test locations instead of four. Two locations were in central Iowa, and the other was in central Indiana.
  • Plant Height PLTHT From ground to tip of tassel Plant Height PLTHT From ground to tip of tassel.
  • GDU Silk GDUSLK Accumulated heat units to the day that 50% of plants in a plot had silks emerged at least one inch.
  • Total DNA can be isolated from various maize tissues (leaves, seedlings, etc.) by any one of several standard methods (for example see Maniatis et al., Molecular Cloning. A Laboratory Manual (1982); Dillon et al., Recombinant DNA Methodology (John Wiley & Sons 1985). Southern Hybridization
  • Agarose gel electrophoresis of restriction enzyme- generated fragments of maize DNA, transfer of the DNA to nylon membranes, and hybridization with a radioactively labeled probe can be done by any of several standard methods (for example, see Maniatis et al., Molecular Cloning, A Laboratory Manual).
  • Total DNA from the maize inbred B73 was purified and digested to completion with the restriction enzyme Pst I and electrophoresed on an agarose gel. Fragments from the size classes 600-1,000 base pairs (bp), 1,000-1,500 bp, 1,500-2,000 bp, and 2,000-2,500 bp were ligated into one of several E. coli vectors at the Pst I site and transformed into one of several laboratory strains of E. coli using standard conditions (for example, see Maniatis et al., Molecular Cloning, A Laboratory Manual). Colonies containing plasmids with single inserts were identified by plasmid minipreps and agarose gel electrophoresis.
  • Plasmid DNA from each previously characterized colony was purified, digested to completion with Pst I, and the two fragments (maize DNA insert and cloning vector) separated by agarose gel electrophoresis. Isolated maize insert DNA, still in the agarose plug, was radioactively labeled with P 32 by either nick translation or random priming.
  • Southern hybridization was done using labeled maize insert DNA as probe and total cell DNA from various maize inbreds digested to completion with one or more restriction enzymes as target. After washing the membrane to remove any nonhybridized probe, the membranes were subjected to autoradiography.
  • Probes were selected for further consideration if (1) 1-3 autoradiographic bands were observed for at least one restriction enzyme, and (2) band patterns were different between at least two maize inbreds.
  • EXAMPLE 3 Determination of Linkage Among RFLP Marker Loci
  • TC F3 top crosses
  • PS F4 bulk tests

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Abstract

L'invention concerne des procédés permettant de trouver des corrélations entre des gènes importants du point de vue agronomique dans les céréales, ainsi que des polymorphismes de longueurs de fragments à restriction et des sondes d'ADN présentées comme révélant des polymorphismes.
PCT/US1989/000709 1988-02-22 1989-02-22 Liaisons genetiques entre des genes importants du point de vue agronomique et polymorphismes de longueurs de fragments a restriction WO1989007647A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004651A1 (fr) * 1988-10-19 1990-05-03 Whitehead Institute For Biomedical Research Topographie de traits quantitatifs en utilisant des marqueurs genetiques
EP0552393A1 (fr) * 1992-01-23 1993-07-28 KWS KLEINWANZLEBENER SAATZUCHT Aktiengesellschaft vorm. Rabbethge & Giesecke Procédé pour identifier plants et graines de betteraves résistants au virus-BNYV et sonde de RFLP pour réaliser ce procédé
EP0566793A1 (fr) * 1992-04-24 1993-10-27 Iowa State University Research Foundation, Inc. Association de l'ADN mitochondrial bovin avec des traits qui ont une importance économique
