WO2006099055A2 - Sequence, polymorphismes et technique par test de marquage concernant la resistance a la maladie et la croissance (nfkb1) - Google Patents

Sequence, polymorphismes et technique par test de marquage concernant la resistance a la maladie et la croissance (nfkb1) Download PDF

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WO2006099055A2
WO2006099055A2 PCT/US2006/008433 US2006008433W WO2006099055A2 WO 2006099055 A2 WO2006099055 A2 WO 2006099055A2 US 2006008433 W US2006008433 W US 2006008433W WO 2006099055 A2 WO2006099055 A2 WO 2006099055A2
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gene
sequence
seq
animal
allele
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WO2006099055A3 (fr
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Christopher K. Tuggle
Shu-Hong Zhao
Martha A. Mellencamp
John Bastiaansen
Lucina Galina-Pantoja
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Iowa State University Research Foundation, Inc.
Pig Improvement Company Uk Limited
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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
    • 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
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • TITLE SEQUENCE, POLYMORPHISMS, AND MARKER TEST TECHNOLOGY FOR DISEASE RESISTANCE AND GROWTH (NFKBl)
  • This invention relates generally to the detection of genetic differences among animals. More particularly, the invention relates to genetic markers which have been identified in several genes indicative of heritable phenotypes associated with improved traits, such as disease resistance. Methods and compositions for use of these markers in genotyping of animals and selection are also disclosed.
  • RFLP analysis has been used by several groups to study pig DNA.
  • SLA swine leukocyte antigen
  • MHC major histocompatibility complex
  • DNA markers have several advantages; segregation is easy to measure and is unambiguous, and DNA markers are co-dominant, i.e., heterozygous and homozygous animals can be distinctively identified. Once a marker system is established, selection decisions could be made very easily, since DNA markers can be assayed any time after a tissue or blood sample can be collected from the individual infant animal, or even an embryo.
  • the present invention provides a genetic markers, based upon the discovery of polymorphisms in the putative porcine NFKBl gene, which correlate with resistance or susceptibility to pathogenic infection in pigs. This will permit genetic typing of pigs for their NFKBl allele and for determination of the relationship of specific RFLPs to resistance to infection and growth traits. It will also permit the identification of individual males and females that carry the gene for improved resistance and growth. Thus, the markers may be selection tools in breeding programs to develop lines and breeds that produce litters containing more resistant offspring. Also disclosed are novel porcine NFKBl genomic sequences, as well as primers for assays to identify the presence or absence of marker alleles.
  • NFKBl gene which is associated with the improved resistance to pathogenic infection.
  • Another object of the invention is to provide a method for identifying genetic markers for improved disease resistance.
  • a further object of the invention is to provide genetic markers for selection and breeding to obtain pigs that will be expected to have a lower susceptibility to infection than those without the favorable allele.
  • a still further object of the invention is to provide genetic markers for selection and breeding to obtain pigs that will be expected to have superior growth traits than those without the favorable allele.
  • Yet another object of the invention is to provide a kit for evaluating a sample of pig DNA for specific genetic markers of disease resistance and growth traits.
  • the present invention provides a method for screening animals to determine those more likely to have beneficial phenotypes such as, or associated with, improved innate immunity, disease resistance or resistance to bacterial infection, as evidenced by one or more of the following: bacterial clearance, leukocyte base line and response to infection leukocyte function and fever response to infection, in a particular population, when bred, or raised or to select against pigs which have alleles indicating unfavorable phenotypes.
  • beneficial phenotypes such as, or associated with, improved innate immunity, disease resistance or resistance to bacterial infection, as evidenced by one or more of the following: bacterial clearance, leukocyte base line and response to infection leukocyte function and fever response to infection, in a particular population, when bred, or raised or to select against pigs which have alleles indicating unfavorable phenotypes.
  • improved or superior disease resistance or innate immunity shall mean a measurable statistically significantly difference in indicators of innate immunity or disease resistance including but not limited to lymphocyte counts, bacterial counts, temperature response to infection, monocyte counts, eosinophil counts, and PMN killing when one allele is present as compared to the same indicator when the alternate allele is present.
  • the traits are not so limited to these specific measurements.
  • the present invention provides a method for screening animals to determine those more likely to have beneficial phenotypes such as, or associated with, growth traits as evidenced by one or more of the following no limiting list of indicia of growth traits: days to 110 Kg, or life time daily gain.
  • Growth traits may be measured by any of a number of indicia and the invention is not limited to these particular traits only. The growth traits mentioned herein are beyond and separate from those which may be attributed to improved innate immunity
  • the present invention provides a method for screening pigs to determine those more likely to have the improved trait of superior disease resistance or growth and/or those less likely to demonstrate those traits which method comprises the steps: 1) obtaining a sample of tissue of genomic DNA from an animal; and 2) analyzing the mRNA or genomic DNA obtained in 1) to determine which NFKBl allele(s) is/are present. Briefly, the sample of genetic material analyzed to determine the presence or absence of a particular allele that is correlated with a desirable trait, or one which is linked thereto.
  • nucleic acid molecules for sequence differences. These include by way of example, restriction fragment length polymorphism analysis, heteroduplex analysis, single strand conformation polymorphism analysis, denaturing gradient electrophoresis and temperature gradient electrophoresis.
