CN1260369C - Retinol binding protein 4 as genetic marker for increased litter size - Google Patents

Abstract

Disclosed herein are genetic markers for superior reproductive performance in animals, such as litter size and weaning weight, methods for identifying such markers and screening animals to identify animals more likely to produce superior reproductive performance, and to preferentially select such animals for future breeding purposes method. This marker is based on whether there is a certain polymorphism in the porcine reproductive gene, retinol binding protein 4.

Description

Retinol-binding protein 4 as a genetic marker of litter size

field of invention

The present invention relates generally to the detection of genetic differences in reproductive efficiency in animals, and more particularly to genetic markers identified in several genes that indicate genetic phenotypes associated with high reproductive performance. The invention also discloses a method and composition for identifying and selecting animal genes using these markers.

Background of the invention

Reproductive efficiency, specifically in relation to litter size, is the major limiting factor in the efficient production of pork as well as most other livestock products. Measurements of several indicators of reproductive performance revealed genetic differences. With average litter sizes of 4 to 16 sows and average developmental ages of 3 to 7 months, such genetic differences among pig breeds suggest that genetic improvements in fecundity are possible. The average number of live piglets born in the United States is around 9.5 per litter. Heritability for litter size is low (10%-15%), and standard genetic methods of selecting breeding sows based on past litter size are no longer effective. Therefore, a cellular level or DNA level approach is needed to deal with the selection of reproductive performance.

Chinese pig breeds are known to reach the age of development early and have large litter sizes. It is also known that American pig breeds grow fast and have a lot of lean meat. It would therefore be desirable to combine the superior characteristics of these two breeds to increase the efficiency of U.S. pork production. These efforts were greatly aided by the discovery of genetic markers associated with high reproductive performance, such as increased piglets per litter.

Several research groups have studied pig DNA using RFLP analysis. Jung et al. (Theor.Appl.Genet.77: 271-274 (1989)) disclosed the use of PFLP technology to display genetic differences between two breeds of pigs, demonstrating that there are multiple swine leukocyte antigen (SLA) class I genes in these two breeds. state phenomenon. Hoganson et al. (Abstract of the American Association of Animal Science Midwest Annual Meeting, March 26-28, 1990) reported the polymorphism of the major histocompatibility complex (MHC) gene in Chinese pigs, and proved it by RFLP analysis. Jung et al. (Animal Genetics, 26:79-91, 1989) reported the results of RFLP analysis of some boar SLAI genes. These authors claim that their findings suggest a possible association between porcine SLA/MHC class I genes and yield and reproductive performance. They also claim that the use of SLAI class restriction fragments as genetic markers may have potential use in improving pig growth performance in the future.

Additionally, US Patent 5,550,024 to Rothschild et al. discloses polymorphisms in the porcine estrogen receptor gene that are associated with greater litter size.

Messer, L. et al. ("Location and survey of candidate genes for litter size in French white pigs" AnimalGenetics, vol.27, no. Suppl 2, 1996) disclosed that retinol binding protein 4 may contribute to increase. Messer, L et al. ("The Retinol Binding Protein 4 (RBP4) gene is linked to porcine chromosome 14" Mammalian Genome, vol.7, no.5, 1996, p396) disclose a method for amplifying polymorphic loci Primers for the porcine RBP4 region of the dot.

Another hormone in pigs associated with superior reproductive performance is prolactin (PRL), an anterior pituitary peptide hormone that is involved in many different endocrine activities and is essential for reproductive success. US Patent No. 5,935,784 describes and discloses the use of polymorphic loci in the prolactin receptor gene as markers of increased litter size.

The genetic markers provided by the present invention are based on the discovery of RBP4 gene polymorphisms which are associated with improved reproductive performance such as litter size and litter size born. This allows the genotyping of reproductive genes in pigs and the determination of correlations between specific genes and markers of reproductive performance. It will also be possible to identify individual boars and sows carrying superior genotypes.

In the case of sows, this allows the sow to be expected to produce more, earlier and healthier piglets per litter than average for the breed. In the case of boars, female offspring can be counted on to have such good properties. Therefore, these markers will be a selection tool in breeding programs to develop pigs capable of producing piglets with superior reproductive phenotypes.

It is an object of the present invention to provide a screening method for determining which pigs are likely to have more piglets.

Another object of the present invention is to provide a method for identifying genetic markers of pig reproductive performance, such as litter size.

Yet another object of the present invention is to provide genetic markers of litter size in pigs.

Yet another object of the present invention is to provide a kit for evaluating unique genetic markers associated with superior reproductive performance in porcine DNA samples.

Other objects and advantages of the present invention will be partially set forth in the following description, and can also be partially understood from the description, or can be understood through the practice of the present invention. The objects and advantages of the invention are attained by means of the measures and combinations thereof pointed out in the appended claims.