EP0570496A1 (fr) * 1991-01-15 1993-11-24 Genmark Marqueurs polymorphes d'adn utilises chez les bovins
EP0577598A1 (fr) * 1990-07-19 1994-01-12 The Regents Of The University Of California Systeme de transformation de plantes sans danger au niveau biologique
US5437697A (en) * 1992-07-07 1995-08-01 E. I. Du Pont De Nemours And Company Method to identify genetic markers that are linked to agronomically important genes
US5492547A (en) * 1993-09-14 1996-02-20 Dekalb Genetics Corp. Process for predicting the phenotypic trait of yield in maize
US5574210A (en) * 1995-06-06 1996-11-12 Zeneca Limited Gray leaf spot resistant corn and the production thereof
WO1998008963A1 (fr) * 1996-08-30 1998-03-05 Regents Of The University Of Minnesota Combinaisons geniques conferant au mais la tolerance aux herbicides
US5746023A (en) * 1992-07-07 1998-05-05 E. I. Du Pont De Nemours And Company Method to identify genetic markers that are linked to agronomically important genes
WO1998030717A2 (fr) * 1996-12-02 1998-07-16 Biocem S.A. Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci
US5792904A (en) * 1993-01-08 1998-08-11 Novartis Finance Corporation Method for breeding disease resisteance into plants
WO1998041655A1 (fr) * 1997-03-20 1998-09-24 E.I. Du Pont De Nemours And Company Methode pour identifier des loci de marqueur genetique associes a des loci de traits de caractere
WO1999032661A1 (fr) * 1997-12-22 1999-07-01 Pioneer-Hi-Bred International, Inc. Etablissement des cartographies des qtl dans des populations vegetales de selection
US6054634A (en) * 1994-01-21 2000-04-25 North Caroline State University Methods for within family selection in woody perennials using genetic markers
US6069298A (en) * 1993-02-05 2000-05-30 Regents Of The University Of Minnesota Methods and an acetyl CoA carboxylase gene for conferring herbicide tolerance and an alteration in oil content of plants
US6107544A (en) * 1993-01-08 2000-08-22 Novartis Finance Corporation Method for breeding disease resistance into plants
US6222099B1 (en) 1993-02-05 2001-04-24 Regents Of The University Of Minnesota Transgenic plants expressing maize acetyl COA carboxylase gene and method of altering oil content
US6232525B1 (en) 1993-01-08 2001-05-15 Novartis Finance Corporation Mutant plants and uses therefor
WO2001055395A1 (fr) * 2000-01-28 2001-08-02 Biogemma Identification de genes associes a un locus qtl de digestibilite du maïs
WO2001073091A2 (fr) * 2000-03-24 2001-10-04 Pioneer Hi-Bred International, Inc. Procede de selection et de mise au point de vegetaux de meilleure qualite racinaire et de meilleure resistance a la verse racinaire
EP1230385A1 (fr) * 1999-10-08 2002-08-14 Pioneer Hi-Bred International, Inc. Identification assistee par marqueur d'un gene associe a un trait phenotypique
US6500616B1 (en) 1998-04-16 2002-12-31 Case Western Reserve University Methods of monitoring genomic integrity and detecting genomic destabilization of plant cells in tissue culture
WO2003074734A2 (fr) * 2002-03-05 2003-09-12 Solexa Ltd. Procedes de detection de variations de sequence a l'echelle du genome associees a un phenotype
KR100443569B1 (ko) * 2000-12-15 2004-08-09 학교법인 인하학원 길이다형성 ｄｎａ 염기서열을 이용한 다중 증폭중합효소연쇄반응 조건의 최적화에 의한 개인식별 및친자감별 방법
US7091398B2 (en) 2001-02-22 2006-08-15 Pioneer Hi-Bred International, Inc. Isolated sucrose sythase polynucleotides and uses thereof
EP2051986A2 (fr) * 2006-08-14 2009-04-29 Monsanto Technology, LLC Polymorphismes de maïs et procédés de génotypage
WO2010071431A1 (fr) 2008-12-19 2010-06-24 Monsanto Invest N.V. Procédé de reproduction de plants de concombre résistants au virus du jaunissement et du rabougrissement des cucurbitacées (cysdv)

