WO1996039538A1 - Evaluating scrotal hernia in swine - Google Patents

Abstract

The present invention encompasses methods and compositions for predicting and decreasing the incidence of scrotal hernias in swine. In particular, an amplification-based detection system employing microsatellite DNA has been developed for use in swine diagnostic and breeding contexts. Two particular microsatellite alleles have been identified, one of which associates with the rupture phenotype and one of which associates with the non-rupture phenotype. Kits for practicing these methods also are provided.

Description

DESCRIPTION

EVALUATING SCROTAL HERNIA IN SWINE

I. FIELD OF THE INVENTION

The invention relates to methods and compositions useful in swine breeding. In particular, a method for determining genetic markers in swine has been used to identify markers associated with decreased or increased incidence of scrotal hernia. These markers can be used to assist traditional breeding programs designed to reduce the incidence of scrotal hernia in pigs.

II. BACKGROUND OF THE INVENTION

Congenital abnormalities in economically important animals can lead to reduced production, disease and even death. Swine breeding is no exception with more than one hundred types of congenital defects having been recorded in swine. Huston et al . , Veterinary Bulletin 48:645-675 (1978) . The defects represent a major economic loss to the industry.

Hernia or rupture is the protrusion of the intestines, or any other organ, through a natural or artificial opening in the body wall. A hernia is classified according to the part of the body in which it is located. The kinds of hernias commonly found in swine are (i) inguinal, in which the inguinal canal serves as the inguinal ring, (ii) scrotal, in which the scrotum is the hernial sac, (iii) umbilical or navel, in which the umbilical or navel opening is the hernial ring, and (iv) ventral, in which the hernial ring is located in the lower part of the abdomen. Warwick, Wisconsin Agricul tural Experiment Station Bulletin 62:1-27 (1926) . Of the foregoing, scrotal hernias (SIR's) have the gravest economic consequences for the swine producer. This stems primarily from the fact that castration is contraindicated in the presence of SIR'S. Secondarily, however, the overall health of the animal may suffer as well. The exact cause of SIR'S is not known, but there is agreement that development of this defect is genetically influenced. Vogt and Ellersieck, Am. J. Vet . Res . 51:1501-1503 (1990) . Nevertheless, no major genetic markers have been identified nor any there clear pattern of inheritance.

One general approach to search for genetic markers is restriction fragment length polymorphism ("RFLP") analysis. The use of RFLP's to create genetic

"fingerprints" has been well-documented for a variety of different organisms, including both higher plants and animals and, in particular, pigs. For example, polymorphisms in the swine leukocyte antigen (SLA) Class I genes have been documented by RFLP. Jung et al . ,

Theor. Appl . Genet . 77:271-274 (1989) . Hoganson et al . , ABSTRACT FOR ANNUAL MEETING OF MIDWESTERN SECTION OF THE AMERICAN SOCIETY FOR ANIMAL SCIENCE, March 26-28, 1990, reported on polymorphisms in the swine major histocompatibility locus. Another report examined SLA

Class I genes in boars using RFLP's. Jung et al . , Animal Genetics 20:79-91 (1989) . Such RFLP patterns could, in theory, be applied to an analysis of animals possessing and lacking various desirable characteristics. In this way, it is possible that markers for the desirable characteristics can be determined.

At present, while tools are available through which genetic analyses may be undertaken, there remains a need for further information on the genetic background of swine that suffer from SIR'S. III. SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to identify swine genetic markers that serve to identify animals that exhibit or do not exhibit a tendency towards scrotal hernias.

It also is an object of the present invention to provide a general method for determining the association of swine genetic markers with scrotal hernias.

It is yet another object of the present invention to provide breeding methods that will assist in reducing the incidence of hernias in swine.

In fulfilling these objects, there is provided a method of screening pigs for a predisposition to exhibit scrotal hernias comprising the steps of (a) obtaining a nucleic acid sample from a pig; and (b) determining the presence of at least one marker in said sample that correlates with low or high incidence of hernias. In another embodiment, the determining step comprises hybridizing a microsatellite primer pair to said nucleic acid sample; (ii) amplifying the nucleic acid between hybridized pairs of microsatellite primers; and (iii) analyzing the amplification products of the previous step. In further embodiments, the amplification is PCR™ and the primer is a microsatellite primer designated SW1129.