Summary of the invention

As exemplified and broadly described herein, in order to achieve and comply with the objects of the invention, the present invention provides a method of screening pigs and other animals for breeding of those likely to have a superior reproductive phenotype (e.g., more litters per litter) animals, or selected to exclude pigs with alleles displaying an adverse phenotype. As used herein, "more litters per litter" means that the litter size is above the population average.

The term "reproductive performance" as used herein includes any performance that demonstrates improved reproductive efficiency, including but not limited to testis size, semen volume, sperm concentration, sperm quality, libido, reproduction, litter size, litter size born, piglets born Body weight, weaning age, puberty age, weaning to puberty interval, calving interval, ovulation rate, uterine capacity and embryo survival rate.

As used herein, the term "reproductive gene" means any gene which, when expressed, encodes a gene product which, when expressed, affects favorable or undesirable reproductive performance. 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, as well as others disclosed and described herein.

Accordingly, the present invention provides a screening method to determine which pigs are more likely to produce superior reproductive performance (eg, higher litter size) and/or which pigs are less likely to produce fewer piglets per litter. The method comprises the following steps: 1) obtaining a genomic DNA sample of a pig or other animals; 2) analyzing the genomic DNA obtained in step 1 to determine which allele or several alleles of RBP4 exist. Briefly, samples of genetic material are obtained and analyzed to determine the presence or absence of polymorphisms in genes associated with desired reproductive performance.

In a preferred embodiment, the polymorphism is a restriction fragment length polymorphism. The test includes: identifying the reproductive gene in the isolated genetic material; exposing the gene to a restriction endonuclease to generate restriction enzyme fragments of different lengths of the gene; separating the restriction enzyme fragments formed by these fragments by electrophoresis or HPLC, etc. Patterns; compare patterns of restriction fragments produced by animal reproductive genes with and without known desired markers. If an animal tests positive for this marker, the animal may be considered for inclusion in a breeding program. The animal was excluded from the population used if it did not test positive for the genotype marker, otherwise it was used.

In a most preferred embodiment, specific regions of the gene containing polymorphisms are amplified with primers and DNA polymerase, the gene fragments thereof are isolated, and the amplified regions are either directly isolated or sequenced or digested with restriction endonucleases and The resulting fragments are isolated. Separated fragments or RFLP patterns were visualized by simple staining of these fragments or using labeled primers or nucleoside triphosphates in amplification.

In another embodiment, the invention includes a method of identifying a genetic marker for reproductive performance, such as litter size in a particular population. Breeding of male and female animals of the same breed or crossbreed or similar genetic strain and determining the number of offspring produced by each female. Polymorphisms in the reproductive genes of each animal are identified and correlated with desired reproductive performance. Polymorphisms are preferably determined using PCR-RFLP analysis.

Linkage can also be established between unique alleles of other DNA markers and alleles of DNA markers known to be associated with a particular gene (previously shown to be associated with a particular trait, such as the reproductive genes discussed herein) . In such cases, it should be possible to acquire specific reproductive genes, at least in the short term, to select pigs or other animals that produce more litters per litter, or by selecting for unique alleles with other chromosomal markers Genetic indirect selection for alleles with markers associated with a particular reproductive gene to weed out pigs that are likely to have fewer litters per litter. For example, markers known to be linked to the retinol-binding protein 4 gene on porcine chromosome 14 include S0007, S0116, and SW210, all of which are microsatellite DNA.

The invention also includes a kit for detecting the presence in a DNA sample of a desired genetic marker located in a reproductive gene indicative of heritable reproductive performance (eg, greater litter size). At a minimum, the kit is a container containing one or more reagents for identifying polymorphisms in the RBP4 gene, preferably, the reagents are a set of oligonucleotide primers capable of amplifying selected polymorphisms containing A segment of a polymorphic reproductive gene. Preferably, the kit also contains a restriction enzyme capable of cleaving the reproductive gene at at least one position, thereby allowing isolation of the fragments and detection of polymorphic loci.

The invention also includes a method of screening pigs to determine which pigs are more likely to have more piglets per litter and/or which pigs are less likely to have fewer piglets per litter, the method comprising the steps of: determining Alleles of the retinol binding protein 4 gene present; said alleles contain the MspI polymorphism; determination of alleles of other markers in genes known to affect litter size; Combined animals and those carrying bad combinations were culled. Preferably, wherein determining the alleles of a gene capable of affecting litter size comprises determining the presence or absence of at least one allele associated with at least one DNA marker linked to said retinol binding protein 4 gene. Preferably the DNA marker is a microsatellite marker.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate examples of the invention and together with the description serve to explain the principle of the invention.

Description of drawings

Fig. 1 is a pattern diagram of expected fragments generated by the second PCR scheme and primers in Example 5.

Figure 2 is a graph comparing the published sequence of human RBP4 with the new sequence of porcine RBP4.

