JP2016077273A - Production method of pig having desired trait - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple production method in which a pig having a desired trait can be produced efficiently in a short term, and the number of times of selection steps in selective mating is reduced.SOLUTION: A production method includes: a step of respectively selecting a boar and sow having a specific genotype associated with the desired trait as a female-based male species pig and a female-based female species pig from a pig group by using a genotypical analysis result associated with a desired trait; a step of obtaining a female species pig by mating the female-based male species pig and the female-based female species pig selected; a step of obtaining a male childbirth of a male species pig candidate by using a genotypical analysis result similarly; a step of analyzing the genetic genotype associated with the desired trait concerning the male childbirth of the male species pig candidate, and selecting an individual having a specific genotype associated with the desired trait as a male species pig from the male childbirth of the male species pig candidate; and a step of mating the male species pig with the female species pig, and obtaining a childbirth.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing a pig having a desired character used as an experimental animal for medical research or the like. Specifically, the present invention relates to a method for producing a pig having a desired trait by combining genetic analysis of pig individuals and mating of individuals selected based on the analysis results.

  In drug development, preclinical studies using animals (dogs, monkeys, pigs, etc.) are conducted before the human clinical trials. This type of test requires that the trait of the animal being tested be constant in order to increase reproducibility. In addition, in order to elucidate the mechanism of a disease, a test using an animal having a trait related to the disease state of the disease (for example, a disease state model lacking a carbohydrate metabolism function in a test for a diabetic drug) is performed. Thus, the experimental animal is required to have a certain character or a specific character.

  On the other hand, among experimental animals, pigs are considered to be suitable for experimental animals because they are anatomically and physiologically close to humans and have few ethical obstacles. However, since pigs generally have a large body weight of 80 to 130 kg at the time of sexual maturity and are expensive to breed and administer, they have not been widely used as experimental animals. However, in recent years, a small ultra-small pig whose weight at the time of sexual maturity is about 10 kg or less has been developed (Patent Document 1). Therefore, in order to use this ultra-small pig as an experimental animal, it is desired to produce a large number of ultra-small pigs having specific traits required for experimental animals.

  In general, animals having specific traits are produced by selective crossing or somatic cell cloning techniques. The selection method by selective mating is to select those with the desired specific traits from the population of animals, and further select the offspring while crossing these, and repeat the mating to have the traits It is a method of creating a new line. If males and females having a desired trait are obtained, an animal having the trait can be obtained by mating them. The somatic cell cloning technology is a method for producing a large number of cloned animals from a somatic cell having a desired trait or a somatic cell into which a gene expressing a desired trait has been introduced by a cloning technique. Is the method.

JP 2010-166819 A

  However, in the production method by selective crossing described above, there is a problem that many generations must be crossed before the desired trait is fixed, and a long time is required until a strain having the desired trait is completed. was there. In addition, it is necessary to select only individuals with the desired trait every time they are bred, especially when trait whose trait does not appear on the outer shape is desired, genetic testing of each individual is required, so selection work Took a lot of effort. In addition, since a large number of individuals that were not selected are generated, the disposal of these individuals has become an ethical problem. In addition, even when an animal group having a desired trait is completed, in order to maintain the group, mating must be performed within the group, so the inbreeding coefficient of the litter increases and inbreeding may occur. There was sex.

  Moreover, in the production method by the somatic cell cloning technique, the success of cloning is low because the cloning operation is delicate and complicated. For example, the success rate of cloning from porcine somatic cells is about 1 to 5%. Therefore, there has been a problem that it is inefficient as a means for producing a large number of experimental animals.

  The present invention has been devised in view of the above points, and its purpose is to easily produce a pig having a desired trait in a short period of time, and to reduce the number of selection steps in selective crossing. The purpose is to provide a production method.

  Another object of the present invention is to provide a method for efficiently and easily producing a large number of ultra-small pigs having a desired character in a short period of time.

  In order to solve the above problems, a method for producing a pig having a desired trait according to the present invention includes a step of determining a desired trait, and a genotype of a gene related to the determined desired trait in an arbitrary pig population. Using the analysis step and the analysis result of the genotype, from the swine population, (a) when the desired trait is a dominant trait, the genotype of the gene associated with the desired trait is arbitrary, (b) If the desired trait is haplotype homozygous, the haplotype of the locus associated with the desired trait is homozygous; (c) if the desired trait is recessive, it is associated with the desired trait Selecting a male pig and a female pig, each of which is a recessive homozygous gene, as a female genital breed pig and a female genital breed pig, and a selected female genital breed pig and female genital breed pig; Crossbreeding to obtain female pigs, and genotype Using the analysis results, from the pig population, (d) when the desired trait is a dominant trait, the genotype of the gene associated with the desired trait is dominant homozygous or heterozygous, and (e) desired Is a haplotype homozygous type, the haplotype of the locus associated with the desired trait is homozygous or heterozygous, and (f) if the desired trait is a recessive trait, the desired trait Selecting male pigs and sows that are recessive homozygous or heterozygous genotypes as male genital pigs and male genital breed pigs, and selected male genital breed pigs And male male sows are bred to obtain male offspring of male pig candidates, and male offspring of male pig candidates are analyzed for genotypes of genes related to desired traits. From male offspring of candidate sows, (A) the desired trait is the dominant form In this case, the genotype of the gene related to the desired trait is dominant homozygous, and (B) when the desired trait is haplotype homozygous, the haplotype of the locus related to the desired trait is (C) if the desired trait is a recessive trait, the step of selecting the individual as a male pig whose genotype of the gene associated with the desired trait is a recessive homozygous type, Crossbreeding a breeding pig and a female breeding pig to obtain a litter.