  • the polymorphism is a restriction fragment length polymorphism and the assay comprises identifying the gene from isolated genetic material; exposing the gene to a restriction enzyme that yields restriction fragments of the gene of varying length; separating the restriction fragments to form a restriction pattern, such as by electrophoresis or HPLC separation; and comparing the resulting restriction fragment pattern from an animal gene that is either known to have or not to have the desired marker. If an animal tests positive for the marker (or allele), such animal can be considered for inclusion in the breeding program. If the animal does not test positive for the marker genotype, the animal can be culled from the group and otherwise used.
  • the gene, or a fragment thereof is isolated by the use of primers and DNA polymerase to amplify a specific region of the gene which contains the polymorphism or a polymorphism linked thereto.
  • the amplified region is either directly separated or sequenced or is digested with a restriction enzyme and fragments are again separated. Visualization of the separated fragments, or RFLP pattern, is by simple staining of the fragments, or by labeling the primers or the nucleoside triphosphates used in amplification.
  • the invention comprises a method for identifying a genetic marker for disease resistance traits, such as bacterial counts, lymphocyte count, neutrophil count, or monocyte count after challenge or for growth traits such as average daily gain, days to 110 Kg., life time daily gain and the like.
  • a genetic marker for disease resistance traits such as bacterial counts, lymphocyte count, neutrophil count, or monocyte count after challenge or for growth traits such as average daily gain, days to 110 Kg., life time daily gain and the like.
  • Male and female animals of the same breed, breed cross, or similar genetic lineage are bred, and the growth/disease resistance traits are determined.
  • a polymorphism in the NFKBl gene of each animal is identified and associated with the desired trait(s).
  • PCR-RFLP analysis is used to determine the polymorphism.
  • NFKBl gene or the NFKBl gene it would be possible, at least in the short term, to select for pigs likely to produce disease resistance/ superior growth or alternatively, against pigs likely to produce susceptible litters/inferior growth indirectly, by selecting for certain alleles of the NFKBl associated marker or the NFKB 1 associated marker through the selection of specific alleles of alternative markers located on chromosome 8 where NFKBl is.
  • 2 different polymorphisms have been identified in the NFKBl gene which are correlated with significant differences in traits associated with disease resistance and innate immunity and/or growth.
  • the first is located in intron 18 of the putative NFKBl gene and is a C/A single nucleotide polymorphism. This polymorphism is expected to show significant differences with respect to innate immunity and disease resistance and may thus be used by a breeder to identify animals which are more likely to demonstrate superior disease resistance or innate immunity compared to an animal which has the alternative gene form.
  • the second is located in the exon 24 of the putative NFKBl gene and is a C/T single nucleotide polymorphism.
  • This polymorphism has shown significant differences with respect to innate host defense including fever response to infection, and bacterial clearance and may thus be used by a breeder to identify animals which are more likely to exhibit superior disease resistance or innate immunity when compared to an animal which has the alternative gene form.
  • the exon 24 SNP has also been shown to be significantly correlated with growth traits. Animals with the favorable allele were shown to have fewer days to reach 110 Kg., and a higher life time daily gain than animals without the favorable allele.
  • Figure 1 shows a combination of the novel porcine NFKBl partial cDNA sequence, intron 18, which has been placed at its correct position between exon 18 and exon 19 to show SNP 1 in the context of the cDNA sequence, and the 3' untranslated region, including exon 24 which encompasses SNP 2 of the invention.
  • Intron 18 is shown in blue text from position 2074 to position 2397 and is 324 base pairs long.
  • SNP 1, located in intron 18 is located at position 2089 of SEQ ID NO: 1, and is designated as M (C or A) as the difference between alleles is at this position.
  • Primers and SNP 2 are highlighted in red text.
  • the SNP 2 is located at position 3082 of SEQ ID NO:1, and is designated as Y (C or T) as the difference between alleles is at this position.
  • Figure 2 is the sequence amplified from primers SEQ ID NO: 2 and 3. Primers are bold italic underlined, the C or A polymorphism is indicated in red text as M (amplified sequence for allele 1 with C nucleotide is SEQ ID NO:8).
  • Figure 3 is the sequence amplified from primers SEQ ID NO: 4 and 5. Primers are bold italic underlined, the C or T polymorphism is indicated in red text as Y (amplified sequence for allele 1 with C nucleotide is SEQ ID NO: 9).
  • Figure 4 is the partial cDNA sequence for porcine NFKBl of 3,314 base pairs (SEQ ID NO: 10). Also shown is the complete open reading frame predicted from SEQ ID NO: 10, and the protein sequence derived (SEQ ID NO: 11). This protein sequence was compared to the known human NFKBl sequence (Genbank Accession number: NM_003998) and our novel sequence (SEQ ID NO: 11) is 92% identical to this sequence.
  • the 3' untranslated region is from position 2,877 (position 2878-2880 is a TAG stop codon) to position 3,314.
