JP6792779B2 - Method for discriminating highly immunocompromised pigs and polymorphism markers for that purpose - Google Patents

Description

The present invention relates to a marker utilizing a polymorphism for discriminating the immunity of a pig, and a method for discriminating a pig using the marker.

Until now, pig breeding has been vigorously carried out with respect to meat quality and the like, which are easy to measure, by statistical methods using phenotypic values of traits. Recent porcine genome sequencing and population genetic analysis using the data have revealed evidence of loss of diversity in the genomic region containing genes related to vertebral bone number and body length, depending on the desired phenotype. Changes in the genomic nucleotide sequence due to selection are becoming clear.

On the other hand, regarding the resistance to infectious diseases such as pneumonia and diarrhea, that is, the anti-disease property, it is difficult to measure the degree of the disease / lesion, and almost no improvement attempt has been made from this point.

In the research group of the applicants, three types of trait values, leukocyte hypophagia, complement alternative pathway activity, and antibody titer after porcine toxin vaccination, were simultaneously increased in the Large White pig species. We have created a highly immune pig population that has been improved over generations. Then, in this population, we obtained data suggesting that the mortality rate during growing on mycoplasma-positive farms is significantly reduced (Non-Patent Document 1).

On the other hand, it is known that a large number of single nucleotide polymorphisms (SNPs) including amino acid polymorphisms are present in the immune system genes of pigs and wild boars, especially Toll-like receptors (TLRs) involved in pathogen recognition. (Non-Patent Document 2), it has been reported that some of these amino acid polymorphisms change the recognition ability of pathogens (Non-Patent Documents 3 and 4). In addition, TLR and porcine leukocyte antigen (SLA) class II genes in pig populations (Durok species) kept in a specific-pathogen-free (SPF) environment where there are no specific diseases that adversely affect pig health. It has been reported that there is a significant correlation between type and antibody response after vaccination (Non-Patent Document 5).

Takahagi et al., 9th International Veterinary Immunology Symposium, 2010 Shinkai, H. et al., Immunogenetics 58: 324-330, 2006 Shinkai, H. et al., Mol. Immunol. 48: 1114-1120. 2011 Shinkai H. et al., DNA Cell Biol. 31: 575-581, 2012 Shinkai H. et al., Comp. Immunol. Microbiol. Infect. Dis. 35: 523-532, 2012

In recent years, in large-scale intensive pig farming, the epidemic of infectious diseases caused by poaching has become a major factor pushing up production costs due to pig wear and drug use, and inheritance of pig disease resistance. Expectations for improvement are high.

The present inventors have investigated the possibility that indirect immune traits that can be measured from blood samples can substitute for direct traits such as lesions. Then, from the data suggesting a decrease in mortality rate at a specific farm obtained for a pig population improved so that indirect trait values such as leukocyte anemia are high, blood-derived indirect immune traits are disease-resistant in pigs. I thought it could be a selection index for improvement. In addition, from the relationship between the presence of SNP in TLR and changes in pathogen recognition ability, and the correlation between genotypes such as TLR in the SPF pig population and antibody response after vaccination, polymorphisms in immune system genes are associated with infectious diseases. We thought that it could be a marker for judging susceptibility / resistance.

On the other hand, the present inventors focused on individual differences in expression changes in macrophages. Macrophages are cells responsible for pathogen recognition and food loss in the early stages of infection and play a vital role in both the innate and adaptive immune systems. We have recently isolated and stimulated macrophages from the blood of the highly immunized pig population and the pig population that was the basis for its formation, and identified immune system genes that showed differences in expression changes after stimulation between the populations. Furthermore, we found a polymorphism that is biased among populations in the promoter region of the gene, and completed the present invention.

The present invention provides:
[1] A method for discriminating highly immunocompromised pigs, in which at least one of the bases at the positions on the chromosomes shown in (1) to (115) below in the genome of the target pig is shown in the corresponding lower row. A method for determining a highly immune pig when it is a base or a base sequence.

