EP0944741A2 - Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci - Google Patents

Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci

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Publication number
EP0944741A2
EP0944741A2 EP97955036A EP97955036A EP0944741A2 EP 0944741 A2 EP0944741 A2 EP 0944741A2 EP 97955036 A EP97955036 A EP 97955036A EP 97955036 A EP97955036 A EP 97955036A EP 0944741 A2 EP0944741 A2 EP 0944741A2
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EP
European Patent Office
Prior art keywords
nucleic acid
allele
sequence
specific oligonucleotide
segment
Prior art date
Legal status (The legal status 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 status listed.)
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EP97955036A
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German (de)
English (en)
Inventor
Alain Biocem S.A. MURIGNEUX
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Groupe Limagrain Holding SA
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BIOCEM SA
Groupe Limagrain Holding SA
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Publication of EP0944741A2 publication Critical patent/EP0944741A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • 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 variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral.
  • a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism.
  • a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form.
  • both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.
  • a restriction fragment length polymorphism means a variation in DNA sequence that alters the length of a restriction fragment as described in Botstein et al., Am. J. Hum . Genet . 32, 314-331 (1980).
  • the restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment.
  • RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; WO 90/11369; Donis-Keller, Cell 51, 319-337 (1987); Lander et al . , Genetics 121, 85-99 (1989)).
  • a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.
  • VNTR variable number tandem repeat
  • Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs .
  • Some single nucleotide polymorphisms occur in proteincoding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein.
  • Other single nucleotide polymorphisms occur in noncoding regions . Some of these polymorphisms may also result in defective or variant protein expression (e.g., as a result of defective splicing) .
  • Other single nucleotide polymorphisms have no phenotypic effects .
  • Single nucleotide polymorphisms can be used in the same manner as RFLPs, and VNTRs but offer several advantages .
  • Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism.
  • the greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms.
  • the different forms of characterised single nucleotide polymorphisms are often easier to distinguish that other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers) .
  • the invention provides nucleic acid segments containing at least 10, 15 or 20 contiguous bases from a vegetal fragment including a polymorphic site notably a single nucleotide polymorphism (SNP).
  • a vegetal fragment does not belong to the Cruciferae family .
  • the segments can be DNA or RNA, and can be double- or single-stranded. Some segments are 10-20 or 10-50 bases long. Preferred segments include a diallelic polymorphic site.
  • the invention concerns nucleic acid segments from a fragment shown in Table I (corn) .
  • the Invention further provides allele-specific oligonucleotides that hybridizes to a segment of a vegetal fragment, for example fragment in Table I. These oligonucleotides can be probes or primers .
  • isolated nucleic acid comprising a sequence of Table I or the complement thereto, in which the polymorphic site within the sequence is occupied by a base other than the reference base shown in Table I .
  • the invention further provides a method of analyzing a nucleic acid from a subject.
  • the method determines which base or bases is/are present at any one of the polymorphic vegetal sites for example of those of Table I.
  • a set of bases occupying a set of the polymorphic sites shown in Table I is determined. This type of analysis can be performed on a plurality of subjects who are tested for the presence of a phenotype. The presence or absence of phenotype can then be correlated with a base or set of bases present at the polymorphic sites in the subjects tested.
  • a nucleic acid such an oligonucleotide
  • oligonucleotide can be DNA or RNA, and single- or double-stranded.
  • Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means.
  • Preferred nucleic acids of the invention include segments of DNA, or their complements including any one of the polymorphic sites shown in Table I. The segments are usually between 5 and 100 bases, and often between 5-10, 5-20, 10-20, 10-50, 20-50 or 20-100 bases. The polymorphic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in Table I. Methods of synthesizing oligonucleotides are found in, for example, Oligonucleotide Synthesis : A Practical ApproacA (Gait, ed., IRL Press, Oxford, 1984).
  • Hybridization probes are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al . , Science 254, 1497-1500 (1991) .
  • primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides . Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template
  • a primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.
  • primer site refers to the area of the target DNA to which a primer hybridizes .