  • NFKB nuclear factor kappa-B
  • TNF- ⁇ tumor necrosis factor alpha
  • LPS bacterial lipopolysaccharide
  • NFKBl functions both as an 1KB in its longer precursor form, pl05, and as an active transcription factor, p50, after proteolytic processing by the proteasome, which removes the IKB-like C-terminal half of pi 05. Processing to p50 occurs in a constitutive, unregulated fashion. However, following cellular stimulation with ligands, such as TNF- ⁇ , two serines in the pl05 PEST domain are rapidly phosphorylated by the 1KB kinase (IKK) complex, triggering complete pl05 degradation with little effect on processing to p50. This degradation results in the release of associated p50 and other NFKB subunits, which can then translocate into the nucleus.
  • ligands such as TNF- ⁇
  • the invention relates to the identification of quantitative trait loci (QTL) for improved disease resistance or resistance to bacterial infection and/or growth traits, including, but not limited to, Salmonellosis, identifiable by traits such as bacterial count, lymphocyte count, neutrophil count, monocyte count post challenge, days to 110 Kg, or life time daily gain, to identify pigs and other animals which have superior bacterial killing or ability to stave off infection and/or growth.
  • QTL quantitative trait loci
  • Salmonellosis identifiable by traits such as bacterial count, lymphocyte count, neutrophil count, monocyte count post challenge, days to 110 Kg, or life time daily gain, to identify pigs and other animals which have superior bacterial killing or ability to stave off infection and/or growth.
  • the invention also relates to a method of screening animals to determine those more likely to have improved resistance/growth traits when bred by identifying the presence or an absence of a polymorphism in certain genes (NFKBl) that are correlated with these traits.
  • NFKBl polymorphism in certain genes
  • the invention relates to genetic markers and methods of identifying those markers in a pig or other animal of a particular breed, strain, population, or group, whereby an animal is more likely to have disease resistance/growth that is superior to that of an animal which has a different allele present as well as for the mean for that particular breed, strain, population, or group.
  • the marker may be identified by any method known to one of ordinary skill in the art which identifies the presence or absence of the particular allele or marker, including, for example, direct sequencing single-strand conformation polymorphism analysis (SSCP), base excision sequence scanning (BESS), RFLP analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, allelic PCR, temperature gradient electrophoresis, ligase chain reaction, direct sequencing, minisequencing, nucleic acid hybridization, and micro-array-type detection of the NFKBl gene, or other linked sequences, and examination for a polymorphic site.
  • SSCP single-strand conformation polymorphism analysis
  • BESS base excision sequence scanning
  • RFLP analysis heteroduplex analysis
  • denaturing gradient gel electrophoresis allelic PCR
  • temperature gradient electrophoresis ligase chain reaction
  • direct sequencing minisequencing
  • nucleic acid hybridization and micro-array-type detection of the NFKBl gene, or other linked sequence
  • Samples of genomic DNA are isolated from any convenient source including saliva, buccal cells, hair roots, blood, cord blood, amniotic fluid, interstitial fluid, peritoneal fluid, chorionic villus, and any other suitable cell or tissue sample with intact interphase nuclei or metaphase cells.
  • the cells can be obtained from solid tissue as from a fresh or preserved organ or from a tissue sample or biopsy.
  • the sample can contain compounds which are not naturally intermixed with the biological material such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • Genomic DNA can also be isolated from cultured primary or secondary cell cultures or from transformed cell lines derived from any of the aforementioned tissue samples.
  • RNA can be isolated from tissues expressing the NFKBl gene as described in Sambrook et al., supra.
  • RNA can be total cellular RNA, rnRNA, poly A+ RNA, or any combination thereof.
  • the RNA is purified, but can also be unpurified cytoplasmic RNA.
  • RNA can be reverse transcribed to form DNA which is then used as the amplification template, such that the PCR indirectly amplifies a specific population of RNA transcripts. See, e.g., Sambrook, supra, Kawasaki et al., Chapter 8 in PCR Technology, (1992) supra, and Berg et al., Hum. Genet. 85:655-658 (1990).
  • PCR polymerase chain reaction
  • Tissues should be roughly minced using a sterile, disposable scalpel and a sterile needle (or two scalpels) in a 5 mm Petri dish. Procedures for removing paraffin from tissue sections are described in a variety of specialized handbooks well known to those skilled in the art.
  • telomere sequence To amplify a target nucleic acid sequence in a sample by PCR, the sequence must be accessible to the components of the amplification system.
  • One method of isolating target DNA is crude extraction which is useful for relatively large samples. Briefly, mononuclear cells from samples of blood, amniocytes from amniotic fluid, cultured chorionic villus cells, or the like are isolated by layering on sterile Ficoll-Hypaque gradient by standard procedures. Interphase cells are collected and washed three times in sterile phosphate buffered saline before DNA extraction.
  • the cells are resuspended (10 6 nucleated cells per 100 ⁇ l) in a buffer of 50 mM Tris- HCl (pH 8.3), 50 mM KCl 1.5 mM MgCl 2 , 0.5% Tween 20, 0.5% NP40 supplemented with 100 ⁇ g/ml of proteinase K. After incubating at 56°C for 2 hr. the cells are heated to 95°C for 10 min to inactivate the proteinase K and immediately moved to wet ice (snap-cool). If gross aggregates are present, another cycle of digestion in the same buffer should be undertaken. Ten ⁇ l of this extract is used for amplification.