[2] The method described in 1.
A method for determining a highly immune pig when the genome of the target pig satisfies at least one of the following (A) to (F).
(A) The bases at the positions on the chromosomes shown in (1) to (9) above are the corresponding bases in the lower row.
(B) The bases at the positions on the chromosomes shown in (10) to (15) above are the corresponding bases in the lower row.
(C) The bases at the positions on the chromosomes shown in (16) to (45) above are the corresponding bases in the lower row.
(D) The bases at the positions on the chromosomes shown in (46) to (65) above are the corresponding bases in the lower row.
(E) The bases at the positions on the chromosomes shown in (66) to (115) above are the corresponding bases in the lower row.
[3] One of the following polynucleotides (a) to (e) to be used for discriminating highly immunocompromised pigs.
(a) The entire base sequence of SEQ ID NO: 1 or a continuous part containing at least one base at the positions shown in (1) to (9) below, and (1) to (9) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(b) The entire base sequence of SEQ ID NO: 2, or a continuous part containing at least one base at the positions shown in (10) to (15) below, and (10) to (15) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(c) The entire base sequence of SEQ ID NO: 3, or a continuous part containing at least one base at the positions shown in (16) to (45) below, and (16) to (45) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(d) The entire base sequence of SEQ ID NO: 4, or a continuous part containing at least one base at the positions shown in (46) to (65) below, and (46) to (65) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(e) The entire base sequence of SEQ ID NO: 5, or a continuous part containing at least one base at the positions shown in (66) to (115) below, and (66) to (115) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

[4] Complementary to a part of the base sequence set forth in any one of SEQ ID NOs: 1 to 5 and having a length of 15 bases or more for use in the discrimination method described in 1 or 2. , Probes or primers.
[5] A method for breeding highly resistant pigs, which comprises selecting highly immune pigs by the discrimination method according to 1 or 2.
[6] A method for breeding highly resistant pigs, which comprises selecting highly immune pigs by the discrimination method according to 1 or 2 and using the selected pigs or their progeny for mating.
[7] A method for producing a highly resistant pig, which comprises selecting a highly immune pig by the discrimination method according to 1 or 2 and using the selected pig or its progeny for mating.

According to the present invention, pigs with high immunity can be efficiently selected.
The highly immunized pig obtained by the present invention can impart high immunity to pigs having other excellent traits, and a new breed of pig having excellent disease resistance can be bred.

A gene in which a difference of 3 times or more was detected in the expression change after stimulation. Gene Ontology analysis was performed, and genes assumed to be related to the immune response ([GO: 0002376] immune system process, [GO: 0001071] Nucleic acid binding transcription factory activity, [GO: 0006914] autophagy, [GO: 0061630] ubiquitin protein ligase activity) was extracted. A gene in which a difference of 3 times or more was detected in the expression change after stimulation. Gene Ontology analysis was performed, and genes assumed to be related to the immune response ([GO: 0002376] immune system process, [GO: 0001071] Nucleic acid binding transcription factory activity, [GO: 0006914] autophagy, [GO: 0061630] ubiquitin protein ligase activity) was extracted.

The numerical range "m to n" includes both ends m and n unless otherwise specified.

[Discrimination method]
The present invention provides a method for discriminating a pig having high immunity (sometimes referred to as a highly immune pig) by detecting a polymorphism existing in the genome of the pig. Immunity includes direct or indirect immune traits. Direct immune traits include less susceptibility to infectious diseases such as pneumonia and diarrhea and lesser disease and lesions. Indirect immune traits do not directly support porcine anti-disease, such as leukocyte hypophagia, complement alternative pathway activity, and antibody titer after erysipeloid vaccination, but the trait and anti-disease Refers to a trait whose relationship can be inferred.

For discrimination, the target pig (the pig used in the discrimination method of the present invention) is determined to be a highly immune pig with technically significant certainty, and the target pig is a non-immunized pig (has high immunity). It includes determining that it is not a pig) with technically significant certainty. It also refers to distinguishing between highly immune pigs and other pigs (eg, black pigs, general pigs, etc.). Discrimination may be expressed as identification, appraisal, discrimination, judgment, inspection, and the like. It is considered that the certainty of discrimination can be improved by examining the type, number, combination, etc. of the polymorphism to be used.

Polymorphism refers to the existence of individuals with different genotypes in a population of the same species, or the gene / DNA sequence of the different genotypes. Polymorphisms include single nucleotide polymorphisms (SNPs), Insertion / Deletion (insertion / deletion) polymorphisms, restricted fragment length polymorphisms, tandem repeats polymorphisms (VNTR), hypervariable regions, and mini. Includes satellite, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, and simple sequence repeats.