There also is provided a method of reducing the incidence of scrotal hernias in pigs comprising the steps of (a)crossing a first pig exhibiting a low predisposition for hernias with a second pig exhibiting at least one other desirable trait; (b) obtaining a nucleic acid sample from progeny of said cross;

(c) determining the presence of at least one marker in said sample that correlates with low or high incidence of hernias; and (d)effecting a breeding decision based on the previous step. In another embodiment, the step of determining comprises (i) hybridizing a microsatellite primer to said nucleic acid sample; (ii) amplifying the nucleic acid between pairs of microsatellite primers; and (iii) analyzing the amplification products of the previous step. In further embodiments, the amplification is PCR™ and the primer is a microsatellite primer designated Swll29.

There also is provided a method of identifying a marker for scrotal hernia in pigs comprising (a) determining the incidence of hernias for a male pig; (b) obtaining a nucleic acid sample from said pig; and (c) determining the presence or absence of at least one marker in said nucleic acid sample; and (d) repeating steps a-c with a sufficient number of male pigs to determine the relationship of said marker with the incidence of hernias. In another embodiment, the step of determining comprises (i) hybridizing a microsatellite primer to said nucleic acid sample; (ii) amplifying the nucleic acid between pairs of microsatellite primers; and (iii) analyzing the amplification products of the previous step. In further embodiments, the amplification is PCR™ and the nucleic acid sample is genomic DNA.

Also provided are kits for use with any of the foregoing compositions or methods including at least one receptacle.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comprehensive (Cytogenetic and Linkage) Map of Swine Chromosome 6.

FIG. 2. Framework Linkage Map of Swine Chromosome 6^a.

V. DETAILED DESCRIPTION OF THE INVENTION

As stated above, the use of RFLP's to create genetic fingerprints has been well-documented for a variety of different organisms, including both higher plants and animals. The principal underlying this technology is that small variations in the genomes of related organisms may reflect important differences in the phenotype of these organisms. The small genetic variations will be those that result in changes in the size of genomic DNA fragments. These changes may be caused by insertions or deletions of genetic material or, alternatively, may result from the addition or loss of recognition sites for restriction enzymes, which cleave DNA a specific points. Any of these alterations can result in a change in the molecular weight of DNA fragments generated by cleavage with a given restriction enzyme.

RFLP analysis is, for a variety of reasons, rather cumbersome and has been supplanted by similar, but more advanced, technology. For example, RAPD ("random amplification of polymorphic DNA") relies on short, randomly generated oligonucleotide primers that hybridize to multiple points within the genome of any organism. When a given primer happens to bind to both strands of the genomic sequence within about 2000 base pairs, such that one primer binds 3' with respect to the other member of the pair, the primers will support polymerase amplification of the intervening sequences.

Another amplification based-approach to genotyping is the semi-random process involving microsatellite DNA. Microsatellite DNA is comprised of long, tandem repetitions of a short nucleotide sequence. The repeat unit in a sequence of this type may be composed of only one or two nucleotides, but most repeats are longer. In mammals, they typically are composed of variants of a short sequence organized into a repeat of a few hundred nucleotides. Generally, these sequences are non- transcribed and located most often in the heterochromatin associated with centromeric regions of chromosomes. Because microsatellite DNA is polymorphic, primers designed to flank these sequences can be used in the same manner as the RAPD primers, discussed above. The pig genome is estimated to have 60,000 to 100,000 microsatellite loci scattered throughout the genome. Archibald et al . , Mammalian Genome 6:157-175 (1995). At present, about 600 microsatellite markers are known.

If the resulting amplification products are integrally labeled, for example, with radio- or chromophore-labeled deoxynucleotides, the products can be identified following size separation, much like restriction fragments. Alternatively, unlabeled products may be visualized with a nucleic acid dye such as ethidium bromide.

The present invention relies on this sort of microsatellite amplification to examine microsatellite sequences for pigs having varying incidences of scrotal ruptures (SIRs) . By analyzing the DNAs of pigs, one can identify microsatellite repeats whose presence or absence correlates with the incidence of SIRs. Because these bands correspond to sequences in the genomes of the animals under study, sequences identified as associating with a trait of interest are designated as "markers. " Subsequently, breeding programs can be implemented in which the identified markers are used to predict the predisposition of progeny to hernias, thereby permitting selection against those animals carrying the hernia^"1" alleles and, hence, offering a way of removing the trait from the population by the breeder.