Detailed description of the invention

Reference in detail to the preferred embodiments of the invention is provided which together with the following examples illustrate the principles of the invention.

The present invention relates to genetic markers for superior reproductive performance (such as litter size) in pigs and other animals. Provides a method for screening animals, by identifying the presence or absence of a certain polymorphism in a reproductive gene (i.e. RBP4 gene) related to certain reproductive performance, to determine those animals in breeding that are likely to give birth earlier, healthier, and each litter Litters are larger.

Accordingly, the present invention relates to genetic markers in particular species, strains, populations, families of pigs or other animals and methods of identifying these markers. With this genetic marker, it is possible to determine which sows are likely to produce significantly more, healthier, and precocious piglets per litter among the above-mentioned specific breeds, strains, populations, and families of pigs. Methods for identifying the presence or absence of the marker can 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 directly sequence reproductive genes and examine certain recognition patterns.

Other possible techniques include non-gel systems such as TaqMan ^™ (Perkin Elmer). In this system, PCR oligonucleotide primers flanking the mutation are designed and PCR amplification of the region is performed. A third oligonucleotide probe is then designed to hybridize to the region containing bases that are prone to change between different alleles of the gene. This probe is labeled at both 5' and 3' ends with two fluorescent dyes. When the two selected fluorescent dyes are close to each other, the fluorescence at one end is quenched by the fluorescence at the other end and cannot be detected. When TagDNA polymerase extends from the PCR primer at the 5' end of the template relative to the probe, the 5' nuclease activity of the enzyme causes the cleavage of the fluorescent dye bound to the 5' end of the annealed probe, thus eliminating the quenching effect, thereby Fluorescence from the dye at the 3' end of the probe can be detected.

If the hybridization of the probe to the template molecule is incomplete, that is, there is some form of mismatch, no cleavage of the dye will occur, thus allowing different DNA sequences to be distinguished. Therefore, quenching is eliminated only when the nucleotide sequence of the oligonucleotide probe is completely complementary to the template molecule to which it binds. The reaction mixture may contain two different probe sequences, each for a different allele that may be present, thus enabling the detection of both alleles in one reaction.

The use of RLFP is the preferred method for detecting polymorphisms, most preferred is PCR-RFLP analysis. However, since the application of RFLP analysis ultimately relies on polymorphisms and DNA restriction sites in nucleic acid molecules, other methods of detecting polymorphisms can also be used. These methods include analysis of polymorphic gene products and detection of polymorphisms by detecting differences in the gene products.

RFLP is a general technique well known to those skilled in the art. See, for example, U.S. Patent Nos. 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 August 1989 No. 4,861,708 awarded to Frossard on the 29th. In summary, the technique involves obtaining the DNA to be studied, digesting the DNA with restriction endonucleases, isolating the resulting fragments and detecting fragments of various genes.

In the present invention, a sample of genetic material is obtained from an animal. Samples may be obtained from blood, tissue, sperm, and the like. Peripheral blood cells are usually used as the source, and the genetic material is DNA. Obtain a sufficient number of cells to provide a sufficient amount of DNA for analysis. The required amount will be known or readily determined by those skilled in the art. DNA is isolated from blood cells using techniques known to those skilled in the art.

In the next step, the region containing the polymorphism is amplified using primers and standard techniques (eg, polymerase chain reaction). This technique is described in the following U.S. Patents: Nos. 4,683,195 and 4,583,202 to Mullis et al., issued July 28, 1987; Nos. 4,800,159 to Mullis et al., issued January 24, 1989; No. 4,889,818 to Gelfaud et al. and No. 4,902,624 issued February 20, 1990 to Clumbus et al. Primer selection is discussed in these references. These primers should amplify the region containing the polymorphism. Several primers for amplifying specific polymorphic regions are disclosed herein. Those skilled in the art can design other primers in combination with the techniques described herein, and these should be included in the present invention.

The isolated DNA is then analyzed and optionally digested with a restriction endonuclease, an enzyme that cleaves or cleaves the DNA at specific nucleotide sequences called restriction sites. Such endonucleases, also known as restriction enzymes, are well known to those skilled in the art. For the purposes of the present invention, a restriction enzyme should be chosen that cleaves at least one place in a selected reproductive gene, resulting in at least two fragments of that gene. Using techniques known in the art in conjunction with the methods herein, it is determined whether these fragments are polymorphic and whether the polymorphism correlates with a desired reproductive performance (eg, litter size). The amount of this enzyme to add to a sample containing porcine DNA, as well as other suitable conditions for processing the sample, can be readily determined by those skilled in the art based on the teachings herein.

The restriction fragments are then analyzed using known techniques. These techniques usually include: separating the fragments, staining or re-blotting and hybridization for visualization to obtain a specific pattern, or to determine the different sizes of the fragments. The latter will allow the identification of one or more fragments (markers) of increased litter size. A preferred separation technique is gel electrophoresis.