  • the amount of the above mentioned buffer with proteinase K may vary according to the size of the tissue sample.
  • the extract is incubated for 4-10 hrs at 50°-60°C and then at 95 0 C for 10 minutes to inactivate the proteinase. During longer incubations, fresh proteinase K should be added after about 4 hr at the original concentration.
  • PCR can be employed to amplify target regions in very small numbers of cells (1000-5000) derived from individual colonies from bone marrow and peripheral blood cultures.
  • the cells in the sample are suspended in 20 ⁇ l of PCR lysis buffer (10 mM Tris- HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20) and frozen until use.
  • PCR When PCR is to be performed, 0.6 ⁇ l of proteinase K (2 mg/ml) is added to the cells in the PCR lysis buffer. The sample is then heated to about 60°C and incubated for 1 hr. Digestion is stopped through inactivation of the proteinase K by heating the samples to 95 0 C for 10 min and then cooling on ice.
  • a relatively easy procedure for extracting DNA for PCR is a salting out procedure adapted from the method described by Miller et al., Nucleic Acids Res. 16:1215 (1988), which is incorporated herein by reference.
  • Mononuclear cells are separated on a Ficoll- Hypaque gradient. The cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM Na 2 EDTA, pH 8.2). Fifty ⁇ l of a 20 mg/ml solution of proteinase K and 150 ⁇ l of a 20% SDS solution are added to the cells and then incubated at 37°C overnight. Rocking the tubes during incubation will improve the digestion of the sample.
  • Kits for the extraction of high-molecular weight DNA for PCR include a Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, Lid.), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher & Schuell, Keene, N.H.), DNA Extraction Kit (Stratagene, LaJoIIa, Calif.), TurboGen Isolation Kit (Invitrogen, San Diego, Calif.), and the like. Use of these kits according to the manufacturer's instructions is generally acceptable for purification of DNA prior to practicing the methods of the present invention.
  • the concentration and purity of the extracted DNA can be determined by spectrophotometric analysis of the absorbance of a diluted aliquot at 260 nm and 280 nm.
  • PCR amplification may proceed.
  • the first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands.
  • the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer.
  • the cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
  • strand separation is achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase (see U.S. Pat. No. 4,965,188, incorporated herein by reference).
  • Typical heat denaturation involves temperatures ranging from about 80°C to 105°C for times ranging from seconds to minutes.
  • Strand separation can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means.
  • Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity.
  • the enzyme RecA has helicase activity in the presence of ATP.
  • reaction conditions suitable for strand separation by helicases are known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of which is incorporated herein by reference.
  • Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering systems.
  • Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis.
  • the target regions may encode at least a portion of a protein expressed by the cell.
  • mRNA may be used for amplification of the target region.
  • PCR can be used to generate a cDNA library from RNA for further amplification, the initial template for primer extension is RNA.
  • Polymerizing agents suitable for synthesizing a complementary, copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase (RT), such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or Thermus thermophilus (Tth) DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer Cetus, Inc.
  • RT reverse transcriptase
  • Tth Thermus thermophilus
  • the genomic RNA template is heat degraded during the first denaturation step after the initial reverse transcription step leaving only DNA template.
  • Suitable polymerases for use with a DNA template include, for example, E.
  • coli DNA polymerase I or its Klenow fragment T4 DNA polymerase, Tth polymerase, and Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus and commercially available from Perkin Elmer Cetus, Inc.
  • the latter enzyme is widely used in the amplification and sequencing of nucleic acids.
  • the reaction conditions for using Taq polymerase are known in the art and are described in GeIf and, 1989, PCR Technology, supra. Allele Specific PCR
  • Allele-specific PCR differentiates between target regions differing in the presence of absence of a variation or polymorphism. PCR amplification primers are chosen which bind only to certain alleles of the target sequence. This method is described by Gibbs, Nucleic Acid Res. 17:12427-2448 (1989). Allele Specific Oligonucleotide Screening Methods
  • Oligonucleotide (ASO) screening methods employ the allele-specific oligonucleotide (ASO) screening methods, as described by Saiki et al., Nature 324:163-166 (1986). Oligonucleotides with one or more base pair mismatches are generated for any particular allele. ASO screening methods detect mismatches between variant target genomic or PCR amplified DNA and non-mutant oligonucleotides, showing decreased binding of the oligonucleotide relative to a mutant oligonucleotide. Oligonucleotide probes can be designed that under low stringency will bind to both polymorphic forms of the allele, but which at high stringency, bind to the allele to which they correspond.
  • ASO allele-specific oligonucleotide
  • stringency conditions can be devised in which an essentially binary response is obtained, i.e., an ASO corresponding to a variant form of the target gene will hybridize to that allele, and not to the wildtype allele.
  • Target regions of a test subject's DNA can be compared with target regions in unaffected and affected family members by ligase-mediated allele detection.
  • Ligase may also be used to detect point mutations in the ligation amplification reaction described in Wu et al., Genomics 4:560-569 (1989).
  • the ligation amplification reaction (LAR) utilizes amplification of specific DNA sequence using sequential rounds of template dependent ligation as described in Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189-193 (1990).