Single nucleotide polymorphisms (SNPs) occur at polymorphic sites consisting of one nucleotide, where one nucleotide is replaced by another (nucleotide, or di-tetranucleotide, oligonucleotide or polynucleotide). Single nucleotide polymorphisms also occur due to the deletion of one nucleotide or the insertion of one nucleotide compared to the reference allele. When expressing a polymorphism, the type of nucleotide involved may be indicated by using the type of base (A, G, T or C) constituting the nucleotide for convenience.

In the discrimination method of the present invention, a polymorphism existing in the porcine genome is used as a marker.

In one of the preferred embodiments, as a polymorphism marker (a marker utilizing the polymorphism), macrophages collected from each of the hyperimmune pig population and the basal population (sometimes referred to as a control population) used for its formation are stimulated. As a result of comparing gene expression, polymorphisms present in the promoter region of a gene whose expression level changes several times, preferably two times or more, more preferably three times or more can be used.

In one of the more preferred embodiments, as the polymorphism marker, a polymorphism that is a gene whose expression level changes and is present in the promoter region of a gene that is supposed to be involved in an immune response can be used. Examples of genes that are supposed to be involved in the immune response are Gene Ontology [GO: 0002376] immune system process, [GO: 0001071] Nucleic acid binding transcription factory activity, [GO: 0006914] autophagy, or [GO: 0061630]. It is a gene belonging to the hierarchy of ubiquitin protein ligase activity. Particularly preferred examples of genes that are supposed to be involved in the immune response are TRIM21 (one of the ubiquitin ligase E3, the causative gene of Sjogren's syndrome), STAT3 (involved in cytokine signaling, etc.), RNASEL (RNA degrading enzyme). One of them. Involved in resistance to RNA virus.), Or SAMHD1 (involved in protection against viral infections such as HIV-1.), And TMEM150C.

In any case, it is preferable to use a polymorphism marker having a higher frequency in the hyperimmune pig population than in the basal population.

Specific examples of the polymorphism marker used in the present invention (may be referred to as "polymorphism marker of the present invention" in the present specification) are shown below. In addition, in this specification and claims, information on chromosomes and positions, and SNP rs numbers are based on information provided by the National Center for Biotechnology Information (NCBI) unless otherwise specified.

Polymorphisms present in the promoter region of RNASEL ((1) to (9) in the table below)
The middle part of the table shows the chromosomes where polymorphisms exist and their positions (polymorphism sites). The lower row shows the bases that frequently appear in the highly immune pig population at that position. That is, when the base at that position is the base shown in the corresponding lower part of the table, it can be determined to be a highly immune pig.

Polymorphisms present in the promoter region of SAMHD1 ((10) to (15) in the table below)
The middle part of the table shows the chromosomes where polymorphisms exist and their positions (polymorphism sites). The lower row shows the bases that frequently appear in the highly immune pig population at that position. That is, when the base at that position is the base shown in the corresponding lower part of the table, it can be determined to be a highly immune pig.

Polymorphisms present in the promoter region of STAT3 ((16)-(45) in the table below)
Chromosomes in which polymorphisms are present and their positions (polymorphism sites) are shown in the middle of each table. The lower row of each shows the bases that frequently appear in the highly immunocompromised pig population at that position. That is, when the base at that position is the base shown in the corresponding lower part of the table, it can be determined to be a highly immune pig.

Polymorphisms present in the promoter region of TMEM150C ((46)-(65) in the table below)
The middle part of the table shows the chromosomes where polymorphisms exist and their positions (polymorphism sites). The lower row shows the bases that frequently appear in the highly immune pig population at that position. That is, when the base at that position is the base shown in the corresponding lower part of the table, it can be determined to be a highly immune pig.

Polymorphisms present in the promoter region of TRIM21 ((66)-(115) in the table below)
Chromosomes in which polymorphisms are present and their positions (polymorphism sites) are shown in the middle of each table. The lower row of each shows the bases that frequently appear in the highly immunocompromised pig population at that position. That is, when the base at that position is the base shown in the corresponding lower part of the table, it can be determined to be a highly immune pig.

In the present invention, one polymorphic marker can be used, and a plurality of polymorphic markers can be used. It is considered that the polymorphism marker used is preferably one in which the frequency difference between the highly immunocompromised pig population and the basal population is large among the above-mentioned polymorphism markers. A person skilled in the art can appropriately design the number and types of polymorphic markers used for discrimination by taking into consideration the description of this specification and common general technical knowledge.