In this technique, digested fragments are separated in size in a support medium under the influence of an applied electric field. Usually, a gel sheet or plate (such as agarose or agarose-allylamine) is used as a supporting medium, and the sample containing the restriction fragment is added to one end of the gel. Run the electrophoresis on the same gel with one or more markers of different sizes as controls in order to estimate the size of the restriction fragments. The method typically resolves isolated fragments that differ in size from each other by as little as 100 base pairs.

In another embodiment, the fragments are denatured and physically transferred from the gel to a solid support (preferably a nylon membrane). This is done by contacting the gel with the membrane under appropriate conditions and in the presence of appropriate reagents to facilitate DNA transfer. These reagents and conditions are well known to those skilled in the art. In this way, the relative position of the individual DNA fragments produced by the separation method is maintained.

The next step involves detecting fragments in different size ranges, or detecting a fragment of a specific size. The latter may be of particular interest because the fragment is a genetic marker associated with desired reproductive performance. The fragments are preferably stained with ethidium bromide or the like and then detected.

Another technique is to use hybridization probes. Such a probe is an oligonucleotide or polynucleotide sufficiently complementary or homologous to the fragment to be hybridized to form a probe-fragment complex. The probes are preferably cDNA probes. The oligonucleotide or polynucleotide is labeled with a detectable molecule, allowing detection of restriction fragments that hybridize to the probe. Standard labeling methods can be used, such as labeling probes with radioactive labels, enzyme labels, fluorescent labels, biotin-avidin labels, and the like. See US Patents: No. 4,711,955, issued December 8, 1987 to Ward et al. and No. 4,868,103 issued September 19, 1989 to Stavrianopoulos et al.

The probe is contacted with the nylon membrane containing the restriction fragment for a sufficient time and under hybridization conditions suitable for hybridization of the probe to the fragment. Wash the nylon membrane to remove unbound probe and other unwanted material.

The probe-fragment complex bound to the nylon membrane is then detected using known techniques. For example, if the probe is radioactively labeled ( ^32P ), detection involves contacting the nylon membrane with a radiosensitive film. After an appropriate time exposure, the fragments of interest and control fragments are displayed.

This detection step provides a pattern of fragments separated by size. By comparing these fragments to a control fragment of known size run on the same gel, the size of each group of fragments can be estimated. The resulting patterns were then compared by similar analysis of DNA from different pigs to identify various polymorphisms in reproductive genes. For some individual animals, the pattern map will differ from the regular pattern map produced by most other animals. This is due to length polymorphism of one or more restriction fragments, ie restriction fragments of different lengths produced by endonuclease cleavage of the reproductive gene. This reflects a different base pair sequence in this pig.

Once a particular RFLP, ie, a restriction fragment of a particular length, has been identified, probes for that fragment can be constructed using known methods. This enables other faster ways to detect such polymorphisms. For example, digested DNA can be detected by sandwich hybridization. This method is disclosed in US Patent Nos. 4,486,539, RanKi et al., issued December 4, 1984 and 4,563,419, RanKi et al., issued January 7, 1986. The sample is contacted with a capture probe immobilized on a solid support, which binds to the fragment, the support is washed, and a labeled detection probe is added. After another wash, the detection probe is assayed to demonstrate the presence of the desired fragment.

In another embodiment, once the RFLP pattern or particular polymorphic segment is determined, it is compared to a second known RFLP pattern or segment that is associated with increased litter size. The second pattern or fragment produced by the reproductive gene can also be determined under the same conditions using the same restriction endonuclease and the same probe or its equivalent as the first.

In another embodiment of the invention, restriction fragments can be detected by solution hybridization. In this method, fragments are hybridized to a probe, separated, and the probe-fragment complex detected as described above. Such complexes are usually detected on gels without transfer to filter paper.

In a preferred embodiment, PCR amplification is used without any probes to detect the polymorphism, methods known to those skilled in the art and see U.S. Patent No. 4,795,699 entitled "DNA Polymerase" and U.S. Patent No. 4,795,699 No. 4,965,188, entitled "Methods for Amplifying, Detecting and/or Cloning Nucleic Acid Sequences Using a Thermostable Enzyme".

This method requires the construction of primers capable of amplifying the region where the polymorphism is located. Therefore, primers of 4-30 bases should be designed according to the sequence around the polymorphism, including the 5' forward primer and the 3' antisense (reverse) primer of the polymorphic region. These primers need not be exactly complementary, and substantially identical sequences are acceptable. A DNA polymerase, such as Taq polymerase (many such polymerases are known and not commercially available), is then added in the presence of the 4 nucleoside triphosphates and often a buffering reagent. Simple staining of the isolated product with, for example, ethidium bromide will facilitate detection for the presence of fragments of the expected size for the length of the amplified region. Reaction times, reagents and primer design are known to those skilled in the art and are described in the literature cited herein. PCR amplification can be combined with single-strand conformation polymorphism (SSCP). See Orita et al. "Detection of polymorphism in human DNA by gel electrophoresis: single-strand conformation polymorphism", PNAS 86(8) Apr. 1989 (2766-70) and Lessa et al. "Detection of allelic variation in DNA sequence Screening technology "Mol Ecol2(2) 119-29 Apr.1993.