  • 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.
  • DNA molecules melt in segments, termed melting domains, under conditions of increased temperature or denaturation. Each melting domain melts cooperatively at a distinct, base-specific melting temperature (TM). Melting domains are at least 20 base pairs in length, and may be up to several hundred base pairs in length.
  • a target region to be analyzed by denaturing gradient gel electrophoresis is amplified using PCR primers flanking the target region.
  • the amplified PCR product is applied to a polyacrylamide gel with a linear denaturing gradient as described in Myers et al., Meth. Enzymol. 155:501-527 (1986), and Myers et al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95-139 (1988), the contents of which are hereby incorporated by reference.
  • the electrophoresis system is maintained at a temperature slightly below the Tm of the melting domains of the target sequences.
  • the target sequences may be initially attached to a stretch of GC nucleotides, te ⁇ ried a GC clamp, as described in Chapter 7 of Erlich, supra.
  • a GC clamp Preferably, at least 80% of the nucleotides in the GC clamp are either guanine or cytosine.
  • the GC clamp is at least 30 bases long. This method is particularly suited to target sequences with high Tm's.
  • the target region is amplified by the polymerase chain reaction as described above.
  • One of the oligonucleotide PCR primers carries at its 5' end, the GC clamp region, at least 30 bases of the GC rich sequence, which is incorporated into the 5' end of the target region during amplification.
  • the resulting amplified target region is run on an electrophoresis gel under denaturing gradient conditions as described above. DNA fragments differing by a single base change will migrate through the gel to different positions, which may be visualized by ethidium bromide staining.
  • Temperature gradient gel electrophoresis is based on the same underlying principles as denaturing gradient gel electrophoresis, except the denaturing gradient is produced by differences in temperature instead of differences in the concentration of a chemical denaturant.
  • Standard TGGE utilizes an electrophoresis apparatus with a temperature gradient running along the electrophoresis path. As samples migrate through a gel with a uniform concentration of a chemical denaturant, they encounter increasing temperatures.
  • An alternative method of TGGE, temporal temperature gradient gel electrophoresis uses a steadily increasing temperature of the entire electrophoresis gel to achieve the same result. As the samples migrate through the gel the temperature of the entire gel increases, leading the samples to encounter increasing temperature as they migrate through the gel. Preparation of samples, including PCR amplification with incorporation of a GC clamp, and visualization of products are the same as for denaturing gradient gel electrophoresis.
  • Target sequences or alleles at the NFKBl locus can be differentiated 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. 85:2766-2770 (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.
  • electrophoretic mobility of single-stranded amplification products can detect base-sequence difference between alleles or target sequences.
  • heterohybrid means a DNA duplex strand comprising one strand of DNA from one person, usually the patient, and a second DNA strand from another person, usually an affected or unaffected family member. Positive selection for heterohybrids free of mismatches allows determination of small insertions, deletions or other polymorphisms that may be associated with alterations in androgen metabolism.
  • Hybridization probes are generally oligonucleotides which bind through complementary base pairing to all or part of a target nucleic acid. Probes typically bind target sequences lacking complete complementarity with the probe sequence depending on the stringency of the hybridization conditions.
  • the probes are preferably labeled directly or indirectly, such that by assaying for the presence or absence of the probe, one can detect the presence or absence of the target sequence. Direct labeling methods include radioisotope labeling, such as with 32P or 35S.
  • Indirect labeling methods include fluorescent tags, biotin complexes which may be bound to avidin or streptavidin, or peptide or protein tags.
  • Visual detection methods include photoluminescents, Texas red, rhodamine and its derivatives, red leuco dye and e, e', 5, 5'-5354amethylbenzidine (TMB), fluorescein, and its derivatives, dansyl, umbelliferone and the like or with horse radish peroxidase, alkaline phosphatase and the like.
  • Hybridization probes include any nucleotide sequence capable of hybridizing to the porcine chromosome where NFKBl resides, and thus defining a genetic marker linked to NFKBl, including a restriction fragment length polymorphism, a hypervariable region, repetitive element, or a variable number tandem repeat.
  • Hybridization probes can be any gene or a suitable analog.
  • Further suitable hybridization probes include exon fragments or portions of cDNAs or genes known to map to the relevant region of the chromosome.
  • Preferred tandem repeat hybridization probes for use according to the present invention are those that recognize a small number of fragments at a specific locus at high stringency hybridization conditions, or that recognize a larger number of fragments at that locus when the stringency conditions are lowered.
  • One or more additional restriction enzymes and/or probes and/or primers can be used. Additional enzymes, constructed probes, and primers can be determined by routine experimentation by those of ordinary skill in the art and are intended to be within the scope of the invention.
  • the methods described herein may be in terms of the use of a single restriction enzyme and a single set of primers, the methods are not so limited.
  • One or more additional restriction enzymes and/or probes and/or primers can be used, if desired. Additional enzymes, constructed probes and primers can be determined through routine experimentation, combined with the teachings provided and incorporated herein.
  • polymorphisms in the NFBKl gene have been identified which have an association with disease resistance and overall innate immunity as well as growth.