In the case of polymorphic markers with large frequency differences between the hyperimmune pig population and the basal population, the use of one or several polymorphic markers provides technically significant certainty of the hyperimmune pigs. It is considered that it is possible to distinguish. Even in the case of polymorphic markers with a relatively small difference in frequency between the hyperimmune pig population and the basal population, by using multiple polymorphism markers, discrimination can be made with the technically significant certainty of the hyperimmune pigs. It is considered possible.

In general, the more polymorphic markers are used, the better the discrimination accuracy. Therefore, in the present invention, it may be preferable to use a plurality of polymorphic markers. The number of polymorphic markers used in the method of the present invention can be 1 or several (1 to 9), for example, 5 or more, 8 or more, 10 or more, and 20. As mentioned above, the number may be 30 or more, 40 or more, 50 or more, 60 or more, or 70 or more.

In one of the preferred embodiments, any one of the bases at the position on the chromosome shown in one of the above tables (which may be one or more; the same shall apply hereinafter) and any other. If the combination with any of the bases at the positions on the chromosome shown in the table is the corresponding base shown in the lower row, it may be determined to be a highly immunocompromised pig. For example, when any of the bases at the positions on the chromosomes shown in (1) to (9) above is the corresponding base shown in the lower row, the positions on the chromosomes shown in (10) to (15) above. Any of the bases at the positions on the chromosomes shown in (16) to (45) above, any of the bases at the positions on the chromosomes shown in (46) to (65) above, or the above If any of the bases at the positions on the chromosomes shown in (66) to (115) is the corresponding base shown in the lower row, it may be determined to be a highly immune pig.

In one of the preferred embodiments, a highly immune pig can be determined if at least one of the following (A) to (E) is satisfied.
(A) The bases at the positions on the chromosomes shown in (1) to (9) above are the corresponding bases in the lower row.
(B) The bases at the positions on the chromosomes shown in (10) to (15) above are the corresponding bases in the lower row.
(C) The bases at the positions on the chromosomes shown in (16) to (45) above are the corresponding bases in the lower row.
(D) The bases at the positions on the chromosomes shown in (46) to (65) above are the corresponding bases in the lower row.
(E) The bases at the positions on the chromosomes shown in (66) to (115) above are the corresponding bases in the lower row.

In one of the preferred embodiments, any two of (A) to (E) are satisfied (eg, (A) and (B), (A) and (C), (A) and (D). , (A) and (E), (B) and (C), (B) and (D), (B) and (E), (C) and (D), (C) and (E) Or (D) and (E)), if any three are met (eg, (A) and (B) and (C), (A) and (B) and (D), (A) and (B) ) And (E), (A) and (C) and (D), (A) and (C) and (E), (A) and (D) and (E), (B) and (C) and When any four of (D), (B) and (D) and (E), (C) and (D) and (E) are satisfied (for example, (A) and (B) and (C)) And (D), (A) and (B) and (C) and (E), (A) and (B) and (D) and (E), (A) and (C) and (D) and ( High immunity when E), (B) and (C) and (D) and (E)), or all, ie (A) and (B) and (C) and (D) and (E) are satisfied It may be determined that it is a Noh pig.

In one of the preferred embodiments, various known methods can be used as methods for determining the base of the desired polymorphic site in the target pig. The method of determining the base of a polymorphic site usually involves the step of amplifying the region containing the polymorphic site. Nucleic acid samples that serve as templates for amplification include, for example, peripheral blood, organ tissue, serum, plasma, blood cells, bone marrow, bone-separated mononuclear cells, oral cells, hair (hair root cells), nails, blood stains, and umbilical band , Cerebral spinal fluid, affected area scrapes, affected area wipes, atrioventricular water, lymph nodes, bronchial alveolar lavage fluid, pleural effusion, ascites, cardiac sac fluid, pancreatic fluid, sputum, bone, pus, urine, feces, vomitus, gastric fluid, It can be obtained from lymph nodes, skin prisoners, intestinal tissue, solid tissue (biopsy surgery), paraffin embedding block, frozen section embedding block, autopsy material (tissue), cultured cells.