Although the methods described herein employ one restriction enzyme and one set of primers, the methods are not limited thereto. One or more additional restriction enzymes and/or probes and/or primers may be employed, if desired. Additional enzymes, construction of probes and primers can be identified by routine experimentation, in conjunction with the techniques provided herein.

Gene markers linked to reproductive genes, especially the RBP4 gene or other genes can be determined as follows. Mating male and female animals of the same breed, hybrid breed or similar genetic strains, measuring the number of offspring with excellent reproductive performance, and measuring the number of offspring per litter produced by each female animal. RFLP analysis of parental DNA was performed as described above to identify polymorphisms in selected reproductive genes for each animal. Correlate this polymorphism with reproductive performance. At least 20, preferably at least 40 female animals are used for this determination. Each female animal has given birth to at least one litter. The best gestation and calving cycle is repeated at least twice, preferably 3 times.

When performing this analysis, as well as using RFLP or other assays to determine polymorphisms, similar known sequences from humans or other closely related animals can be used to design amplification primers. Many germline sequences share homology. Primers can also be designed using known gene sequences exemplified by Genbank, or even sequences obtained from linkage data closely surrounding these genes. The inventors have selected several sets of primers to identify polymorphic regions in reproductive genes. These polymorphic segments have been shown to be allelic segments. Each segment was shown to be associated with superior reproductive performance (eg, increased litter size) in various breeds. The genotypes associated with this trait often differ among other breeds. This result is similar to that described in US Patent No. 5,374,523 "Allelic Variation in the Bovine Somatotropin Gene, Genetic Marker for Super Dairy Cows". The inventors of this patent discovered that one allelic polymorphism is the somatotropin gene, one allelic form is beneficial to Jersey cows and the other form is beneficial to Holstein cows.

Suitable reagents for carrying out the methods of the invention can be packaged in conventional kits. The kit provides the necessary materials packaged in appropriate containers. The kit contains at least reagents capable of identifying selected reproductive gene polymorphisms associated with a reproductive performance (eg, increased litter size). The reagent is preferably a set of PCR reagents (a set of primers, DNA polymerase and 4 nucleoside triphosphates) that can hybridize to reproductive genes or fragments thereof. The kit preferably includes a PCR series and a restriction enzyme that cleaves the germline at at least one site. Kits also suitably include other materials, such as reagents for detecting or assaying detectable molecules or controls. Other reagents for hybridization, prehybridization, DNA extraction, visualization, etc. may also be included as desired.

The materials and methods of the present invention can be used more generally for evaluating animal DNA, genotyping individual animals and detecting genetic differences among animals. In particular, a genomic DNA sample can be evaluated against one or more controls to determine the presence or absence of polymorphisms in reproductive genes. RFLP analysis should be performed on reproductive genes and the results compared to controls. Controls are the results of RFLP analysis of reproductive genes of different animals with known reproductive gene polymorphisms. Similarly, the animal's reproductive genotype can be determined by obtaining the animal's genomic DNA, performing RFLP analysis of the reproductive genes in the DNA, and comparing the results to controls. Also, the controls are the results of RFLP analysis of reproductive genes of different animals. The results determine the genotype of the pig by determining polymorphisms in selected reproductive genes of the pig. Ultimately, genetic differences between animals can be detected by obtaining at least two samples of genomic DNA from the animals, identifying the presence or absence of reproductive polymorphisms, and comparing the results. Figure 2 is a comparison of the published human RBP4 sequence and the new porcine RBP4 sequence.

As noted above, these assays can be used to identify genetic markers associated with litter size, to identify other polymorphisms in reproductive genes that may be associated with other traits, and for overall scientific analysis of genotypes and phenotypes.

The genetic markers, methods and kits of the invention may also be used in breeding programs to increase litter size in animal breeds, strains and populations. For polymorphisms associated with superior reproductive performance (eg, increased litter size), continued selection and breeding of animals that are at least heterozygous (preferably homozygous) will result in breeds with higher litter sizes in dams , strains and populations. In this way, these markers become selection tools.

The examples and methods herein disclose a reproductive gene identified as having a polymorphism that is positively or negatively associated with a superior reproductive performance (litter size) that affects the reproductive efficiency of the animal. A polymorphism in one gene (RBP4) was identified, often a single base change that resulted in a restriction site in an allele. However, as demonstrated and discussed herein, an allele can have many base changes associated with it. Those bases can be tested to indicate the same polymorphism, and other genetic markers or genes can also be linked to the polymorphisms described herein, so that the test can include identification of other genes or gene fragments, but the test ultimately relies on the same polymorphism. genetic traits of animals. Any assay that selects and identifies animals based on allelic differences is within the scope of this invention. Once a polymorphism has been identified and proven to be associated with a particular trait, each of skill in the art will understand that there are a myriad of ways to genotype an animal for that polymorphism. as fully described herein. These modified experimental designs merely optimize parameters known to those skilled in the art and are intended to be included within the scope of the present invention.