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Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004651A1 (fr) * 1988-10-19 1990-05-03 Whitehead Institute For Biomedical Research Topographie de traits quantitatifs en utilisant des marqueurs genetiques
EP0577598A4 (en) * 1990-07-19 1994-05-18 Univ California Biologically safe plant transformation system
EP0577598A1 (fr) * 1990-07-19 1994-01-12 The Regents Of The University Of California Systeme de transformation de plantes sans danger au niveau biologique
EP0570496A1 (fr) * 1991-01-15 1993-11-24 Genmark Marqueurs polymorphes d'adn utilises chez les bovins
EP0570496A4 (en) * 1991-01-15 1996-06-12 Genmark Polymorphic dna markers in bovidae
US5981832A (en) * 1991-02-19 1999-11-09 Dekalb Genetics Corp. Process predicting the value of a phenotypic trait in a plant breeding program
US6455758B1 (en) 1991-02-19 2002-09-24 Dekalb Genetics Corporation Process predicting the value of a phenotypic trait in a plant breeding program
EP0552393A1 (fr) * 1992-01-23 1993-07-28 KWS KLEINWANZLEBENER SAATZUCHT Aktiengesellschaft vorm. Rabbethge & Giesecke Procédé pour identifier plants et graines de betteraves résistants au virus-BNYV et sonde de RFLP pour réaliser ce procédé
EP0566793A1 (fr) * 1992-04-24 1993-10-27 Iowa State University Research Foundation, Inc. Association de l'ADN mitochondrial bovin avec des traits qui ont une importance économique
US5437697A (en) * 1992-07-07 1995-08-01 E. I. Du Pont De Nemours And Company Method to identify genetic markers that are linked to agronomically important genes
US5746023A (en) * 1992-07-07 1998-05-05 E. I. Du Pont De Nemours And Company Method to identify genetic markers that are linked to agronomically important genes
US6107544A (en) * 1993-01-08 2000-08-22 Novartis Finance Corporation Method for breeding disease resistance into plants
US6232525B1 (en) 1993-01-08 2001-05-15 Novartis Finance Corporation Mutant plants and uses therefor
US5792904A (en) * 1993-01-08 1998-08-11 Novartis Finance Corporation Method for breeding disease resisteance into plants
US6268550B1 (en) 1993-02-05 2001-07-31 Regents Of The University Of Minnesota Methods and a maize acetyl CoA carboxylase gene for altering the oil content of plants
US6222099B1 (en) 1993-02-05 2001-04-24 Regents Of The University Of Minnesota Transgenic plants expressing maize acetyl COA carboxylase gene and method of altering oil content
US6146867A (en) * 1993-02-05 2000-11-14 Regents Of The University Of Minnesota Methods for expressing a maize acetyl CoA carboxylase gene in host cells and encoded protein produced thereby
US6069298A (en) * 1993-02-05 2000-05-30 Regents Of The University Of Minnesota Methods and an acetyl CoA carboxylase gene for conferring herbicide tolerance and an alteration in oil content of plants
US5492547A (en) * 1993-09-14 1996-02-20 Dekalb Genetics Corp. Process for predicting the phenotypic trait of yield in maize
US6054634A (en) * 1994-01-21 2000-04-25 North Caroline State University Methods for within family selection in woody perennials using genetic markers
US5574210A (en) * 1995-06-06 1996-11-12 Zeneca Limited Gray leaf spot resistant corn and the production thereof
WO1998008963A1 (fr) * 1996-08-30 1998-03-05 Regents Of The University Of Minnesota Combinaisons geniques conferant au mais la tolerance aux herbicides
WO1998030717A3 (fr) * 1996-12-02 1999-04-29 Biocem S A Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci
WO1998030717A2 (fr) * 1996-12-02 1998-07-16 Biocem S.A. Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci
US6219964B1 (en) 1997-03-20 2001-04-24 E. I. Du Pont De Nemours And Company Method for identifying genetic marker loci associated with trait loci
WO1998041655A1 (fr) * 1997-03-20 1998-09-24 E.I. Du Pont De Nemours And Company Methode pour identifier des loci de marqueur genetique associes a des loci de traits de caractere
US6399855B1 (en) 1997-12-22 2002-06-04 Pioneer Hi-Bred International, Inc. QTL mapping in plant breeding populations
WO1999032661A1 (fr) * 1997-12-22 1999-07-01 Pioneer-Hi-Bred International, Inc. Etablissement des cartographies des qtl dans des populations vegetales de selection
US6773889B2 (en) 1998-04-16 2004-08-10 Case Western Reserve University Method for detecting genomic destabilization arising during tissue culture of plant cells
US6500616B1 (en) 1998-04-16 2002-12-31 Case Western Reserve University Methods of monitoring genomic integrity and detecting genomic destabilization of plant cells in tissue culture
EP1230385A4 (fr) * 1999-10-08 2004-12-08 Pioneer Hi Bred Int Identification assistee par marqueur d'un gene associe a un trait phenotypique
EP1230385A1 (fr) * 1999-10-08 2002-08-14 Pioneer Hi-Bred International, Inc. Identification assistee par marqueur d'un gene associe a un trait phenotypique
FR2804970A1 (fr) * 2000-01-28 2001-08-17 Biogemma Fr Identification de genes associes a un locus qtl de digestibilite du mais
WO2001055395A1 (fr) * 2000-01-28 2001-08-02 Biogemma Identification de genes associes a un locus qtl de digestibilite du maïs
WO2001073091A2 (fr) * 2000-03-24 2001-10-04 Pioneer Hi-Bred International, Inc. Procede de selection et de mise au point de vegetaux de meilleure qualite racinaire et de meilleure resistance a la verse racinaire
WO2001073091A3 (fr) * 2000-03-24 2002-06-27 Pioneer Hi Bred Int Procede de selection et de mise au point de vegetaux de meilleure qualite racinaire et de meilleure resistance a la verse racinaire
KR100443569B1 (ko) * 2000-12-15 2004-08-09 학교법인 인하학원 길이다형성 ｄｎａ 염기서열을 이용한 다중 증폭중합효소연쇄반응 조건의 최적화에 의한 개인식별 및친자감별 방법
US7091398B2 (en) 2001-02-22 2006-08-15 Pioneer Hi-Bred International, Inc. Isolated sucrose sythase polynucleotides and uses thereof
WO2003074734A2 (fr) * 2002-03-05 2003-09-12 Solexa Ltd. Procedes de detection de variations de sequence a l'echelle du genome associees a un phenotype
WO2003074734A3 (fr) * 2002-03-05 2004-02-19 Manteia S A Procedes de detection de variations de sequence a l'echelle du genome associees a un phenotype
EP2051986A2 (fr) * 2006-08-14 2009-04-29 Monsanto Technology, LLC Polymorphismes de maïs et procédés de génotypage
EP2051986A4 (fr) * 2006-08-14 2010-03-17 Monsanto Technology Llc Polymorphismes de maïs et procédés de génotypage
WO2010071431A1 (fr) 2008-12-19 2010-06-24 Monsanto Invest N.V. Procédé de reproduction de plants de concombre résistants au virus du jaunissement et du rabougrissement des cucurbitacées (cysdv)

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AU4030289A (en) 1989-09-06
EP0402401A1 (fr) 1990-12-19
EP0402401A4 (en) 1991-09-11

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