The nucleic acid contained in the sample thus obtained can be used as a template and amplified to detect a polymorphism. Existing methods can be appropriately utilized for the detection of polymorphisms. For example, PCR method, SDA method, TMA method, NASBA method, TRC method, ICAN method, SmartAmp method, RCA method, LAMP method, and their improved methods can be mentioned.

[Polymorphic marker and probe for detecting it]
The present invention also comprises the polymorphism marker according to any one of (1) to (115) above, and a polynucleotide containing such a polymorphism marker for use in discriminating highly immune pigs, and such. Auxiliary products of such polynucleotides, compositions containing such polynucleotides (eg, including such polynucleotides and buffers), kits, reagents, products and the like are provided.

In the sequence listing of the present specification, the nucleotide sequence of 5000 bp upstream from the transcription start point of each gene for which the polymorphism search was performed in the section of the examples of the present specification is listed as the sequence of SEQ ID NO: 1 to 5. Specifically, it is as follows.
SEQ ID NO: 1 Nucleotide sequence of 5000 bp upstream from the transcription start point of RNACSEL (Sscrofa 10.2: 9: 136224411: 136229410)
SEQ ID NO: 2 Nucleotide sequence of 5000 bp upstream from the transcription start point of SAMHD1 (Sscrofa 10.2: 17: 45580068: 45585067)
SEQ ID NO: 3 Nucleotide sequence of 5000 bp upstream from the transcription start point of STAT3 (Sscrofa 10.2: 12: 20775572: 20780571)
SEQ ID NO: 4 Nucleotide sequence of 5000 bp upstream from the transcription start site of TMEM150C (Sscrofa 10.2: 8: 145098791: 145103790)
SEQ ID NO: 5 Nucleotide sequence of 5000 bp upstream from the transcription start point of TRIM21 (Sscrofa 10.2: 9: 6531121: 6536120)
The numbers in parentheses are the chromosome numbers and base positions in the latest version of the published porcine genome sequence (Sscrofa 10.2).

The present invention provides the following polynucleotides (a) to (e) containing polymorphic markers.
(a) The entire base sequence of SEQ ID NO: 1 or a continuous part containing at least one base at the positions shown in (1) to (9) below, and (1) to (9) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(b) The entire base sequence of SEQ ID NO: 2 or a continuous part containing at least one base at the positions shown in (10) to (15) below, and (10) to (15) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(c) The entire base sequence of SEQ ID NO: 3, or a continuous part containing at least one base at the positions shown in (16) to (45) below, and (16) to (45) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(d) The entire base sequence of SEQ ID NO: 4, or a continuous part containing at least one base at the positions shown in (46) to (65) below, and (45) to (65) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

(e) The entire base sequence of SEQ ID NO: 5, or a continuous part containing at least one base at the positions shown in (66) to (115) below, and (66) to (115) below. A polynucleotide in which at least one of the bases at the position shown in) is the corresponding base or base sequence shown in the lower row.

The length of the polynucleotide containing the above polymorphic marker is not particularly limited, but is, for example, 4000 bases or less, 3000 bases or less, 2000 bases or less, 1000 bases or less, 500 bases or less, 300 bases or less, It may be 100 bases or less, 50 bases or less, 30 or 20 bases or less.

The present invention also provides a distinguishing or discriminating reagent for use in a method for discriminating highly immunocompromised pigs. A preferred embodiment of the reagent is a test agent for discriminating highly immunocompromised pigs, which comprises any of the following substances (a) or (b) as an active ingredient.
(A) An oligonucleotide that hybridizes to the base sequence set forth in any of SEQ ID NOs: 5 to 10 or its complementary strand under stringent conditions, and is present in the base sequence described above (1) to (1) to ( An oligonucleotide for a PCR primer for amplifying a region containing any of the polymorphic sites of 115).
(B) An oligonucleotide that hybridizes to the base sequence set forth in any of SEQ ID NOs: 5 to 10 or its complementary strand under stringent conditions, and is present in the base sequence described above (1) to (1) to ( An oligonucleotide for a probe, which hybridizes with a DNA region containing any of the polymorphic sites of 115).

As used herein, "hybridizing" refers to normal hybridization conditions, preferably stringent hybridization conditions (eg, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd Edition). Under the conditions described in 1989), it means that cross-hybridization with DNA other than the intended one does not occur significantly. If specific hybridization is possible, the polynucleotide need not be completely complementary to the nucleotide sequence to be detected.