  • primer pair means a set of primers including a 5 ' upstream primer that hybridizes with the 5 ' end of the DNA sequence to be amplified and a 3 ' , downstream primer that hybridizes with the complement of the 3 ' end of the sequence to be amplified.
  • Linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome, and can be measured by percent recombination between the two genes, alleles, loci or genetic markers.
  • Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • a polymorphic locus may be a" small as one base pair.
  • Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatelli tes , dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu .
  • allelic form is arbitrarily designated as a the reference form and other allelic forms are designated as alternative or variant alleles.
  • allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms.
  • a diallelic polymorphism has two forms.
  • a triallelic polymorphism has three forms.
  • a single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in 1QSS than 1/100 or 1/1000 members of the populations) .
  • a single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site.
  • a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine or vice versa.
  • Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C
  • conditions of 5X SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4 and a temperature of 25-30°C are suitable for allele-specific probe hybridizations.
  • Nucleic acids of the invention are often in isolated form.
  • An isolated nucleic acid means an object species that is the predominant species present (i.B., on a molar basis it is more abundant than any other individual species in the composition) .
  • an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.
  • the object species is purified to essential homogeneity (contaminant specie" cannot be detected in the composition by conventional detection methods) .
  • Novel Polymorphisms of the Invention provides for example oligonucleotides containing polymorphic sequences isolated from graminae species for example maize.
  • the invention also includes various methods for using those novel oligonucleotides to identify, distinguish, and determine the relatedness of individual strains or pools of nucleic acids from plants .
  • DNA was extracted from maize lines as described in Rogers and Bendich (1988 Plant Mol Biol Manual A6 : 1- 10) with modification described in Murigneux et al (1993 theo Appl Genet 86 : 837-842) .
  • PCR amplification was done on six maize lines representing a wide range of genetic variability and including both European flint material and US dent germplasm. Those six maize lines have been choosen to maximize the genetic variability of cultivated maize. Doing so, optimize the chance of finding polymorphism in the allelic sequences. For example Gl, an european flint line and G3 , an US Corn Belt Stiff Stalk line, are completly unrelated. Their genetic distance (coefficient of dissimilarity) calculated with our standard approach (89 RFLP probe/enzyme combinations and Nei-li distance) is 0.69. This value is close to the maximum distance between two cultivated maize lines .
  • Genotypes Among the 15 genetic distance between couple of these 6 lines : 8 are superior to 0.6, 6 superior to 0.5 and only one inferior to 0.5. This shows that the choice of the lines avoided as much as it was possible the potential redudancy (or similarity) of allele at the locus sequenced. With the same effort of sequencing we should therefore have collected the maximum number of polyphomism. Genotypes :
  • the markers have been chosen with the following criteria.
  • MAP Pos map position, given by the bin location of the
  • 5.01 means that it is located in the bin 5.01 (on chromosome
  • S01R is the reverse primer for probe 1
  • Genbank/ EMBL Genbank/ EMBL number TABLE A
  • Adh2 S17 4.03 S17F2 TGCCTGCTGCATCTCTAGCC X02915 S17R2 CAAGCCCGAAAATCGCCAC X02915
  • PCR amplification was done with primer designed using the DNA sequences of the markers listed above.
  • the sequences for all markers/genes were available on Genbank/ EMBL.
  • Polymerisation was done in a perkin Elmer 9600 : 1' at 95°C, followed by 35 cycles of (30" at 94°C, 30" at 60°C, 1'30" at 72°C) followed by 1'30" at 72°C.
  • Column 1 (Bt2) represents the name of the marker or gene .
  • SNP single nucleotide polymorphism
  • Column 3 represents : similar to column 2, but with the codification of the marker/gene.
  • /G CSU61-G1/G5-1A means deletion of the base pair G in Gl compared to G5.