Virtually any variety of pig may be used for the present invention. Exemplary breeds are Meishan, Fengjing, Minzhu, Duroc, Hampshire, Landrace, Large White, Yorkshire, Spotted Poland China, Berkshire, Poland China, Pietrain and Chester White. These breeds can be used to construct resource families in which specific genes of biological and/or economic importance are segregated, i . e. , heterozygous. There are a number of different approaches to maximizing parental heterozygosity for efficient linkage mapping. Segregation can be followed in backcross or F2 progeny. For this application, ruptured boars can be (i) crossed with full sibs to produce F2's or (ii) backcrossed to female parent. Schook et al . , Gene Mapping Techniques and Applications, Marcel Deker, New York (1991) .

Alternatively, "selective genotyping" can be performed where random samples of animals lacking a given trait are compared genotypically to random samples from animals possessing the phenotype. A variety of hybrid lines were used for the experiments described below including DEKALB lines DK87, DK89, DK97, DK99, BL, U and Y.

Genomic DNA from swine tissue, blood or semen is used as the sample template for the amplification.

Genomic DNA can be prepared by standard methods using a commercial preparatory reagent such as Triazol™ (Life Technologies, Inc., Gaithersburg, MD) . For example, Triazol™-treated tissue samples are extracted into a Tris-saline-EDTA-SDS buffer and the extract treated with RNAse A and Proteinase K to digest RNA and proteins. After phenol :chloroform extraction, the DNA is ethanol precipitated by standard methods and resuspended in TE buffer. For a general guide, see Kawasaki, In PCR™ PROTOCOLS, Academic Press, New York (1990) pp. 142-152.

Another tool necessary for analysis is an appropriate oligonucleotide primer pair. The primer used in this research project was a microsatellite primer pair designated Swll29. Rohrer et al . , Genetics 136:231-245 (1994) . Swll29 was obtained from Ransom Hill Bioscience, Inc., Ramona, CA. The primers (25 nM) were shipped lyophilized and reconstituted with distilled water, aliquoted in 100 μl amounts and stored at -20°C.

In theory, any microsatellite primer may be used according to the present invention. While the size of a particular primer may vary somewhat, a desirable size range is 8-15 base-pairs; the preferred size is 10 base- pairs. The optimal length for each primer will be determined empirically. Once the amplification conditions have been adjusted to achieve the appropriate amplification, it is then possible to examine the resulting amplification products from target animals to determine any identifiable differences between animals having low or high incidence of SIR'S.

The present invention may take the form of a kit in which particular primers are provided. Typically, the primers are provided in at least one receptacle, preferably in a storage stable solution or lyophilized. Kits also may comprises enzymes useful in template dependent amplification or nucleic acid labeling procedures. Nucleotide and labels also may be included. A number of template dependent-processes are available to amplify the target sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Patents 4,683,195, 4,683,202 and 4,800,159, and in Innis et al . , PCR™ Protocols, Academic Press, Inc., San Diego CA, 1990, each of which is incorporated herein by reference in its entirety. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e. g. , Taq polymerase. If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated. Preferably a reverse transcriptase PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPA No. 320 308, incorporated herein by reference in its entirely. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5' - [alpha-thio] -triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention. Walker et al . , Proc. Nat 'l Acad. Sci . USA 89:392-396 (1992) , incorporated herein by reference in its entirety.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR) , involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR) . In CPR, a probe having a 3' and 5' sequences of non-specific DNA and middle sequence of specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe identified as distinctive products which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR™ like, template and enzyme dependent synthesis. The primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety ( e. g. , enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) , including nucleic acid sequence based amplification (NASBA) and 3SR. Kwoh et al . , Proc . Nat 'l Acad. Sci . USA 86:1173 (1989); Gingeras et al . , PCT Application WO 88/10315, incorporated herein by reference in their entirety. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

Davey et al . , EPA No. 329 822 (incorporated herein by reference in its entirely) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA") , ssDNA, and double-stranded DNA (dsDNA) , which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase) . The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA) . The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homoiogy to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I) , resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. Miller et al . , PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" and "one-sided PCR™." Frohman, M.A. , In: PCR™ PROTOCOLS : A GUIDE TO

METHODS AND APPLICATIONS, Academic Press, N.Y. (1990) and Ohara et al . , Proc . Nat ' l Acad. Sci . USA, 86:5673-5677 (1989) , each herein incorporated by reference in their entirety.

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention. Wu et al . , Genomics 4:560 (1989), incorporated herein by reference in its entirety.