Hybridization conditions can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringent conditions. The low stringent condition is, for example, 42 ° C., 0.1 × SSC, 0.1% SDS, preferably 50 ° C., 0.1 × SSC, 0.1% SDS in the washing after hybridization. More preferable hybridization conditions include high stringent conditions. High stringent conditions are, for example, 65 ° C., 5 × SSC and 0.1% SDS. Under these conditions, it can be expected that DNA having higher homology is efficiently obtained as the temperature is raised. However, a plurality of factors such as temperature and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. ..

Such a polynucleotide can be used as a probe or a primer in the discrimination method of the present invention. When a polynucleotide is used as a primer, its length is usually 15 to 100 bases, preferably 17 to 30 bases. The primer is not particularly limited as long as it can amplify the DNA containing the polymorphism marker of the present invention. When a polynucleotide is used as a probe, the probe is not particularly limited as long as it specifically hybridizes to the DNA region containing the polymorphism marker of the present invention. The probe may be a synthetic polynucleotide and usually has a length of at least 15 bases or more. The primer and probe can be appropriately designed with reference to the conventional primer design method and probe design method. Software is commercially available for methods of designing primers and can also be used for the present invention.

The polynucleotide used as a primer or probe can be produced, for example, by a commercially available polynucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.

When the polynucleotide of the present invention is used as a probe, it is preferably used by appropriately labeling it. As a labeling method, a method of labeling by phosphorylating the 5'end of the polynucleotide with ³² P using T4 polynucleotide kinase, and a method of labeling a random hexamer polynucleotide or the like using a DNA polymerase such as Klenow enzyme are used. Examples of a method (random prime method, etc.) of incorporating an isotope such as ³² P, a fluorescent dye, or a substrate base labeled with biotin or the like as a primer can be exemplified.

In the above-mentioned identification or discrimination reagent (test agent of the present invention), in addition to the polynucleotide which is an active ingredient, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, etc. A protein stabilizer (BSA, gelatin, etc.), a preservative, etc. may be mixed as required.

A kit for discriminating highly immunocompetent pigs containing at least the reagent of the present invention is also included in the present invention. In addition to the substance described in either (a) or (b) above, the kit includes, for example, various reagents, reaction solutions, control preparations, reaction vessels, and operating instruments for use in the method of the present invention. Etc. can be included.

[Breeding method]
As described above, the present invention provides a method for discriminating highly immunocompromised pigs. Therefore, according to the present invention, it is possible to predict a pig having a desired trait (high immunity or anti-disease) at an early stage, and thus efficiently create a pig population or lineage having the desired trait. Helps to do. Therefore, the present invention provides a method for breeding highly resistant pigs, which comprises selecting highly immunocompetent pigs by the discrimination method of the present invention.

As a breeding method for pigs in which pigs having a predetermined trait are selected and raised or bred, for example, a highly immunized pig or a progeny selected by the present invention is applied to a certain strain to a certain preferred trait (for example, meat-producing ability). By using it for mating with pigs having high, robustness, high fertility, etc.), it is possible to create a strain of pigs having a certain trait and having anti-disease properties. Therefore, the present invention comprises a method for raising highly resistant pigs, which comprises selecting highly immunocompetent pigs by the discrimination method of the present invention and using the selected pigs or their progeny for mating, and a method for discriminating the present invention. To provide a method for producing highly resistant pigs, which comprises selecting highly immune pigs and using the selected pigs or their progeny for mating. It should be noted that, according to the discrimination method of the present invention, highly immunocompetent pigs are selected, and pigs produced by a method for producing pigs with high disease resistance, including using the selected pigs for mating, are propagated to obtain a progeny. Also, regardless of whether or not highly immunocompetent pigs are selected for the progeny by the discrimination method of the present invention, as long as a progeny with high disease resistance associated with the polymorphic marker provided by the present invention can be obtained, the present invention Included in the category of production methods.

[Verification of polymorphic effects, etc.]
It can be verified by various methods that the polymorphism disclosed by the present application has the desired effect. For example, an expression vector into which a polymorphism has been introduced is prepared, and the gene is introduced into porcine-derived cultured cells, HEK293 cells, or the like, and then a reporter assay is performed to obtain a ligand such as lipopolysaccharide (Lipid A, TLR4 ligand) and polyinosinic-polycytidylic. This can be verified by analyzing changes in responsiveness to pathogen stimuli using acid (poly (I: C), ligands of RNA sensor molecules such as TLR3 and RIG-I / MDA-5). It can also be verified by investigating the association between immunocompetence and disease-related traits (eg, pneumonia prevalence, etc.) and polymorphisms using populations other than the hyperimmune pig population and the basal population. Such verification means are well known to those skilled in the art, and those skilled in the art can appropriately plan and carry out experiments.