  • ASG12 ASG12-G1/G3-1 S6 G1/G3-1 CTGGTRGAAATGTGTTGAAG[CA]TACTAGTGATGAACTGCTTG
  • ASG12 ASG12-G1/g3-3B S6 G1/g3-3B CGCGCCGAAAAAGGAAAAAG[G ⁇ TGAAGGTCCT ⁇ ACTCACCGA
  • Sh2 Sh2-G3 G6-1 S63G3G6-1 TCTGTGATTGGAGTCTGCTC[G/A]CGTGTCAGCTCTGGATGTGA
  • CSU61 CSU61-G5A36-2B S 5G5 ⁇ 36-2B CCTAAACGCTGACCGCCACAIGAJACGGCGGCGGCTGCCAAATC
  • Wx1 Wx1-G2/G6-2B S43G2/G6-2B CTTGATTGCATTGCAGCTAC[A G]AGAAGCCCGTGGAAGGCCGG
  • Wx1 WX1-G6/G1-1 S43G6K31-1 CTCCTACCAGGGCCGGTTCG ⁇ T/ICCTTCTCCGACTACCCGGAG
  • UMC80 UMC80-G2 ⁇ 33-1 S34G2 G3-1 GAGACAT ⁇ C «TACTCV ⁇ TA( ⁇ AA ⁇ ATTTGATGAAATTATT
  • ASG 9 ASG49-G3/G5-2 S32G3 G5-2 ATGAATTGAAGCTCTGAATA[(VnAGAATCCACCATTCTTCCGA
  • ASG 9 ASG49-G3/G5-3 S32G3 G5-3 GAATCCACCATTCTTCCGAAIA GICTGCTTCCTACAAAACTCGA
  • ASG8 ASG8-G3 G4-1 S31G3 G4-1 TGTATCGGCTAGTCTGGATG[G/AJTCGCACTGGCACTCAGTGCT csnpld
  • U C 2 1 UMC21-G5/G6-1 S 2 8G5/G6-1 CAAGAAGCCTCTTCAGTGTC[A C]GTCGTAGCTTCCTCAAGACC
  • UMC21 UMC21-G5/G6-3 S28G5/G6-3 CTAATCCATGGAGCAGGGAG[G A]AAGGGGCGAGGGGCAGCAAG
  • Ae1 Ae1-G1/G6-1 S23G1/G6-1 TGATTCGGGTCTGTATGCGAtG T GTTGTGGTGGTGAACTGGT
  • Ae1 Ae1-G1/G4-1 S23G1/G4-1 GTTCGCGGTTTCTGGGGCCG[G ⁇ GGGCGGTGCTCGGTGGGGCC
  • UMC90 UMC90-G5 ⁇ 36-1 S22G5/G6-1 CAGA ⁇ GGTGTCGT ⁇ rACTAIA/GJAATTCAGTTCTGTCCATTTG
  • ASG24 ASG24-G5 G6-1 S13G5/G6-1 TTTCACAACTCAACTGATTGIANTCTTGCTTTGATGTGGATTCT
  • UMC131 UMC131-G4/G6-1 S10G4 G6-1 AGCGACAGGGATGTCGAGCA[G TTCTACGGAAGGCAATAATGAG csnpld
  • UMC131 UMC131-G3G6-1 S10G3G6-1 GCATGGCGGAGTGAGGGAGG(TG/]TGTGTGTGTGGCTCCACA
  • UMC131 UMC131-G3/G6-2B S10G3G6-2B GGCCGCTACGCCATTTAGCG[G/A1ATTTGGGAAAATCAATGCAC
  • UMC53 UMC53-G4G6-1 UMC53-G4G6-1 GCAGCCTCAGGTACACGGGG[/A]AAGTCGGAGTGGTTCTTCAC
  • UMC161 UMC161-G2G3-2 S06G2G3-2 ACGGTGAGGAGTGGCACATG[AC]GATGGAAAGTTCCTGTAGAC
  • UMC76 UMC76-G2G5-1B S02G2G5-1B TAGTTTCTCGGCAATTGGCAfG ⁇ TGTGGAATGACCATCTCGTC
  • UMC76 UMC76-G2/G5-2B S02G2G5-2B GTGTGGAATGACCATCTCGT[G/C]GTGATGCCAGCATGCTACTA
  • Target nucleic acids can be genomic or cDNA.
  • Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally PCR Technology :
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dSDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dSDNA double stranded DNA
  • A Detection of Polymorphisms in Target DNA
  • the first type of analysis is sometimes referred to as de novo characterization. This analysis compares target sequences in different individual plants to identify points of variation, i.e., polymorphic sites.
  • de novo identification of the polymorphisms of the invention is described in the Examples section.
  • the second type of analysis is determining which form(s) of a characterized polymorphism is (are) present in plants under test. There are a variety of suitable procedures, which are discussed in turn.
  • Allele-specific probes for analyzing polymorphisms is described by e.g., Saiki et al . , Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one member of a species but do not hybridize to the corresponding segment from another member due to the presence of different polymorphic forms in the respective segments from the two members . Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.
  • Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence .
  • the polymorphisms can also be identified by hybridization to nucleic acid arrays, some example of which are described by Wo 95/11995 (incorporated by reference in its entirety for all purposes) .
  • One form of such arrays is described in the Examples section in connection with de novo identification of polymorphisms.
  • the same array or a different array can be used for analysis of characterized polymorphisms.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant forms of a precharacterized polymorphism. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence.
  • the second group of probes is designed by the same principles as described in the Examples except that the probe" exhibit complementarity to the second reference sequence.
  • the inclusion of a second group (or further groups) can be particular useful for analysing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (i.e., two or more mutations within 9 to 21 bases) .
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res . 1 7 , 2427-2448 (1989) .
  • This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present.
  • a control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • the direct analysis of the sequence of polymorphisms of the present invention can bo accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al . , Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al . , Recombinant DNA Labora tory Manual , (Acad. Press, 1988) ) .
  • Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution, Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7.
  • Alleles of target sequences can be differantiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al . , Proc, Nat . Acad . Sci . 86, 2766-2770 (1989) .
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of target sequences.
  • Example 5 Methods of Use After determining polymorphic form(s) present in a subject plant at one or more polymorphic sites, this information can be used in a number of methods .
  • a genetic fingerprint for an individual strain can be made by determining the nucleic acid sequence possessed by that individual strain that corresponds to a region of the genome known to contain polymorphisms. For a discussion of genetic fingerprinting in the animal kingdom, see, for example, Stokening et.al., Am. J. Hum . Genet . 48:370-382 (1991). The probability that one or more polymorphisms in an individual strain is the same as that in any other individual strain decreases as the number of polymorphic sites is increased.
  • the comparison of the nucleic acid sequences from two strains at one or multiple polymorphic sites can also demonstrate common or disparate ancestry. Since the polymorphic sites are within a large region in the genome, the probability of recombination between these polymorphic sites is low. That low probability means the haplotype (the set of all the disclosed polymorphic sites) set forth in this application should be inherited without change for at least several generations.
  • Knowledge of plant strain or ancestry is useful, for example, in a plant breeding program or in tracing progeny of a proprietary plant.
  • Fingerprints are also used to identify an individual strain and to distinguish or determine the relatedness of one individual strain to another. Genetic fingerprinting can also be useful in hybrid certification, the certification of seed lots, and the assertion of plant breeders rights under the laws of various countries .
  • polymorphisms of the invention may contribute to the phenotype of a plant in different ways. Some polymorphisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. Other polymorphisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymorphism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymorphisms in different genes.
  • polymorphisms predispose a plant to a distinct mutation that is causally related to a certain phenotype .
  • Phenotypic traits include characteristics such as growth rate, crop yield, crop quality, resistance to pathogens, herbicides, and other toxins, nutrient requirements, resistance to high temperature, freezing, drought, requirements for light and soil type, aesthetics, and height.
  • Other phenotypic traits include susceptibility or resistance to diseases, such as plant cancers. Often polymorphisms occurring within the same gene correlate with the same phenotype.
  • Correlation is performed for a population of plants, which have been tested for the presence or absence of a phenotypic trait of interest and for polymorphic markers sets.