PCR™ amplification products are analyzed by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al . , MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) . In a preferred embodiment, the gel is a 1% agarose gel that is stained with ethidium bromide and visualized under UV light. Alternatively, the amplification products can be integrally labeled with radio- or fluorometrically- labeled nucleotides. Gels can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, respectively. As stated above, resource families can be generated by crossing siblings produce F2's. The incidence of SIR'S in male F2's is then measured and the microsatellite profiles for these animals determined and compared. An association is then made between the banding pattern of the microsatellite-associated amplification products and the phenotype. If particular microsatellite alleles consistently associate with a phenotype, then there is evidence that the microsatellite is closely linked to the trait. Also as stated above, the trait can be tested further using a resource family.

Alternatively, selective genotyping can be used to detect a marker linked to the trait. Selective genotyping involves the random sampling of animals with and without the trait. These animals are then genotyped to identify markers associating with the trait. Once a microsatellite allele is identified that appears to associate with the trait of interest, it is tested statistically to determine if it is linked to the trait locus and, therefore, a valid marker. Such markers can be used in a variety of breeding contexts to identify those animals carrying the alleles causing the trait and remove from or select for the breeding population.

As discussed below in the examples, two markers were tested using a selective genotyping approach. Two microsatellite primer pairs, designated Swl22 and Swll29, were used to screen for SIR-related alleles in SIR^4" boars. Initially, Swl22 appeared to identify an allele that associated with SIRs. More rigorous studies revealed, however, that the association did not hold up. Swll29 also was initially determined to be an accurate indicator of the SIR⁺ phenotype. Further analysis with this primer pair was consistent with the early results and Swll29 was determined to identify a marker segregating with the SIR⁺ phenotype. Swll29 is comprised of two primers having the following sequences:

Forward primer: 5'-GATCATATGAGGAAAAGAATGTGT-3' Reverse primer: 5' -CACAGGGGGAACACCTTAAT-3'

The amplified sequence is found on swine chromosome 6 and 6^a in a region described as 6ql.1-2.1. Within this region is the gene for transforming growth factor-B binding protein (TGFB) . Derynck et al . , Nature 316:701 (1985). Seven different alleles were identified ranging from smallest (#1) to largest (#7) . The approximate molecular weights for the amplification products are as follows:

allele #1 - 127 base pairs allele #2 - 137 base pairs allele #3 - 141 base pairs allele #4 - 145 base pairs allele #5 - 151 base pairs allele #6 - 155 base pairs allele #7 - 161 base pairs

Alleles #1-6 have previously been reported but allele #7 appears to be a new allele.

The markers identified as correlating with low and high SIR incidence can be used to identify associated genes that can be cloned and sequenced. Methods by which cloning and sequencing may be accomplished are well known to those of skill in the art. With the identification of sequences flanking the microsatellite markers, different probes may be designed for use in accordance with the present invention. This also may permit the identification of polymorphisms that more accurately correspond to the trait of interest. There are several candidate genes that may harbor a genetic defect related to SIRs. In humans, fibrillin has been found to be a defective gene product in Marfan's syndrome. Marfan's is an autosomal dominant disorder of connective tissue. Pyeritz, "Marfan Syndrome," in

PRINCIPLES AND PRACTICE OF MEDICAL GENETICS, 2nd Ed. , ed. Emery et al . , Churchill-Livingstone, New York (1990) pp. 1047-1063. People afflicted with this disease will, on occasion, develop scrotal hernias, presumably due to patent connective tissue near the scrotal canal in males. Interestingly, it has been reported that fibrillin contains numerous cysteine-rich sequences homologous to peptide motifs of epidermal growth factor (EGF) and TGFB. Pereira et al . , Human Mol . Genet . 2:961-968 (1993) ; Dietz et al . , Nature 352:337-339 (1991) . The pig fibrillin gene has not yet been mapped.

Another potential target is the swine version of procollagen III (pCIII) . The human pCIII gene has a mutation, probably in its promoter, that is linked to scrotal hernia. Friedman et al . , Annals of Surgery 218:754-760 (1993) . The results showed a statistical difference (p<0.0001) between pCIII and pCI (procollagen I) expression in patients with scrotal hernia. The ration of pCI:pCIII was 6.3 in normal patients and 3.0 in patients exhibiting scrotal hernias. This difference, based on RNA expression, suggested that an increase in secretion of pCIII may make the scrotal ring region more patent or less firm, thus leading to herniation.

