EP1141390A1 - Proteine de liaison 4 de retinol comme marqueur genetique associe a une amelioration des naissances par accouchement - Google Patents

Proteine de liaison 4 de retinol comme marqueur genetique associe a une amelioration des naissances par accouchement

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Publication number
EP1141390A1
EP1141390A1 EP99902277A EP99902277A EP1141390A1 EP 1141390 A1 EP1141390 A1 EP 1141390A1 EP 99902277 A EP99902277 A EP 99902277A EP 99902277 A EP99902277 A EP 99902277A EP 1141390 A1 EP1141390 A1 EP 1141390A1
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EP
European Patent Office
Prior art keywords
gene
polymorphism
reproductive
fragments
binding protein
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.)
Withdrawn
Application number
EP99902277A
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German (de)
English (en)
Inventor
Max F. Rothschild
Christopher K. Tuggle
Lori A. Messer
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University of Iowa Research Foundation UIRF
Iowa State University Research Foundation ISURF
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University of Iowa Research Foundation UIRF
Iowa State University Research Foundation ISURF
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Application filed by University of Iowa Research Foundation UIRF, Iowa State University Research Foundation ISURF filed Critical University of Iowa Research Foundation UIRF
Publication of EP1141390A1 publication Critical patent/EP1141390A1/fr
Withdrawn legal-status Critical Current

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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
    • 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

  • This invention relates generally to the detection of genetic differences for reproductive efficiency among animals. More particularly the invention relates to genetic markers which have been identified in several genes indicative of heritable phenotypes associated with improved reproductive traits. Methods and compositions for use of these markers in genotyping of animals and selection are also disclosed.
  • Reproductive efficiency is the major limiting factor in the efficient production of pork as well as most other livestock animals.
  • Genetic variability exists for several reproductive measures. Average litter size among breeds pigs varies from 4-16 pigs per litter. Mean age at puberty varies from 3 to 7 months of age. This genetic variability within breeds suggests that genetic improvement in reproduction is possible. The number of pigs born alive in the United States averages approximately 9.5 pigs per litter. Heritability for litter size is low (10%-15%), and standard genetic methods of selecting breeding females on the basis of past litter size have not been effective. Therefore, there is a need for an approach that deals with selection for reproductive traits at the cellular or DNA level.
  • SLA swine leukocyte antigen
  • MHC major histocompatibility complex
  • Jung et al. Animal Genetics, 26:79-91 (1989), incorporated herein by reference reports on RFLP analysis of SLA Class I genes in certain boars.
  • United States Patent 5,550,024 to Rothschild et al. discloses a polymorphism in the pig estrogen receptor gene which is associated with larger litter size, the disclosure of which is incorporated herein by reference.
  • Another pig hormone related to beneficial reproductive traits is Prolactin.
  • Prolactin (PRL) is an anterior pituitary peptide hormone involved in many different endocrine activities, but is essential for reproductive success.
  • PRL Prolactin
  • PRL is an anterior pituitary peptide hormone involved in many different endocrine activities, but is essential for reproductive success.
  • Use of polymorphic loci in the prolactin receptor gene as markers for increased litter size is described and disclosed in United States Patent Application Serial No. 08/812,208, the disclosure of which is hereby incorporated by reference.
  • the present invention provides genetic markers, based upon the discovery of polymorphisms in the RBP4 gene, which relate to improved reproductive traits such as litter size and number born alive. This will permit genetic typing of pigs for their reproductive genes and for determination of the relationship of specific genes and markers to reproductive traits. It will also permit the identification of individual males and females that carry beneficial genotypes. In the case of females it would permit that a female would be expected to produce a litter size larger than the average earlier than average or healthier than average for their breed, or in the case of males, for their female offspring to have the beneficial traits. Thus, the markers will be selection tools in breeding programs to develop lines and breeds that produce litters with favorable reproductive phenotypes.
  • Another object of the invention is to provide a method for identifying genetic markers for reproductive traits such as pig litter size.
  • a further object of the invention is to provide genetic markers for pig litter size.