  • the presence or absence of the markers in one embodiment may be assayed by PCR-RFLP analysis using the restriction endonucleases and amplification primers may be designed using analogous human, pig or other NFBKl sequences due to the high homology in the region surrounding the polymorphisms, or may be designed using known NFBKl gene sequence data as exemplified in GenBank or even designed from sequences obtained from linkage data from closely surrounding genes based upon the teachings and references herein.
  • sequences surrounding the polymorphism will facilitate the development of alternate PCR tests in which a primer of about 4-30 contiguous bases taken from the sequence immediately adjacent to the polymorphism is used in connection with a polymerase chain reaction to greatly amplify the region before treatment with the desired restriction enzyme.
  • the novel sequence disclosed herein, SEQ ID NO: 1 may be useful in this regard as well.
  • the primers need not be the exact complement; substantially equivalent sequences are acceptable.
  • the design of primers for amplification by PCR is known to those of skill in the art and is discussed in detail in Ausubel (ed.), Short Protocols in Molecular Biology, 4th Edition, John Wiley and Sons (1999).
  • primer design The following is a brief description of primer design.
  • PCR polymerase chain reaction
  • Designing oligonucleotides for use as either sequencing or PCR primers requires selection of an appropriate sequence that specifically recognizes the target, and then testing the sequence to eliminate the possibility that the oligonucleotide will have a stable secondary structure. Inverted repeats in the sequence can be identified using a repeat-identification or RNA-folding program such as those described above. If a possible stem structure is observed, the sequence of the primer can be shifted a few nucleotides in either direction to minimize the predicted secondary structure.
  • the sequence of the oligonucleotide should also be compared with the sequences of both strands of the appropriate vector and insert DNA. Obviously, a sequencing primer should only have a single match to the target DNA.
  • primer sequence should be compared to the sequences in the GenBank database to determine if any significant matches occur. If the oligonucleotide sequence is present in any known DNA sequence or, more importantly, in any known repetitive elements, the primer sequence should be changed.
  • the methods and materials of the invention may also be used more generally to evaluate pig DNA, genetically type individual pigs, and detect genetic differences in pigs.
  • a sample of pig genomic DNA may be evaluated by reference to one or more controls to determine if a polymorphism in the NFBKl gene is present.
  • RFLP analysis is performed with respect to the pig NFBKl gene, and the results are compared with a control.
  • the control is the result of a RFLP analysis of the pig NFBKl gene of a different pig where the polymorphism(s) of the pig NFBKlgene is/are known.
  • the NFBKl genotype of a pig may be determined by obtaining a sample of its genomic DNA, conducting RFLP analysis of the NFBKl gene in the DNA, and comparing the results with a control. Again, the control is the result of RFLP analysis of the NFBKl gene of a different pig. The results genetically type the pig by specifying the polymorphism(s) in its NFBKl genes. Finally, genetic differences among pigs can be detected by obtaining samples of the genomic DNA from at least two pigs, identifying the presence or absence of a polymorphism in the NFBKl gene, and comparing the results.
  • assays are useful for identifying the genetic markers relating to disease resistance and/or innate immunity, as discussed above, for identifying other polymorphisms in the NFBKl gene and for the general scientific analysis of pig genotypes and phenotypes.
  • the examples and methods herein disclose certain gene(s) which has been identified to have a polymorphism(s) which is associated either positively or negatively with a beneficial trait that will have an effect on disease resistance or innate immunity for animals carrying this alternate allele.
  • the identification of the existence of a polymorphism within a gene is often made by a single base alternative that results in a restriction site in certain allelic forms.
  • a certain allele may have a number of base changes associated with it that could be assayed for which are indicative of the same polymorphism (allele).
  • genes may be linked to the polymorphisms disclosed herein so that assays may involve identification of other genes or gene fragments, but which ultimately rely upon genetic characterization of animals for the same polymorphism. Any assay which sorts and identifies animals based upon the allelic differences disclosed herein are intended to be included within the scope of this invention.
  • One of skill in the art once a polymorphism has been identified and a correlation to a particular trait established will understand that there are many ways to genotype animals for this polymorphism. The design of such alternative tests merely represents optimization of parameters known to those of skill in the art and is intended to be within the scope of this invention as fully described herein.
  • genes for genes are determined as follows. Male and female animals of the same breed or breed cross or derived from similar genetic lineages are mated. The offspring with the beneficial trait are determined. RPLP analysis of the parental DNA is conducted as discussed above in order to determine polymorphisms in the selected gene of each animal. The polymorphisms are associated with the traits.
  • amplification primers may be designed using analogous human or other closely related animal known sequences. The sequences of many of the genes have high homology. Primers may also be designed using known gene sequences as exemplified in Genbank or even designed from sequences obtained from linkage data from closely surrounding genes. According to the invention, the gene here was identified using primers designed from mouse and human sequences. According to the invention, sets of primers have been selected which identify regions in polymorphic genes. The polymorphic fragments have been shown to be alleles, and each was shown to be associated with beneficial traits, such as disease resistance, for various breeds. Often genotype associated with this trait alternates for different breeds.
  • the methods and materials of the invention may also be used more generally to evaluate animal DNA, genetically type individual animals, and detect genetic differences in animals.
  • a sample of genomic DNA may be evaluated by reference to one or more controls to determine which allele of the gene is present.