The present invention has identified polymorphisms in the RBP4 gene that are associated with increased litter size. Blastocysts express retinol-binding protein 4 (RBP4) during elongation, which presumably transports and regulates the amount of retinol received by the embryo. The present invention identifies a polymorphism and maps the genes of RBP4. Hybridization of SacI digestion product with RBP4 showed a polymorphism (fragment) with two alleles, and its linkage analysis found that it was obviously linked with several loci on pig chromosome 14. Further refinement studies included a secondary PCR assay with primers to determine the MSPI polymorphism, which showed a difference of 1.05 pigs per litter between the homozygous genotypes.

It should be understood that it is within the ability of one of ordinary skill in the art to implement the teachings of the present invention to a particular problem or environment in light of the teachings herein. The examples of products and methods of the invention shown in the following examples are not meant to limit the scope and content of the invention.

Example 1

   Linkage map of retinol-binding protein 4 (RBP4) gene and pig chromosome 14

Location shown in the diagram: The locus sequence of the distal part of chromosome (Chr) 14: -ACTN2-1.7-ACTAl-2.7-PLAU-0-SW210-8.2-S0169-9.9-S0072-11.1-S0007-7.3-RBP4-16.2- S0116-20.0-Sw761-36.1-S0015.

Mapping Methods: Three-Generation PiGMaP Families of Six Meishan x Large White and European Wild Boar x Large White, Archibald et al., 1995. Mamm. Genome 6: 157-175

Molecular reagent: the porcine RBP4 gene probe was obtained by amplifying the 311-bp fragment of the 12-day porcine blastocyst with primers designed according to the porcine cDNA sequence. The 5' primer (5'-TTCCGAGTCAAAGAGAACTTCG-3', SEQ ID NO: 1) 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. The amplified product was purified and radiolabeled with ³² P using random primer method. ,

Allele detection: SacI polymorphism in pig genomic DNA was detected by Southern blot hybridization with the labeled 011bp porcine RBP4 fragment. The stringency of the final wash was set at 65°C, 0.7xSSC and 0.2% SDS. Mendelian inheritance (regulation) was found in the two polymorphic fragments detected at 12.1 kb and 7.8 kb. The RBP4 genotypes of 62 unrelated pigs from 8 breeds were determined. The frequency of the 12.1 kb segment was 0.55 in Landrace pigs (n=10) and 0.75 in Duroc pigs (n=10). Yorkshire pig (n=5) is 1.0, Chester white pig (n=4) is 0.5, Large White pig (n=11) is 0.59, Hampshire pig (n=5) is 1.0, Meishan pig (n=15) is 0.80, 1.0 for wild boars (n=2).

Previously identified homology: human RBP4 is located at 10q23-24 (Rocchi. M et al. 1989, Somat. Cell Mol. Genet., 15: 185-190) and mouse Rbp-4 is located at the distal end of chromosome 19 (Chainani , M. et al., Genomics. 1991, 9: 376-379).

Discussion: Retinol-binding protein is a major secretory product of the pig pre-implantation conception. RBP4 production is increased during the rapid morphological development of porcine blastocyst extension, a critical period for embryo survival, suggesting that RBP4 may be an interesting candidate gene for the study of porcine reproductive quantitative trait loci (QTL).

Linkage analysis was performed with the CRIMAP version 2.4 software package. Two-point linkage analysis obtained the significance Lod score (>3.0) between RBP4 and loci S0007, S0116 and SW210 on porcine chromosome 14 (Table 1). The order of this locus on our map is consistent with the new locus arrangement of KapKe and colleagues' PiGMaP map on chromosome 14 (KapKe.P et al. 1995. Anim Genet., to be published), with the exception of loci S0007 and S0072 rearrange. The addition of RBP4 to the reworked PiGMaP map further increased the length of the sex balance map from 193 cM to 202 cM. Replacing RBP4 on chromosome 14 strengthens the homology between porcine chromosome 14 and human chromosome 10. (Johansson et al. 1995, Genomics, 25:682-690).