  • Yet another object of the invention is to provide a kit for evaluating a sample of pig DNA for specific genetic markers associated with favorable reproductive traits. Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objects and advantages of the invention will be attained by means of the instrumentality's and combinations particularly pointed out in the appended claims.
  • the present invention provides a method for screening pigs and other animals to determine those more likely to have beneficial reproductive phenotypes such as a larger litter, when bred or to select against pigs which have alleles indicating unfavorable phenotypes.
  • larger litters means a significant increase in litter size above the mean of a given population.
  • the term “reproductive trait” shall include any trait which is indicative of improved reproduction efficiency including but not limited to testicular size, sperm volume, sperm concentration, sperm quality, libido, breeding aggressiveness, litter size, number born alive, litter birth weight, number weaned, age at puberty, weaning to oestrus interval, farrowing interval, ovulation rate, uterine capacity, and embryo survival.
  • the term "reproductive gene” shall mean any gene which encodes a gene product which, upon expression, influences either favorably or negatively a reproductive trait. Examples of such genes include but are not limited to the estrogen receptor gene, the prolactin receptor gene and the retinol binding protein 4 gene and other genes disclosed and described herein.
  • the present invention provides a method for screening pigs to determine those more likely to produce beneficial reproductive traits such as larger litters, and/or those less likely to produce smaller litters, which method comprises the steps 1) obtaining a sample of genomic DNA from a pig or other animal; and 2) analyzing the genomic DNA obtained in 1) to determine which allele(s) of the retinol binding protein 4 is/are present.
  • the sample of genetic material is obtained and is analyzed to determine the presence or absence of a polymorphism in a gene that is correlated with a desirable reproductive trait.
  • the polymorphism is a restriction fragment length polymorphism and the assay comprises identifying the reproductive 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 reproductive gene that is either known to have or not to have the desired marker. If an animal tests positive for the marker, such animal can be considered for inclusion in the breeding program.
  • the animal can be culled from the group and otherwise used.
  • the gene of 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.
  • 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 reproductive traits such as litter size in a particular population.
  • Male and female animals of the same breed or breed cross or similar genetic lineage are bred, and the number of offspring produced by each female is determined.
  • a polymorphism in the reproductive gene of each animal is identified and associated with the desired reproductive trait.
  • PCR-RFLP analysis is used to determine the polymorphism. It is also possible to establish linkage between specific alleles of alternative DNA markers and alleles of DNA markers known to be associated with a particular gene (e.g. the reproductive genes discussed herein), which have previously been shown to be associated with a particular trait.
  • markers known to be linked to retinol binding protein 4 gene on porcine chromosome 14 includes S0007, S0116, and
  • the invention further comprises a kit for evaluating a sample of DNA for the presence in genetic material of a desired genetic marker located in the reproductive gene indicative of the inheritable reproductive trait such as large litter size.
  • the kit is a container with one or more reagents that identify a polymorphism in the RBP4 gene.
  • the reagent is a set of oligonucleotide primers capable of amplifying a fragment of the selected reproductive gene that contains a polymorphism.
  • the kit further contains a restriction enzyme that cleaves the reproductive gene in at least one place, allowing for separation of fragments and detection of polymorphic loci.
  • Figure 1 is a schematic depicting the expected fragment pattern using the second PCR protocol and primers in Example 5.
  • Figure 2 is a schematic depicting a comparison of the human published retinol binding protein 4 sequence and the new pig retinol binding protein 4 sequence.
  • the invention relates to genetic markers for beneficial reproductive traits such as litter size in pigs and other animals. It provides a method of screening animals to determine those more likely to produce earlier, healthier, or larger litters when bred by identifying the presence or an absence of a polymorphism in certain reproductive genes (namely the RBP4 gene) that are correlated with these reproductive traits.
  • 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 the female is more likely to produce a litter that is significantly larger (number) is healthier, is earlier to mature, etc. above the mean for that particular breed, strain, population, or group.
  • Any method of identifying the presence or absence of this marker may be used, including for example single-strand conformation polymorphism (SSCP) analysis, RFLP analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, and temperature gradient electrophoresis, ligase chain reaction or even direct sequencing of the reproductive gene and examination for the certain recognition patterns.