Once cloning of flanking regions is accomplished, it will be of interest to look for open reading frames within the cloned fragment and, further, to compare any significant open readings frames with those known through databases such as GenBank. With additional sequences from the marker available, it also will be possible to design other primers that will be specific for the marker. Primer sets are prepared in both the sense and antisense orientation. Suitable oligonucleotide primers can be synthesized using commercial synthesizers, such as those supplied by Applied Biosystems (Foster City, CA) . PCR™-type methods can be used to sequence regions from the genome that are adjacent to the marker and, further, to clone the associated genomic sequences.

Once the DNA sequence encoding an entire coding region is known, it can be used to prepare non-degenerate primers corresponding to that sequence, optionally containing restriction enzyme recognition sequences to aid in cloning of various DNA products. Alternative methods for carrying out this PCR™ analysis include use of the 5' or 3' RACE methods using commercially available kits, such as those manufactured by Life Technologies (Gaithersburg, MD) or Clontech (Palo Alto, CA) . Primers for this method are selected according to the manufacturer's directions.

Gene fragments can be excised from the cloning vector by restriction enzyme digestion, labeled with ³²P by conventional methods and used as probes to identify the complete gene encoding the marker-associated polypeptide from within a cDNA library. In a preferred embodiment, the probe is chosen such that it is long enough to ensure hybridization specificity, while remaining short enough to allow reasonable rates of hybridization to the target gene. Such probes can be used to screen genomic or cDNA libraries of related or unrelated organisms.

Once the entire coding sequence of a marker- associated gene has been determined, various probes and primers can be designed around that sequence. Primers may be of any length but, typically, are 10-20 bases in length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all primers can be proposed:

n to n + y

where n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one (9 to 19) , where n + y does not exceed the last number of the sequence. Thus, for a 10-mer, the probes correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and so on. For a 15-mer, the probes correspond to bases 1 to 15, 2 to 16, 3 to 17 ... and so on. For a 20-mer, the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and so on.

Once the entire coding sequence of a marker- associated gene has been determined, the gene can be inserted into an appropriate expression system. The gene can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used to vaccinate animals to generate antisera with which further studies may be conducted.

Examples of expression systems known to the skilled practitioner in the art include bacteria such as E. coli , yeast such as Pichia pastoris, baculovirus, and mammalian expression systems such as in Cos or CHO cells. In a preferred embodiment, polypeptides are expressed in E. coli and in baculovirus expression systems. A complete gene can be expressed or, alternatively, fragments of the gene encoding portions of polypeptide can be produced.

In a preferred embodiment, the gene sequence encoding the polypeptide is analyzed to detect putative transmembrane sequences. Such sequences are typically very hydrophobic and are readily detected by the use of standard sequence analysis software, such as MacVector (IBI, New Haven, CT) . The presence of transmembrane sequences is often deleterious when a recombinant protein is synthesized in many expression systems, especially E. coli , as it leads to the production of insoluble aggregates which are difficult to renature into the native conformation of the protein. Deletion of transmembrane sequences typically does not significantly alter the conformation of the remaining protein structure.

Moreover, transmembrane sequences, being by definition embedded within a membrane, are inaccessible. Antibodies to these sequences will not, therefore, prove useful in in vivo or in si tu studies. Deletion of transmembrane-encoding sequences from the genes used for expression can be achieved by standard techniques. See Ausubel et al . , supra, Chapter 8. For example, fortuitously-placed restriction enzyme sites can be used to excise the desired gene fragment, or PCR™-type amplification can be used to amplify only the desired part of the gene.

In a preferred embodiment, computer sequence analysis is used to determine the location of the predicted major antigenic determinant epitopes of the polypeptide. Software capable of carrying out this analysis is readily available commercially, for example MacVector (IBI, New Haven, CT) . The software typically uses standard algorithms such as the Kyte/Doolittle or Hopp/Woods methods for locating hydrophilic sequences which are characteristically found on the surface of proteins and are, therefore, likely to act as antigenic determinants.

Once this analysis is made, polypeptides can be prepared which contain at least the essential features of the antigenic determinant and which can be employed in the generation of antisera against the polypeptide. Minigenes or gene fusions encoding these determinants can be constructed and inserted into expression vectors by standard methods, for example, using PCR™ cloning methodology.

The gene or gene fragment encoding a polypeptide can be inserted into an expression vector by standard subcloning techniques. In a preferred embodiment, an E. coli expression vector is used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of the protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, NJ) , the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT) , and the 6xHis system (Qiagen, Chatsworth, CA) .