Table 1: Results of two-point linkage analysis of RBP4 mark 1 mark 2 recombinant fragment Lod score RBP4RBP4RBP4 S0007S0116SW210 0.070.210.27 17.024.633.50

Example 2

Association of polymorphisms with litter size

RFLP assays were performed using partial cDNA fragments of the RBP4 gene on Southern membranes containing digested family DNA references. The SacI membrane was probed with RBP4, revealing two allelic fragments of 12.1 kb and 7.8 kb. RBP4 map of chromosome 14 by linkage analysis. To illustrate the role of this gene, two Fronch Large White pigs were genotyped at these loci. The former line consisted of French high yielding (LMH) pigs (216 piglets recorded from 32 sows) and the latter line consisted of French control (LW) pigs (242 piglets recorded from 27 sows). The average addition effect of this gene on genotype was assessed as a linear regression of litter size. The additive gene effect of RBP4 on the 7.8 kb allele was 0.52 ± 0.30 in LMH pigs and 0.45 ± 0.43 in LW pigs. The substitution effect of alleles on litter size ranged from 5 to 17% of the phenotypic STD

Example 3 (RBP4)

PCR-RFLP test of MSDI polymorphism

Based on the identification of polymorphisms in the RBP4 gene, a PCR assay was developed

Primer

LM24195'-GAGCAAGATGGAATGGGTT-3' SEQ ID NO: 4

LM24205'-CTCGGTGTCTGTAAAGGTG-3' SEQ ID NO: 5

PCR conditions 25 μL reaction

Mixture No. 1

10X Promega buffer 2.5 μL

25mM _MgCl2 1.5μL

10mM dNTP's 0.5μL

16pMol LM2419(100ng/μL) 0.5μL

16pMol LM2420(100ng/μL) 0.5μL

Double distilled sterile water 12.5μL

25ng genomic DNA (12.5ng/μL) 2.0μL

Taq mixture

Double distilled sterile water 4.9μL

0.6U Taq polymerase (Promega) 0.12 μL

Mix 18 μl of Mix 1 and DNA in a reaction tube. Top with mineral oil. Preheat at 85°C on a heating cycler. Add 5μl Tag mixture and run the following PCR program:

Step 1 93°C for 3 minutes

Step 2 93°C for 30 seconds

Step 3 56°C for 45 seconds

Step 4 72°C for 45 seconds

Step 5 Back to step 2, more than 39 rounds

Step 6 72°C for 5 minutes

Step 7 Keep at 4°C

Check 5 μl of the PCR reaction on a standard 1% agarose gel to confirm successful amplification and a clear negative control. The product size is approximately 550 base pairs and can be digested directly in a PCR tube.

MSPI digestion reaction 30 μL reaction

Remaining PCR product 20.0μL

10X NE Buffer 2 3.0 μL

10U MSP I enzyme (20U/μL) 0.1μL

Double distilled sterile water 6.9μL

Prepare a mixture of buffer, enzyme and water and add 10 μl to each reaction tube containing the DNA. Incubate overnight at 37°C. The loading stain was added directly to the digestion reaction and the entire volume was loaded on a 3% NuSieve gel. The main bands of AA genotype were 190bp, 154bp and 136bp. The BB genotype bands are 154bp, 136bp and 125bp.

Analysis of French pig lines revealed that MSPI allele A = (old) SacI allele 2 for this new PCR assay.

The high-yield pig strains and control strains of the University of Nebraska were tested by PCR-RFLP, and the results are as follows: A B index control .50.47 .50.53

Therefore, the frequency of A is higher in this index.

Nineteen animals of the PIC strain (with appropriate records) were analyzed with the MSPI assay and the results are shown in Tables 2 and 3.

Table 2 AVE NB Genotype N average standard deviation AAABBBB 939818 10.1789.2929.722 0.2370.2750.511

table 3 EBV TNB Genotype N average standard deviation AAABBBB 939818 0.1600.0340.108 0.0620.0490.112

The data is grouped by litter size as follows AAA AB BB A B More litters per litter Fewer litters .62.24 .36.64 .04.12 .78.56 .22.44

Example 4

A second RCR assay was developed and is shown below.

PIC RBP4 assay protocol

RBP4-FOR2 5’ACT GTG CTC TTT GTG CTG 3’SEQ ID NO: 6

RBP4-REV 5'CTC GGT GTC TGT AAA GGT G 3'SEQ ID NO: 7

method

10x PCT buffer 1.2μl

2Mm dNTP's 1.2μl

25mM Mg2 ⁺ 1.2μl

RBP4-FOR 2(5μM) 1.2μl

RBP4-REV(5μM) 1.2μl

Amplitag Gold 0.12μl

QH ₂ O 4.88 μl

1.0 μl cut

                                                                               

PE9700

  94°C 30 seconds}

94°C 12 minutes 58°C 30 seconds}x30→72°C 4 minutes→6°C

                                           

(9600Ramp)

Digestion : 3 hours at 37°C

PCR product 12.01

MSP1(10μ/l) 0.5μl

QH ₂ O 1.0μl

                                                                   

After loading, electrophoresis was performed on 5% NuMe agarose at 150V for about 45 minutes to 1 hour. Figure 1 shows the expected band sizes.