  • SSCP single-strand conformation polymorphism
  • oligonucleotide PCR primers are designed that flank the mutation in question and allow PCR amplification of the region.
  • a third oligonucleotide probe is then designed to hybridize to the region containing the base subject to change between different alleles of the gene. This probe is labeled with fluorescent dyes at both the 5' and 3' ends. These dyes are chosen such that while in this proximity to each other the fluorescence of one of them is quenched by the other and cannot be detected.
  • Extension by Taq DNA polymerase from the PCR primer positioned 5' on the template relative to the probe leads to the cleavage of the dye attached to the 5' end of the annealed probe through the 5' nuclease activity of the Taq DNA polymerase. This removes the quenching effect allowing detection of the fluorescence from the dye at the 3' end of the probe.
  • the discrimination between different DNA sequences arises through the fact that if the hybridization of the probe to the template molecule is not complete, i.e. there is a mismatch of some form, the cleavage of the dye does not take place.
  • a reaction mix can contain two different probe sequences each designed against different alleles that might be present thus allowing the detection of both alleles in one reaction.
  • RFLPs are the preferred method of detecting the polymorphism most preferred PCR-RFLP analysis.
  • RFLP analysis depends ultimately on polymorphisms and DNA restriction sites along the nucleic acid molecule, other methods of detecting the polymorphisms can also be used. Such methods include ones that analyze the polymorphic gene product and detect polymorphisms by detecting the resulting differences in the gene product.
  • RFLP analysis in general is a technique well-known to those skilled in the art. See, for example, U.S. Patents 4,582,788 issued April 15, 1986 to Erlich and 4,666,828 issued May 19, 1987 to Gusella, 4,772,549 issued September 20, 1988 to Frossard, and 4,861,708 issued August 29, 1989 to Frossard, the disclosures of which are incorporated herein by reference. Broadly speaking, the technique involves obtaining the DNA to be studied, digesting the DNA with restriction endonucleases, separating the resulting fragments, and detecting the fragments of various genes.
  • a sample of genetic material is obtained from an animal.
  • Samples can be obtained from blood, tissue, semen, etc.
  • peripheral blood cells are used as the source, and the genetic material is DNA.
  • a sufficient amount of cells are obtained to provide a sufficient amount of DNA for analysis. This amount will be known or readily determinable by those skilled in the art as explained in the material incorporated herein.
  • the DNA is isolated from the blood cells by techniques known to those skilled in the art.
  • the region containing the polymorphism is amplified by the use of primers and standard techniques, such as the polymerase chain reaction. This technique is described in U.S.
  • the selection of primers is discussed in the references mentioned and incorporated herein.
  • the primers should amplify the region containing the polymorphism.
  • primers for specific polymorphisms are disclosed herein. Other such primers are designable by those of skill in the art combined with the teachings herein, and are intended to be encompassed by the invention.
  • the isolated DNA is then analyzed and optionally digested with a restriction endonuclease that cleaves or cuts DNA hydrolytically at a specific nucleotide sequence, called a restriction site.
  • a restriction endonuclease that cleaves or cuts DNA hydrolytically at a specific nucleotide sequence, called a restriction site.
  • endonucleases also called restriction enzymes
  • RFLP polymorphism
  • the restriction fragments are then analyzed by known techniques that generally involve either the separation of the fragments and visualization by staining or subsequent blotting and hybridization to obtain a particular pattern or the determination of different sizes of the fragments.
  • the latter permits the identification of one or more fragments (markers) for increased litter size.
  • the preferred separation technique is gel electrophoresis.
  • the digested fragments are separated in a supporting medium by size under the influence of an applied electric field. Gel sheets or slabs, such as agarose or agarose-acrylamide, are typically used as the supporting medium.
  • the sample, which contains the restriction fragments is added to one end of the gel.
  • One or more size markers are run on the same gel as controls to permit an estimation of the size of the restriction fragments. This procedure generally permits a degree of resolution that separates fragments that differ in size from one another by as little as 100 base pairs.