Some of these systems produce recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the antigenic ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the polypeptide to its native conformation. Other fusion systems produce polypeptide where it is desirable to excise the fusion partner from the desired polypeptide. In a preferred embodiment, the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA) . In another preferred embodiment, the expression system used is one driven by the baculovirus polyhedron promoter. The gene encoding the polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector. See Ausubel et al. , supra . A preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, CA) . The vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols, and the cells are cultured and processed to produce the recombinant antigen. See Summers et al . , A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station.

As an alternative to recombinant polypeptides, synthetic peptides corresponding to the antigenic determinants can be prepared. Such peptides are at least six amino acid residues long, and may contain up to approximately 35 residues, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA) . Use of such small peptides for vaccination typically requires conjugation of the peptide to an immunogenic carrier protein such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin. Methods for performing this conjugation are well known in the art.

In a preferred embodiment, amino acid sequence variants of the polypeptide can be prepared. These may, for instance, be minor sequence variants of the polypeptide which arise due to natural variation within the population or they may be homologues found in other species. They also may be sequences which do not occur naturally but which are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence variants can be prepared by standard methods of site-directed mutagenesis such as those described above for removing the transmembrane sequence.

Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above.

Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. An example of the latter sequence is the SH2 domain, which induces protein binding to phosphotyrosine residues.

Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Insertional variants include fusion proteins such as those used to allow rapid purification of the polypeptide and also can include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide. For example, an insertional variant could include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants can include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site.

In a preferred embodiment, major antigenic determinants of the polypeptide are identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response. For example, PCR™ can be used as described above to prepare a range of peptides lacking successively longer fragments of the C-terminus of the protein. The immunoprotective activity of each of these peptides then identifies those fragments or domains of the polypeptide which are essential for this activity. Further experiments in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide.

Another preferred embodiment for the preparation of the polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules which mimic elements of protein secondary structure. See, for example, Johnson et al . , "Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al . , Eds., Chapman and Hall, New York (1993) . The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule.

Successful applications of the peptide mimetic concept have thus far focused on mimetics of /3-turns within proteins, which are known to be highly antigenic. Likely β-turn structure within an polypeptide can be predicted by computer-based algorithms as discussed above. Once the component amino acids of the turn are determined, peptide mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains, as discussed in Johnson et al . , supra .

All of the references mentioned above, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

VI. EXAMPLES

Example 1 : Preparation of Swine DNA Blood is collected and, if desired, samples may be stored at 4°C prior to use. Blood collection tubes are placed on a orbital mixer for at least three minutes at room temperature to mix whole blood completely. Nine hundred μl of cold, sterile TE buffer (0.009 g EDTA and 0.395 g Tris/HCl in 250 ml of sterile H₂0) is added to a 1.5 ml microfuge tube. One hundred μl of whole blood is added to the tubes, which are then capped and labeled.

After five minutes, the tubes are rocked gently twice by hand. Tubes are spun at 10K g for fifteen seconds and the supernatant decanted. One ml of cold, sterile TE buffer and the tube is then vortexed for 2-5 sec to resuspend the pellet. The process is repeated at least one more time or until red color is absent from the pellet.

The pellet produced according to the preceding protocol is treated with PCR™ buffer including a nonionic detergent and protease K. PCR™ buffer comprises, in 100 ml of sterile H₂0:

0.37 g KCl 1.0 ml Tris/HCl (pH 8.3)

0.024 g MgCl₂ 0.01 g Gelatin 0.45 ml NP40 0.45 ml Tween 20

The tube is gently mixed or vortexed for five sec. The tubes are then incubated in a 37°C drying on an orbital mixed for 15-18 hours, or 56°C for 90 min. The protease is deactivated in a 97°C water bath for 10 min. The sample is spun down and frozen at -20°C prior to use.

Where solid tissues are used as the source for genomic DNA, the "EASY-DNA"™ kit (Invitrogen, San Diego, CA) is employed.

Example 2 : Polymerase Chain Reaction A 25 μl PCR™ reaction mixture is prepared containing 25 ng genomic DNA (template) of the DNA isolated from pigs. To the template is added 0.2 μM of Swll29 primer pair, 2 mM MgCl₂, 50 μM each of dATP, dTTP, dGTP, dCTP, lOmM Tris-HCl pH 8.3 (at 25°C) , 50mM KCl, 0.001% gelatin, 5% DMSO and 1 unit of Tag Polymerase. The reaction mixture is first heated to 94°C for 10 min., then subjected to 45 cycles of PCR™-type amplification of the following regime: 1 minute at 94°C, 1 minute at 35°C and 2 minutes at 72°C.