Example 5

European results

RBP4 genotypes were determined using the protocol of Example 4 on 264 Landrace sows with litter size records recorded on European PIC breeding farms. Two alleles 1 and 2 were identified. The genotype distribution of markers in all lines followed the Hardy Weinerg equilibrium principle.

Two properties were analyzed - total born size (TNB) and born alive (NBA)

Data were analyzed using a mixed model with sire as a random effect (h2 = 0.09)

Fixed effects include: year, month of calving, sow line, boar line, breeding farm (3A1 breeding pigs) cycle 1, 2, 3 ⁺ . Boars are mated to sows (1, 2, 3 ⁺ ). All services were A1 boars to sows.

RBP4 effects between all parity and sow lines Genotype 111222 Number of litters 21222387 TNB10.8410.289.93 NBA9.939.669.36 ad ^* P<0.1 +.46 ^* -.11 +0.29+0.02

RBP4 results over 7509 litters suggested a difference between homozygous genotypes of 1.05 pigs per litter. Figure 1 shows the expected fragmentation pattern using the second PCR protocol and primers described above.

Example 6

RBP4 genotyping was performed on 154 Dam line sows (consisting of the Landrace and Large White/Duroc combined lines) with record litter sizes on US breeding farms. Genotype 111222 Number of litters 297350103 TNB10.7410.049.66 P<0.02

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Claims (17)

1. A method of screening pigs to determine which pigs are more likely to have increased litter size, comprising: determining the presence or absence of a polymorphism in the retinol binding protein 4 gene in a sample of genetic material, said polymorphism is associated with increased litter size, Wherein determining the presence or absence of said polymorphism comprises the steps of: Amplifying a certain amount of reproductive genes or fragments thereof containing said polymorphism; digesting said genetic material with an MspI restriction enzyme that cleaves said retinol binding protein 4 gene at at least one position; Separation of fragments resulting from digestion; Determining a restriction pattern map generated by fragments produced by said digestion; This profile is compared to another restriction profile of reproductive genes obtained using the restriction enzymes, wherein the other profile correlates with increased litter size. 2. The method according to claim 1, wherein the step of determining a restriction pattern is selected from the group consisting of: direct sequencing analysis, restriction fragment length polymorphism analysis, heteroduplex analysis, single-strand conformation polymorphism Sexual analysis, denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis. 3. The method of claim 1, wherein the separation is gel electrophoresis. 4. The method of claim 1, wherein the step of comparing the restriction pattern maps comprises identifying fragments of a particular size and comparing the sizes of the fragments. 5. The method of claim 4, wherein the step of determining the size of the fragments comprises: In the presence of control DNA fragments of known size, the fragments are separated by size using gel electrophoresis; contacting said isolated fragment with a probe capable of hybridizing to said fragment to form a probe-fragment complex; determining the size of the isolated fragments by detecting the presence or absence of probe-fragment complexes; Determine their relative positions to the control DNA fragments. 6. The method of claim 1, wherein the amplification is performed with Taq polymerase. 7. The method of claim 1, wherein said amplifying includes the step of selecting forward and reverse primer sequences that amplify said germline region containing a polymorphic site. 8. The method of claim 7, wherein the primers are SEQ ID NO: 4 and SEQ ID NO: 5. 9. The method of claim 1, wherein the polymorphism is a 190bp restriction length polymorphism. 10. The method of claim 1, wherein the polymorphism is a 125bp restriction length polymorphism. 11. The method of claim 7, wherein the primers are SEQ ID NO: 6 and SEQ ID NO: 7. 12. A primer for determining the presence or absence of a polymorphic site associated with an increase in litter size in the porcine retinol-binding protein 4 gene, wherein the primer is selected from the group consisting of SEQ ID NO: 4 , SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. 13. A genetic marker associated with increased litter size in pigs, wherein said marker comprises the retinol binding protein 4 gene, said marker being amplified by using SEQ ID NO: 6 and SEQ ID NO: 7 The MSP I polymorphism in the region of the gene added was identified. 14. A genetic marker associated with increased litter size in pigs, wherein said marker comprises the retinol binding protein 4 gene, said marker being amplified by using SEQ ID NO: 4 and SEQ ID NO: 5 The MSP I polymorphism in the region of the gene added was identified. 15. A method of screening pigs to determine which pigs are more likely to have more piglets per litter and/or which pigs are less likely to have fewer piglets per litter, characterized in that the method comprises the steps of: determining Alleles of the retinol binding protein 4 gene present in pigs; said alleles contain the MspI polymorphism; determination of alleles of other markers in genes known to affect litter size; selection of alleles with Pigs with good genetic combinations and those carrying bad combinations were culled. 16. The method of claim 15, wherein determining the allele of a gene that affects litter size comprises determining the presence or absence of at least one DNA linked to the retinol binding protein 4 gene At least one allele associated with the marker. 17. The method of claim 16, wherein the DNA marker is a microsatellite marker.