  • a set of polymorphisms i.e. a polymorphic set
  • the alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest.
  • Correlation can be performed by standard statistical methods such as a K-squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted.
  • Yijk n ⁇ + YSi + Pj; + X k ⁇ 13 ⁇ + ... ⁇ 17 + PE Struktur + a n +e p
  • Yij k pn is the milk, fat, fat percentage, SNF , SNF percentage, energy concentration, or lactation energy record
  • ⁇ is an overall mean
  • YSi is the effect common to all cows calving in year-season
  • X ⁇ is the effect common to cows in either the high or average selection line
  • ⁇ i to ⁇ are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms
  • PE n is permanent environmental effect common to all records of cow n
  • a n is effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect
  • e p is a random residual. It was found that eleven of seventeen polymorphisms tested influenced at least one production trait. Bovines having the best polymorphic forms
  • a DNA pool is constructed from plants of a segregating population that are resistant and another pool is constructed from plants that are sensitive to the disease.
  • Those two DNA pools are identical except for the DNA sequences at the resistance gene locus and in the surrounding genomic area. Hybridization of such DNA pools to the DNA sequences listed in Table 1 allows the simultaneous testing of several hundreds of loci for polymorphisms . Allelic polymorphism-detecting sequences that show differences in hybridization patterns between such DNA pools will represent loci linked to the disease resistance gene .
  • nucleic acid pools are constructed from several individuals of a large population.
  • the nucleic acid pools are hybridized to nucleic acids having the polymorphism-detecting sequences listed in Table I.
  • the detection of a rare hybridization profile will indicate the presence of a rare allele in a specific nucleic acid pool.
  • RNA pools are particularly suited to identify differences in gene expression.
  • the markers are used to select, in back-cross populations, the plant that have the higher percentage of recurrent parent, while still remaining the genes given by the donor plant .
  • the invention further provides variant forms of nucleic acids and corresponding proteins.
  • the nucleic acids comprise at least 10 contiguous amino acids of one of the sequences for example as described in Table I, in any of the allelic forms shown. Some nucleic acid encode full-length proteins .
  • Genes can be expressed in an expression vector in which a gene is operably linked to a native or other promoter.
  • the promoter is an eukaryotic promoter for expression in a eukaryotic cell.
  • the transcription regulation sequences typically include an heterologous promoter and U optionally an enhancer which is recognized by the host.
  • the selection of an appropriate promoter for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected.
  • Commercially available expression vectors can be used.
  • Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
  • the means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra .
  • a wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E . coli , yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof, and plant cells. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide . Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like .
  • the DNA fragments are introduced into cultured plant cells by standard methods including electroporation
  • the Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Horsch et al . , Science, 233, 496-498 (1984); Fraley et al . , Proc . Natl . Acad. Sci . USA 80, 4803 (1983)).
  • the protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymol ogy Volume 104, Academic Press, New York (1984); Sc:ope ⁇ , Protein Purification, Principles and Practice ' , 2nd Edition, Springer-Verlag, New York (1987); and Deutscher (ed) , Guide to Protein Purification ' Me thods in Enzymology, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.
  • the invention further provides transgenic plants capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated. Plant regeneration from cultural protoplasts is described in Evans et al . , "Protoplasts Isolation and Culture," Handbook of Plant Cell Cul tures 1 , 124-176 (MacMillan Publishing Co., New York, 1983); Davey, “Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, (1983) - pp. 12-29, (Birkhauser, Basal 1983); Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," Protoplasts (1983) - pp.
  • a variant gene responsible for a disease-resistant phenotype can be introduced into the plant to simulate that phenotype.
  • Expression of an exogenous variant gene is usually achieved by operably linking the qene to a promoter and optionally an enhancer.
  • Inactivation of an exogenous variant genes can be achieved by forming a transgene in which a cloned variant genes is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989) .
  • transgenic plant are useful in a variety of screening assays.
  • the transgenic plant can then be treated with compounds of interest and the effect of those compounds on the disease resistance can be monitored.