Example 3 : Analysis of Amplification Products The amplified reaction mixture is then loaded onto an electrophoresis gel consisting of 1.4% agarose in a buffer of 0.089 M Tris base, 0.089 M boric acid and 0.002 M EDTA. The gel is run using the same buffer and an electric field is applied with the amplified bands moving in the direction of the anode. The gel is run until the bromophenol blue tracking dye has migrated at least 5 cm. For detection of bands, the gel is then examined with a UV light box after the gel is stained in an ethidium bromide solution (0.5 ml of EtBr (1 mg/ml in dH₂0) added to 100ml of gel buffer) .

Example 4 : Analysis of Randomly Selected Boars with

Swll29

Using randomly selected boars as a sample source, the DNA' s from ruptured and non-ruptured boars were compared according to the preceding protocol. It was found that one allele, #4, associated with the SIR⁺ phenotype. The results are provided in TABLE 1:

TABLE 1

SR ALLELES non-SR ALLELES

101 4 and 4 600 1 and 7

102 6 and 6 601 3 and 7

103 2 and 2 602 1 and 7

104 5 and 7 591 3 and 3

105 1 and 7 593 5 and 6

106 5 and 7 594 1 and 5

108 4 and 5 595 1 and 5

109 5 and 7 596 1 and 2

110 3 and 4 597 1 and 2

111 4 and 4 598 6 and 6

112 4 and 4 599 1 and 6

113 3 and 4 621 2 and 2

114 4 and 4 186 6 and 6 115 4 and 4 187 3 and 3

116 3 and 6 188 5 and 6

117 3 and 4 190 6 and 6

251 4 and 6 191 1 and 6

252 4 and 6 193 3 and 4

253 1 and 4 194 3 and 6

254 1 and 6 195 3 and 5

255 4 and 4 197 6 and 6

256 3 and 4 198 6 and 7

257 6 and 6 199 3 and 5

258 4 and 4 200 3 and 5

259 4 and 6

038 4 and 7

5722 4 and 4

850012 2 and 3 sdlO 7 and 7

2175 4 and 4

491 4 and 4

492 4 and 4

493 4 and 4

494 5 and 5

496 4 and 4

498 4 and 4

499 4 and 4

500 2 and 2

627 4 and 4

646 4 and 4 total 28 out of 41 are total 1 out of 24 is

4+ 4+ and and

18 out of 41 are 0 out of 24 are

4+\4+ 4+\4+ The data shown in Table 1 were analyzed via a chi-square analysis and a significant difference was demonstrated between the allele frequency for allele #4 in ruptured and non-ruptured boars - a chi-square value of 27.34 for allele #4 was observed for SIR boars. The critical value (p<0.005) is 12.8 (for 3 degrees of freedom) and the chance of getting a value higher than 12.8 is 1 in 200.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: DEKALB SWINE BREEDERS

(B) STREET: 3100 Sycamore Road

(C) CITY: DeKalb

(D) STATE: Illinois (E) COUNTRY: United States of America

(F) POSTAL CODE (ZIP) : 60115

(ii) TITLE OF INVENTION: EVALUATING SCROTAL HERNIA IN SWINE

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/466,586

(B) FILING DATE: 06-JUN-1995

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GATCATATGA GGAAAAGAAT GTGT 24 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CACAGGGGGA ACACCTTAAT

20

VII. REFERENCES

Archibald et al . , Mammalian Genome 6:157-175 (1995)

Davey et al., EPA No. 329 822

Derynck et al., Nature 316:701 (1985)

Dietz et al., Nature 352:337-339 (1991) Emery et al., Churchill-Livingstone, New York (1990) pp. 1047-1063

Friedman et al., Annals of Surgery 218:754-760 (1993) Frohman, M.A. , In: PCR™ PROTOCOLS : A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y. (1990)

Gingeras et al., PCT Application WO 88/10315 Hoganson et al . , ABSTRACT FOR ANNUAL MEETING OF

MIDWESTERN SECTION OF THE AMERICAN SOCIETY FOR ANIMAL SCIENCE, March 26-28, 1990

Huston et al. , Veterinary Bulletin 48:645-675 (1978)

Innis et al., PCR™ Protocols, Academic Press, Inc., San Diego CA, 1990

Johnson et al., "Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY

Jung et al., Theor. Appl . Genet. 77:271-274 (1989)