Conventionally, since it is difficult to measure the traits of porcine anti-disease, few attempts have been made to improve heredity. According to the present inventors, indirect immune traits may be a selection index for improving anti-disease, and polymorphisms related to immune system genes may be markers of susceptibility / resistance to infectious diseases. Found. The present invention made on the basis of the present invention provides a polymorphism marker for discriminating highly immunocompetent pigs and its use, and is considered to be useful for the realization of anti-disease breeding.

The present invention also provides highly immunocompetent or highly resistant pigs with the novel polynucleotides provided by the present invention. Furthermore, the present invention provides meat and processed meat products (eg, ham, sausage, bacon, roasted pork, etc.) obtained from the highly immunocompromised or highly resistant pigs obtained by the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Analysis of changes in gene expression in a highly immune pig population]

(Isolation of macrophages from kidney)
Kidneys were collected from four experimental pigs of the same sex belonging to the highly immune pig population and its basal population. Macrophages were isolated by a method capable of repeated cell recovery using a mixed culture system (Non-Patent Document 6). Specifically, when human tissue is dispersed into glomerular / tubular units by collagenase treatment and then seeded in a culture flask, macrophages proliferate in the upper layer of human parenchymal cells, which are feeders, after 2 to 3 weeks. Since it came, I collected it and used it for the experiment. By adding the medium to the flask after kidnapping and culturing, macrophages could be repeatedly proliferated and recovered, and the number of cells required for the experiment was secured.

(Expression analysis of immune system molecules)
Macrophages derived from all eight experimental pigs were analyzed in a three-point time series of 0 (unstimulated), 1 and 4-hour stimulation. Stimulation of RNA sensor molecules such as lipopolysaccharide (Lipid A, TLR4 ligand) and polyinosinic-polycytidylic acid (poly (I: C), TLR3 and RIG-I / MDA-5), assuming bacterial and viral infections. It was done by ligand). Total RNA was extracted from cells after stimulation for 4 hours, and comprehensive expression analysis of mRNA was performed by microarray.

Gene Ontology analysis was performed on the dataset for expression changes, and genes assumed to be related to the immune response ([GO: 0002376] immune system process, [GO: 0001071] Nucleic acid binding transcription factory activity, [GO: 0006914] autophagy, [GO: 0061630] ubiquitin protein ligase activity) was extracted. The genes in which a difference of 3 times or more was detected between the highly immunized pig population and the basal population are shown in FIGS. 1A and 1B.

In the highly immunized pig population, the expression was increased by more than 3 times compared with the basal population in 42 genes with Lipid A stimulation and 27 genes with poly (I: C) stimulation. Twenty-five of these genes were detected in duplicate, suggesting that the difference due to stimulation is not large. Promoter polymorphisms were detected in 42 of the detected genes, excluding the FOXS1 gene and PLSCR1 gene, for which information is scarce.

[Detection of promoter polymorphisms of immune system molecules with different expression]
(Polymorphism detection)
A polymorphism search of the promoter region of 5 kb was performed on 10 animals from each population. Using the porcine genome nucleotide sequence (Sscrofa10.2) (Non-Patent Document 7) decoded by the International Pig Genome Sequencing Consortium, about 5 kb upstream from the transcription start site was extracted, and primers for sequencing were synthesized.

As a result, a total of 1654 SNPs and 577 Insertions / Deletions were detected in the promoter region 200.48 kb of 42 genes. Of these, 50, 30, 20, 9, and 6 of TRIM21, STAT3, TMEM150C, RNASEL, and SAMHD1, respectively, were identified as polymorphisms with significantly different distribution patterns among populations. The table below summarizes the promoter polymorphisms whose distribution was biased between the highly immunized pig population and the basal population. All rows are the number of all polymorphisms detected, Complete genotype rows are the number of polymorphisms for which data could be obtained for all 20 animals, and rows P <0.01 and P <0.001 are significant among the populations. Shows the number of polymorphisms shown to have different distribution patterns (chi-square independence test).