  • RFLP analysis is performed with respect to the gene, and the results are compared with a control.
  • the control is the result of a RFLP analysis of the gene of a different animal where the polymorphism of the gene is known.
  • the genotype of an animal may be determined by obtaining a sample of its mRNA or genomic DNA, conducting RFLP analysis of the gene in the DNA, and comparing the results with a control. Again, the control is the result of RPLP analysis of the same gene of a different animal.
  • the results genetically type the pig by specifying the polymorphism in its selected gene.
  • genetic differences among animals can be detected by obtaining samples of the mRNA or genomic DNA from at least two animals, identifying the presence or absence of a polymorphism in the gene, and comparing the results.
  • assays are useful for identifying the genetic markers relating to disease resistance, as discussed above, for identifying other polymorphisms in the gene that may be correlated with other characteristics, and for the general scientific analysis of genotypes and phenotypes.
  • the genetic markers, methods, and novel sequence of the invention are also useful in a breeding program to improve disease resistance in a breed, line, or population of animals. Continuous selection and breeding of animals that are at least heterozygous and preferably homozygous for a polymorphism associated with a beneficial trait such as disease resistance would lead to a breed, line, or population having higher numbers of offspring in each litter of the females of this breed or line. Thus, the markers are selection tools.
  • the examples and methods herein disclose certain genes which have been identified to have a polymorphism which is associated either positively or negatively with a beneficial trait that will have an effect on disease resistance of that animal.
  • the identification of the existence of a polymorphism within a gene is often made by a single base alternative that results in a restriction site in certain allelic forms.
  • a certain allele may have a number of base changes associated with it that could be assayed for which are indicative of the same polymorphism.
  • other genetic markers or genes may be linked to the polymorphisms disclosed herein so that assays may involve identification of other genes or gene fragments, but which ultimately rely upon genetic characterization of animals for the same polymorphism. Any assay which sorts and identifies animals based upon the allelic differences disclosed herein are intended to be included within the scope of this invention.
  • NFKBl different marker systems have been identified in the porcine NFKBl gene which are associated with different resistance to pathogens as well as growth.
  • Novel NFKBl sequence has identified as well as exemplary primers for assaying for the polymorphisms.
  • the markers are associated with innate immunity traits which are non pathogen specific, so while bacteria is used for challenge it is expected that these traits will improve animal health against a wide variety of diseases or challenges such as stress, viral pathogen, etc.
  • novel primer sequences those of skill in the art will readily be able to design a multitude of alternative primers to amplify the region of the polymorphism using the sequence data already known in the art and are available in sources such as Genbank and disclosed herein.
  • NFKB 1 different polymorphisms have been identified in the NFKB 1 gene which are postulated to be correlated with significant differences in traits associated with disease resistance and innate immunity as well as growth.
  • the first is located in intron 18 of the NFKBl gene and is a C/A single nucleotide polymorphism.
  • This polymorphism is postulated to show significant differences with respect to innate immunity and disease resistance and may thus be used by a breeder to identify animals which are more likely to demonstrate superior disease resistance or innate immunity compared to an animal which has the alternative gene form.
  • Novel primers have been designed to amplify this region SEQ ID NO: 2 and 3, and the identification of which allele is present can be assayed by use of a Tsp 5091 restriction enzyme. Allele 1 is the no-cut and allele 2 is the cut.
  • the second is located in the exon 24 of NFKBl gene and is a C/T single nucleotide polymorphism.
  • This polymorphism has shown significant differences with respect to associated with, improved innate immunity, disease resistance or resistance to bacterial infection, as evidenced by bacterial clearance (ileocecal lymph node count, fecal bacteria counts challenge, % lymphocytes after challenge, % monocytes after challenge, %PMN after challenge, % eosinophils , including leukocyte function and fever response to infection.
  • This exon 24 polymorphism has also been shown to be significantly correlated with overall growth including days to 110 Kg, and life time daily gain.
  • This marker may thus be used by a breeder to identify animals which are more likely to exhibit superior disease resistance or innate immunity and or growth when compared to an animal which has the alternative gene form.
  • Novel primers have been designed to amplify this region SEQ ID NO: 4 and 5, and the identification of which allele is present can be assayed by use of a Fnu4Hl restriction enzyme. Allele 1 is the cut and allele 2 is no cut.
  • the traits of disease resistance and innate immunity may be measured by a number of different indicators.
  • the indicators are associated with bacterial clearance, innate host defense, fever response to infection, and leukocyte function. It is expected that different indicators may be used to identify the same associations disclosed herein and these are intended to be within the scope of the invention.
  • Such indicators may include but are not limited to: % Lymphocytes at various days, % Lymphocytes difference % PMN, % PMN difference % Monocytes day, % Monocytes difference % Eosinophils, % Eosinophils difference, Lymphocyte SI y with Sal heat extract day -2 (pre-challenge, Lymphocyte SI with Sal heat extract day 7 (post-challenge); PMN uptake (number of bacteria/PMN), PMN killing (%), temperature at each particular day am or pm, Ileocecal lymph node bacterial count (loglO colony-forming units/gram tissue), Fecal bacterial counts, Total white blood cell count before challenge, Total white blood cell count after challenge, Difference in total white blood cell count before and after challenge, Lymphocyte count before challenge, Lymphocyte count after challenge, Difference in lymphocyte count before and after challenge, Monocyte count before challenge, Monocyte count after challenge, Difference in monocyte count before and after challenge.