  • the transgenic plant can be exposed to a variety of environmental conditions to determine the effect of those conditions on the resistance to the disease.
  • the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides .
  • biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding.
  • Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
  • Antibodies that specifically bind to one allelic gene products but not to a second allelic gene product are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual , Cold Spring Harbor Press, New York (1988) ; Goding, Monoclonal antibodies, Principles and Practice (2d ed. ) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.
  • kits comprising at least one allele-specific oligonucleotide as described above. Often, the kits contain one or more pairs of 2? allele-specific oligonucleotides hybridizing to different forms of a polymorphism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all of the polymorphisms shown in Table I.
  • kits include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates , means used to label ( or example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin) , and the appropriate-buffers for reverse transcription, PCR, or hybridization reactions.
  • the kit also contains instructions for carrying out the methods .

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Abstract

L'invention concerne un segment d'acides nucléiques qui comprend au moins 10 nucléotides contigus et que l'on obtient à partir d'une séquence végétale comportant un site polymorphe; elle concerne également le complément de ce segment.
EP97955036A 1996-12-02 1997-12-02 Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci Withdrawn EP0944741A2 (fr)

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AU7206698A (en) * 1996-12-02 1998-08-03 Biocem S.A. Vegetal sequences including a polymorphic site and their uses
CA2349127A1 (fr) * 1998-10-06 2000-04-13 Emory University Diagnostic moleculaire de galactosemie
US6670464B1 (en) * 1998-11-17 2003-12-30 Curagen Corporation Nucleic acids containing single nucleotide polymorphisms and methods of use thereof
AU1928801A (en) * 1999-11-24 2001-06-04 Curagen Corporation Nucleic acids containing single nucleotide polymorphisms and methods of use thereof
JP2004508003A (ja) * 1999-11-30 2004-03-18 キュラジェン　コーポレイション 一ヌクレオチド多型を含む核酸およびその使用方法
CA2395786A1 (fr) * 1999-12-27 2001-07-05 Curagen Corporation Acides nucleiques contenant des polymorphismes de nucleotides simples, et procedes d'utilisation correspondants
EP1250456A2 (fr) * 2000-01-07 2002-10-23 Curagen Corporation Acides nucleiques renfermant des polymorphismes de nucleotide simple et procede d'utilisation associe
EP2147012A4 (fr) * 2007-05-17 2011-03-02 Monsanto Technology Llc Polymorphismes de maïs et procédés de génotypage
US20130244936A1 (en) 2010-06-04 2013-09-19 Vincent Goffin Constitutively active prolactin receptor variants as prognostic markers and therapeutic targets to prevent progression of hormone-dependent cancers towards hormone-independence

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CA1323553C (fr) * 1987-11-03 1993-10-26 Timothy George Helentjaris Methode et appareillage pour l'analyse amelioree du polymorphisme de la longueur des fragments de restriction
AU631562B2 (en) * 1988-02-22 1992-12-03 Pioneer Hi-Bred International, Inc. Genetic linkages between agronomically important genes and restriction fragment length polymorphisms
EP0509089A4 (en) * 1990-11-06 1993-03-10 The Lubrizol Corporation Compositions and methods for analyzing genomic variation
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
US5332408A (en) * 1992-08-13 1994-07-26 Lakeside Biotechnology, Inc. Methods and reagents for backcross breeding of plants
CZ296930B6 (cs) * 1995-07-28 2006-07-12 Sapporo Breweries Ltd. Zpusob identifikace genetických druhu chmelu
US6114116A (en) * 1996-12-02 2000-09-05 Lemieux; Bertrand Brassica polymorphisms
AU7206698A (en) * 1996-12-02 1998-08-03 Biocem S.A. Vegetal sequences including a polymorphic site and their uses
CA2286864A1 (fr) * 1997-01-10 1998-07-16 Pioneer Hi-Bred International, Inc. Amplification et analyse genetiques sur la base d'une hybridation

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CA2274317A1 (fr) 1998-07-16
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WO1998030717A3 (fr) 1999-04-29

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