  • the fragments are denatured and transferred physically from the gel onto a solid support, preferably a nylon membrane, by contacting the gel with the filter in the presence of appropriate reagents and under appropriate conditions that promote the transfer of the DNA.
  • appropriate reagents and conditions are well-known to those skilled in the art.
  • the next step involves the detection of the various categories of sizes of the fragments or, alternatively, the detection of a fragment of a particular size.
  • the latter may be of particular interest because it is a genetic marker associated with a desired reproductive trait. This is preferably accomplished via staining of the fragments with ethidium bromide or the like.
  • a hybridization probe is an oligonucleotide or polynucleotide that is sufficiently complementary or homologous to the fragments to hybridize with them, forming probe -fragment complexes.
  • the probe is a cDNA probe.
  • the oligonucleotide or polynucleotide is labeled with a detectable entity. This permits the detection of the restriction fragments, to which the probes are hybridized.
  • the probes are labeled by standard labeling techniques, such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, and the like. See U.S. patent nos. 4,711,955 issued December 8, 1987 to Ward et al.
  • the probes are contacted with the nylon membrane that contains the restriction fragments for a sufficient period of time and under appropriate hybridizing conditions for the probes to hybridize to the fragments.
  • the filter is then preferably washed to remove unbound probes and other unwanted materials.
  • the probe-fragment complexes, which are bound to the filter are then detected by known techniques. For example, if the probe has been radioactively labeled (32p), detection involves contacting the nylon membrane paper with a piece of radiosensitive film. Following an appropriate exposure period, the fragments of interest, including control fragments, are visualized.
  • the detection step provides a pattern, resulting from the separation of the fragments by size. Comparison of these fragments with control fragments of known size that have also been run on the same gel permits the estimation of the size of the various groups of fragments.
  • the various polymorphisms in the reproductive genes are then determined by comparison of the patterns produced by similar analysis of DNA from a number of different pigs. For some of the individual animals, the patterns will differ from the usual pattern produced by most of the other animals. This will be due to one or more restriction fragment length polymorphisms, i.e., restriction fragments of a different length produced by the endonuclease that cuts the reproductive gene. This indicates different base pair sequences in such pigs.
  • a probe to this fragment may be constructed by the use of known techniques. This permits alternative and faster formats for detecting such polymorphism. For example, once the DNA is digested, a sandwich hybridization format can be used. Such an assay is disclosed in US. Patents 4,486,539 issued December 4, 1984 to Ranki, et al., and 4,563,419 issued January 7, 1986 to Ranki, et al., both of which are incorporated herein by reference. The sample is brought into contact with a capture probe that is immobilized on a solid carrier. The probe binds the fragment. The carrier is then washed, and a labeled detection probe is added. After additional washing, the detection probe is detected, thereby demonstrating the presence of the desired fragment.
  • the RFLP pattern is compared to a second, known RFLP pattern or fragment that is correlated with increased litter size.
  • This second pattern or fragment has also been determined from the reproductive gene, using the same restriction endonuclease as the first and the same probe or an equivalent thereof under the same conditions.
  • the restriction fragments can be detected by solution hybridization.
  • the fragments are first hybridized with the probe and then separated.
  • the separated probe- fragment complexes are then detected as discussed above. Generally, such complexes are detected on the gel without transfer to filter paper.
  • the polymorphism is detected by PCR amplification without any probe.
  • This procedure is known to those of skill in the art and is disclosed in U.S. Patents 4,795,699 entitled “DNA Polymerase” and U.S. Patent 4,965,188 "Process for Amplifying, Detecting, and/or Cloning Nucleic Sequences Using a Thermostable Enzyme” both of which are incorporated herein by reference.
  • primers are constructed to amplify the region in which the polymorphism lies. Accordingly primers which are preferably 4-30 bases are designed based upon the sequence surrounding the polymorphism including a forward 5', primer and a reverse or anti-sense primer 3' of the polymorphism. The primers need not be the exact complement, and substantially equivalent sequences are also acceptable.