  • the traits of overall growth may be measured by a number of different indicators such as average daily gain, overall weight gain, life time daily gain, days to 110 Kg and the like.
  • the use of one particular indicator is not critical to the invention and it is expected that many indicia exist for identifying the same correlation.
  • the description herein of a particular indicator of the general traits of disease resistance and growth shall not be limited to that particular trait measurement only, but shall be interpreted to include any similar measurement that evidences the same effect on disease resistance/growth.
  • Partial cDNA sequence and exon 24 sequence of NFKBl were obtained by PCR amplification using primers designed from the conserved region between human and mouse NFKBl sequence.
  • the sequence comparison from animals of Yorkshire, Meishan and a commercial population revealed polymorphisms, 1 in intron 18 and 1 in exon 24. Two of them were used to detect segregation in Salmonella challenge studies.
  • the porcine NFKBl was mapped to Chromosome 8 q23-27 by testing a somatic cell hybrid panel and PigMaP family (Archibald et al., 1995).
  • Cycle profile 94C 3 min, 40 x (94C 45s, 63C 45s, 72C 45s), 72C 5min
  • PCR- Annotation Amplification was performed using 25 ng of genomic DNA and 0.3 ⁇ M of each primer in a 10 ⁇ l of reaction volume.
  • Cycle profile 94C 3 min, 40 x (94C 45s, 55C 45s, 72C 45s), 72C 5min
  • PCR- Annotation Amplification was performed using 25 ng of genomic DNA and 0.3 ⁇ M of each primer in a 10 ⁇ l of reaction volume.
  • SNPl PCR product size is 427bp
  • SNP2 PCR product size is 281bp
  • SNP2 Fnu4HI digestion was used to reveal allele 1 and 2 :
  • NFKBl was physically mapped on the porcine chromosome 8 q21-q23 by using pig/rodent somatic cell hybrid panel (SCHP) comprising 27 cell lines.
  • the primers used to map the porcine NFKBl are:
  • Fragment 2 (partial sequence of exon 24) is disclosed in Figure 3
  • Phenotypic and genotype data were available on 154 challenged animals from PIC lines (Tables 3a-3c). Phenotypic data that were measured quantitatively, were tested with SAS proc Univariate for deviations from normality. Log transformed data were used for the FMPND6 trait since the range of values was so large; this is a standard approach with this type of data structure.
  • Statistical association is defined as a comparison between phenotype and genotype that show a P value less that 0.1; i.e., a statistical term that indicates the declared statistically significant difference between classes being compared has a confidence of greater than 90% of being correct.
  • Several associations have even higher levels of confidence, such as P values less than 0.05 or even less than 0.01 (see individual Results in Tables 3a-3b below).
  • a negative value for the "allele contrasts" means that the allele that is mentioned first (for example, allele 2 in the 2-3 contrast) is associated with a smaller value than the allele mentioned second.
  • Table 2 shows the frequencies of the Tsp5091 andFnu4Hl alleles in all challenge populations used for the trait associations.
  • Tables 3a-3c show the trait associations for Salmonellosis and innate immunity for the Fnu4Hl polymorphism in combined and individual lines for the NADC challenges.
  • Table 4 shows association data for the Fnu4Hl polymorphism in the Example 3 Compton families experiment..
  • results are the product of evaluation of the NFKBl Exon 24 marker using a sire model and single marker analysis.
  • the growth traits evaluated were: days to 110 Kg and Lifetime daily gain (Table 5).
  • the probabilities are the significance of the genotypic effect from zero.
  • the brown cells are those effects with the greatest significance.
  • Four commercial lines were tested: Line A, Line B, Line C and Line D. The number of genotypes done for each line varied from 707- 790 pigs and the effects were evaluated on all available animals.

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Abstract

L'invention concerne une méthode permettant de déterminer une résistance à la maladie ou une croissance meilleures chez les animaux. Cette méthode passe par des essais propres à faire apparaître des différences génétiques dans le gène NFKB1 de l'animal, lequel gène est associé à une résistance supérieure à la maladie et à des caractéristiques de croissance globalement plus favorables. Cette invention concerne des séquences, des essais et des compositions permettant d'identifier la présence ou l'absence de ces allèles.
PCT/US2006/008433 2005-03-09 2006-03-09 Sequence, polymorphismes et technique par test de marquage concernant la resistance a la maladie et la croissance (nfkb1) WO2006099055A2 (fr)

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Citations (1)

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US20030219427A1 (en) * 1998-08-18 2003-11-27 Allen Hamish J. TPL-2/COT kinase and methods of use

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Publication number Priority date Publication date Assignee Title
US20030219427A1 (en) * 1998-08-18 2003-11-27 Allen Hamish J. TPL-2/COT kinase and methods of use

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DATABASE NCBI [Online] 09 July 2004 OGUMA K. ET AL., XP003008459 Database accession no. (AB183419) *

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