(Haplotype estimation)
The haplotype composed of promoter polymorphisms was estimated and shown in the table below.

[References cited in the examples]
Non-Patent Document 6: Kitani, H., T. Takenouchi, M. Sato, M. Yoshioka, M. Yamanaka, J. Vis Exp.51 e2757, 2011
Non-Patent Document 7: Groenen, MAM, AL Archibald, H. Uenishi, et al., Nature 491: 393-398, 2012

SEQ ID NO .: 1 Nucleotide sequence of 5000 bp upstream from the transcription start site of RNASEL
SEQ ID NO .: 2 Nucleotide sequence of 5000 bp upstream from the transcription start site of SAMHD1
SEQ ID NO .: 3 Nucleotide sequence of 5000 bp upstream from the transcription start point of STAT3
SEQ ID NO .: 4 Nucleotide sequence of 5000 bp upstream from the transcription start site of TMEM150C
SEQ ID NO .: 5 Nucleotide sequence of 5000 bp upstream from the transcription start site of TRIM21

Claims (7)

A method for discriminating highly immunocompromised pigs, in which at least one of the bases at the positions on the chromosomes shown in (1) to (115) below in the genome of the target pig is the base or base shown in the corresponding lower row. A method of determining a hyperimmune pig if it is a sequence.

The method according to claim 1.
A method for determining a highly immune pig when the genome of the target pig satisfies at least one of the following (A) to (F).
(A) The bases at the positions on the chromosomes shown in (1) to (9) above are the corresponding bases in the lower row.
(B) The bases at the positions on the chromosomes shown in (10) to (15) above are the corresponding bases in the lower row.
(C) The bases at the positions on the chromosomes shown in (16) to (45) above are the corresponding bases in the lower row.
(D) The bases at the positions on the chromosomes shown in (46) to (65) above are the corresponding bases in the lower row.
(E) The bases at the positions on the chromosomes shown in (66) to (115) above are the corresponding bases in the lower row. One of the following polynucleotides (a) to (e) to be used for discriminating highly immune pigs.
(a) The entire base sequence of SEQ ID NO: 1 or a continuous part containing at least one base at the positions shown in (1) to (9) below, and (1) to (9) below. ), At least one of the bases is the corresponding base or base sequence shown in the lower row, and the polynucleotide has a length of 500 bases or less.

(b) The entire base sequence of SEQ ID NO: 2, or a continuous part containing at least one base at the positions shown in (10) to (15) below, and (10) to (15) below. ), At least one of the bases is the corresponding base or base sequence shown in the lower row, and the polynucleotide has a length of 500 bases or less.

(c) The entire base sequence of SEQ ID NO: 3, or a continuous part containing at least one base at the positions shown in (16) to (45) below, and (16) to (45) below. ), At least one of the bases is the corresponding base or base sequence shown in the lower row, and the polynucleotide has a length of 500 bases or less.

(d) The entire base sequence of SEQ ID NO: 4, or a continuous part containing at least one base at the positions shown in (46) to (65) below, and (46) to (65) below. ), At least one of the bases is the corresponding base or base sequence shown in the lower row, and the polynucleotide has a length of 500 bases or less.

(e) The entire base sequence of SEQ ID NO: 5, or a continuous part containing at least one base at the positions shown in (66) to (115) below, and (66) to (115) below. ), At least one of the bases is the corresponding base or base sequence shown in the lower row, and the polynucleotide has a length of 500 bases or less.

It is possible to hybridize to a part of the base sequence according to any one of SEQ ID NOs: 1 to 5 under stringent conditions and 15 bases for use in the discrimination method according to claim 1 or 2. having the above lengths, a probe or primer,
A PCR primer for amplifying a region containing either the base or the base sequence shown in the corresponding lower row at the position on the chromosome shown in claims 1 (1) to (115), or
A probe that hybridizes to a region containing either the base or the base sequence . A method for breeding pigs with high disease resistance, which comprises selecting highly immune pigs by the discrimination method according to claim 1 or 2. A method for breeding highly resistant pigs, which comprises selecting highly immunocompetent pigs by the discrimination method according to claim 1 or 2 and using the selected pigs or their progeny for mating. A method for producing a highly resistant pig, which comprises selecting a highly immunocompromised pig by the discrimination method according to claim 1 or 2 and using the selected pig or its progeny for mating.

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