  • a DNA polymerase is then added such as Taq polymerase (many such polymerases are known and commercially available) in the presence of the four nucleoside triphosphates and often a buffering agent. Detection is facilitated by simple staining, such as with ethidium bromide, of separated products to detect for predicted sizes based upon the length of the region amplified.
  • PCR amplification may be used in combination with Single Strand Confirmation Polymorphism (SSCP).
  • SSCP Single Strand Confirmation Polymorphism
  • 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.
  • Genetic markers for reproductive genes or particularly for the RBP4 gene or other genes linked therewith are determined as follows. Male and female animals of the same breed or breed cross or derived from similar genetic lineages are mated. The number of offspring with the beneficial reproductive trait is determined. For litter size the number of offspring produced by each female is determined. RFLP analysis of the parental DNA is conducted as discussed above in order to determine polymorphisms in the selected reproductive gene of each animal. The polymorphisms are associated with the traits. At least 20 and preferably at least 40 females are used in making these determinations. The number of times each female produces a litter (i.e., the parity) is at least 1 time. Preferably, the cycle of breeding and giving birth is repeated at least 2 times and most preferably 3 times. When this analysis is conducted and the polymorphism is determined by
  • RFLP or other analysis amplification primers may be designed using analogous human or other closely related animal known sequences. The sequences of many of the reproductive 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 sets of primers have been selected which identify regions in polymorphic reproductive genes. The polymorphic fragments have been shown to be alleles, and each was shown to be associated with beneficial reproductive traits, such as increased fitter size, for various breeds. Often genotype associated with this trait alternates for different breeds. This outcome is similar to the situation disclosed in U.S.
  • the reagents suitable for applying the methods of the invention may be packaged into convenient kits.
  • the kits provide the necessary materials, packaged into suitable containers.
  • the kit contains a reagent that identifies a polymorphism in the selected reproductive gene that is associated with a reproductive trait such as increased litter size.
  • the reagent is a PCR set (a set of primers, DNA polymerase and 4 nucleoside triphosphates) that hybridize with the reproductive gene or a fragment thereof.
  • the PCR set and a restriction enzyme that cleaves the reproductive gene in at least one place are included in the kit.
  • the kit further comprises additional means, such as reagents, for detecting or measuring the detectable entity or providing a control. Other reagents used for hybridization, prehybridization, DNA extraction, visualization etc. may also be included, if desired.
  • 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 if a polymorphism in the reproductive gene is present.
  • RFLP analysis is performed with respect to the reproductive gene, and the results are compared with a control.
  • the control is the result of a RFLP analysis of the reproductive gene of a different animal where the polymorphism of the reproductive gene is known.
  • the reproductive genotype of an animal may be determined by obtaining a sample of its genomic DNA, conducting RFLP analysis of the reproductive gene in the DNA, and comparing the results with a control.
  • the control is the result of RFLP analysis of the reproductive gene of a different animal.
  • the results genetically type the pig by specifying the polymorphism in its selected reproductive gene.
  • genetic differences among animals can be detected by obtaining samples of the genomic DNA from at least two animals, identifying the presence or absence of a polymorphism in the reproductive gene, and comparing the results.
  • assays are useful for identifying the genetic markers relating to fitter size, as discussed above, for identifying other polymorphisms in the reproductive gene that may be correlated with other characteristics, and for the general scientific analysis of genotypes and phenotypes.
  • the genetic markers, methods, and kits of the invention are also useful in a breeding program to improve litter size in a breed, line, or population of animals.
  • markers are selection tools.
  • the examples and methods herein disclose certain reproductive genes which have been identified to have a polymorphism which is associated either positively or negatively with a beneficial reproductive trait (litter size) that will have an effect on reproductive efficiency of that animal.
  • the identification of the existence of a polymorphism within a gene (RBP4) is often made by a single base alternative that results in a restriction site in certain allelic forms.
  • a certain allele, however, as demonstrated and discussed herein 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.
  • Sad digests hybridized with RBP4 revealed a polymorphism with two alleles and the linkage analysis found significant linkages with several loci on pig chromosome 14. Further refinement includes 2 PCR tests with primers for detection of a MSP I polymorphism showing a difference between homozygous genotypes of 1.05 pigs per litter.