Jung et a2., Animal Genetics 20:79-91 (1989)

Kawasaki, In PCR™ PROTOCOLS, Academic Press, New York (1990) pp. 142-152

Kwoh et al., Proc. Nat'l Acad. Sci. USA 86:1173 (1989)

Miller et al . , PCT Application WO 89/06700

Ohara et al . , Proc. Nat'l Acad. Sci. USA, 86:5673-5677 (1989)

Pereira et al., Human Mol. Genet. 2:961-968 (1993);

Pezzuto et al. , Eds., Chapman and Hall, New York

(1993)Pyeritz, "Marfan Syndrome," in PRINCIPLES AND PRACTICE OF MEDICAL GENETICS, 2nd Ed., ed.

Rohrer et al., Genetics 136:231-245 (1994) Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) Schook et al., Gene Mapping Techniques and Applications, Marcel Deker, New York (1991)

Summers et al., A MANUAL OF METHODS FOR BACULOVIRUS

VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station

Vogt and Ellersieck, Am. J. Vet. Res. 51:1501-1503 (1990)

Walker et al., Proc. Nat'l Acad. Sci. USA 89:392-396 (1992)

Wu et al., Genomics 4:560 (1989)

Warwick, Wisconsin Agricultural Experiment Station Bulletin 62:1-27 (1926) .

Wu et al., Genomics 4:560 (1989)

Claims

VIII. CLAIMSWhat is claimed is:

1. A method of screening pigs for a predisposition to exhibit scrotal hernias comprising the steps of:

(a) obtaining a nucleic acid sample from a pig; and

(b) determining the presence of at least one marker in said sample that correlates with low or high incidence of hernias.

2. The method according to claim 1, wherein said determining comprises the steps of:

(i) hybridizing a microsatellite primer pair to said nucleic acid sample;

(ii) amplifying the nucleic acid between hybridized pairs of microsatellite primers; and

(iii) analyzing the amplification products of step ii.

3. The method of claim 2, wherein said nucleic acid sample is genomic DNA and said amplifying comprises PCR™ amplification.

4. The method of claim 3, wherein said PCR™ amplification employs a microsatellite primer designated Swll29.

5. A method of reducing the incidence of scrotal hernias in pigs comprising the steps of: (a) crossing a first pig exhibiting a low predisposition for hernias with a second pig exhibiting at least one other desirable trait;

(b) obtaining a nucleic acid sample from progeny of said cross;

(c) determining the presence of at least one marker in said sample that correlates with low or high incidence of hernias; and

(d) effecting a breeding decision based on step c.

6. The method according to claim 5, wherein said determining comprises the steps of :

(i) hybridizing a microsatellite primer to said nucleic acid sample;

(ii) amplifying the nucleic acid between pairs of microsatellite primers; and

(iii) analyzing the amplification products of step ii.

7. The method of claim 6, wherein said nucleic acid sample is genomic DNA and said amplifying comprises PCR™ amplification.

8. The method of claim 7, wherein said PCR™ amplification employs a microsatellite primer designated Swll29.

9. The method of claim 7, wherein said progeny are F2 resulting from FI full-sibling crosses.

10. A method of identifying a marker for scrotal hernia in pigs comprising the steps of:

(a) determining the incidence of hernias for a male pig;

(b) obtaining a nucleic acid sample from said pig; and

(c) determining the presence or absence of at least one marker in said nucleic acid sample; and

(d) repeating steps a-c with a sufficient number of male pigs to determine the relationship of said marker with the incidence of hernias.

11. The method of claim 10, wherein said pig is the progeny of a cross of a pig exhibiting a low incidence of hernias with a second pig exhibiting at least one other desirable trait.

12. The method of claim 10, wherein said determining comprises the steps of:

(i) hybridizing a microsatellite primer to said nucleic acid sample;

(ii) amplifying the nucleic acid between pairs of microsatellite primers; and

(iii) analyzing the amplification products of step ii.

13. The method of claim 12, wherein said nucleic acid sample is genomic DNA and said amplifying comprises PCR™ amplification.

14. The method of claim 11, wherein said progeny are F2 resulting from FI full-sibling crosses.

15. A kit comprising:

(a) a nucleic acid comprising Swll29 DNA;

(b) at least one receptacle.

16. A kit according to claim 15, further comprising dATP, dCTP, dGTP and dTTP.

17. A kit according to claim 16, further comprising a polymerase.