  • the porcine RBP4 gene probe was obtained by RT-PCR amplification of a 311-bp fragment from day 12 porcine blastocysts with primers designed based on pig cDNA sequences.
  • the 5' primer (5'- TTCCGAGTCAAAGAGAACTTCG-3' SEQ ID NO:l) represents nucleotides 79- 100
  • the 3' primer (5'-TCATAGTCCGTGTCGATGATCC-3' SEQ ID NO:2) represents nucleotides 368-389. Trout, W., et al. 1991, Mol. Endocrinol. 5:1533-1540. Amplified product was purified and radiolabeled with 32 P by random priming.
  • Retinol-binding protein is a major secretory product of the pig conceptus prior to implantation. Increased production of RBP4 during the rapid morphological developmental period of pig blastocyst elongation, which is a critical period for embryonic survival, suggests that RBP4 may be an interesting candidate gene for investigation of QTL for reproduction in pigs.
  • Partial cDNA fragments ofthe RBP4 gene was used for RFLP detection on Southern membranes containing digested reference family DNA. Sad membranes probed with RBP4 showed two allelic fragments at 12.1 kb and 7.8 kb. RBP4 was mapped by linkage analysis to chromosome 14. To illustrate effects of this gene, two French Large White lines were genotyped at these loci. The first line consisted of French hyperprolific (LWH) pigs (32 sows with 216 litter records). The second line consisted of French control (LW) pigs (27 sows with 242 litter records). The average additive effect of the gene was estimated as the linear regression of litter size on genotype.
  • LWH French hyperprolific
  • LW French control
  • RBP4 had an additive gene effect of 0.52 ⁇ .30 in LWH and .45 ⁇ .43 in LW for the 7.8 kb allele.
  • the allelic substitutive effect for litter size ranged from 5 to 17% of the phenotypic STD.
  • Step 5 Go to step 2 for 39 more cycles
  • the major bands for the AA genotype are 190 bp, 154 bp, and 136 bp;
  • the BB genotype bands are 154 bp, 136 bp and 125 bp.
  • TBA total number born
  • DAGK diacylglycerol kinase gene
  • the ovine Booroola fecundity gene (Fec B ) is linked to markers from a region of human chromosome 4q. Nature Genet. 4, 410-414. Nunes, M., Peelman, L., Vaiman, M., Bourgeaux, N., Chardon, P. (1994). Characterization of six new loci within the swine major histocompatibility complex class III region. Mammal. Genome 5, 616-

Abstract

L'invention concerne des marqueurs génétiques permettant d'améliorer les caractéristiques de reproduction chez les animaux, notamment les naissances à l'accouchement et le poids des animaux sevrés. L'invention concerne aussi des procédés d'identification de ces marqueurs et des procédés de criblage d'animaux permettant de déterminer ceux supposés avoir une reproduction plus favorable et de sélection, de préférence, des animaux à des fins de reproduction future. Le marqueur, la protéine 4 de liaison de rétinol, dépend de la présence ou de l'absence de certains polymorphismes dans le gène de reproduction du cochon.
EP99902277A 1999-01-15 1999-01-15 Proteine de liaison 4 de retinol comme marqueur genetique associe a une amelioration des naissances par accouchement Withdrawn EP1141390A1 (fr)

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CA2421754C (fr) * 2000-09-08 2012-04-10 Iowa State University Research Foundation, Inc. Nouveaux alleles de prkag3 et utilisation de ceux-ci comme marqueurs genetiques de caracteristiques de reproduction et de qualite de viande
CN1321196C (zh) * 2005-08-31 2007-06-13 中国农业大学 一种检测猪产仔数性状的方法
KR101735762B1 (ko) * 2014-10-24 2017-05-16 경남과학기술대학교 산학협력단 유전자의 발현프로필을 이용한 돼지의 산자수 예측방법
CN105368926B (zh) * 2015-08-27 2018-10-19 安徽农业大学 猪基因多态性及其与母猪繁殖性状关联性的测定方法
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