  In the present invention, a genotype of a gene related to a desired trait in an arbitrary pig population is analyzed, and an individual having a predetermined genotype with respect to the desired trait is selected from the pig population as a male breed pig and a female breed pig. To select as. At this time, the female breed pig may have any genotype if the desired trait is a dominant trait, but if the desired trait is a homozygous type of a specific gene Individuals having a homozygous type are selected. On the other hand, with regard to male breed pigs, the gene associated with the desired trait is homozygous or heterozygous, regardless of whether the desired trait is a dominant trait or a homozygous type of a specific gene. Select individuals. In addition, for male offspring obtained by mating male breeder pigs, the gene related to the desired trait is homozygous regardless of whether the desired trait is a dominant trait or a homozygous type of a specific gene. The mating type individuals are selected as male pigs. By selecting male breed pigs, female breed pigs, and male breed pigs in this way, all female offspring obtained by mating female breed pigs can be used as female breed pigs. All the offspring obtained by crossing are obtained as individuals having a desired trait.

  Moreover, in the method for producing pigs of the present invention, the female genital pig and / or the female genital pig used in the step of obtaining the female pig use the somatic cells of individuals selected from the pig population. It is also preferable that it is a cloned pig produced. As a result, a large number of individuals having the same genotype as the selected female original breed pig can be obtained. In addition, even if the selected female protozoan pig individuals are not alive or are difficult to mate or give birth, it is possible to obtain individuals having the same genotype. It becomes possible to create. In particular, in the present invention, female pigs produced from female breeder pigs do not require genetic analysis and selection, and all female offspring of female breeder pigs can be used as female pigs. By obtaining a pig as a cloned pig, it is possible to stably produce a large number of female pigs, and therefore, it is possible to obtain a large number of female sows.

  In the selection of female female breed pigs in the present invention, female female breed pigs are selected so that the average number of offspring per birth is higher than the average value of the pig population. It is also preferable. By selecting a prolific pig as a female progenitor pig, a prolific trait can also be inherited by the female pig that is a child of this female progenitor pig. Therefore, the number of offspring per birth of female pigs is increased, and a large number of pigs having a desired trait can be obtained by one birth.

  Furthermore, in the method for producing pigs of the present invention, the female genital pigs and females are such that the inbred coefficient of the offspring obtained by mating male and female pigs is less than 15%. It is also preferable to select a female genital pig, a male genital pig, and a male genital pig. As a result, the inbreeding coefficient of the pig obtained by mating the female and male pigs is maintained below a certain value, so that inbred degeneration does not occur and a healthy and well-developed pig is obtained. .

  Further, in the method for producing a pig of the present invention, the male male and / or male female pig used in the process of obtaining male offspring of male male pigs is an individual selected from a pig population. It is also preferable that it is a cloned pig produced using these somatic cells. As a result, individuals having the same genotype can be obtained even when the selected male protozoan pig individuals do not exist or are difficult to mate. Moreover, it is possible to obtain a plurality of selected male protozoan pigs and to stably produce pigs having a desired character.

  Furthermore, in the pig production method of the present invention, it is also preferable that the desired traits include two types of traits, white hair and SLA haplotype homozygous. Thereby, the pig which has a suitable trait as an experimental pig for medical research is produced efficiently.

  Furthermore, in the method for producing pigs of the present invention, it is also preferable that the pig population is a population of ultra-small pigs. Thereby, since a small-sized pig having a desired character is produced, a pig more suitable as an experimental pig for medical research can be obtained.

ADVANTAGE OF THE INVENTION According to this invention, the production method of the pig which has the following outstanding effects and has the desired character can be provided.
(1) Since the offspring (3rd generation) of the breeding pig (2nd generation) produced by crossing the original breed pig (1st generation) is obtained as a pig having the desired trait, in a short period of 3 generations Many pigs with the desired traits can be obtained.
(2) For selection of breeding pigs, it is only necessary to select at least one of the male offspring obtained by mating male breeding pigs as male breeding pigs, and females obtained by mating female breeding pigs. Since all litters can be used as female pigs, the labor of selection is greatly reduced.
(3) All the offspring obtained by crossing male and female pigs have the desired traits, so there is no need for selection work such as genetic analysis, and there is no need to dispose of individuals that have missed the selection. It becomes.
(4) The same genotype as the individual selected by the cloning technique from the somatic cells of the selected individual even when the selected individual in the pig population is not alive or the mating and birth of the selected individual is difficult Therefore, it is possible to obtain a plurality of selected original breed pigs and to stably produce pigs having a desired character. In addition, by producing the original pig by the cloning technique, it is possible to produce a pig having a desired character (commercial pig) at any time according to the needs of the customer, or to produce it permanently.
(5) Female pigs are selected by selecting individuals whose inbred coefficient of the target pig finally obtained when selecting each female breed pig and each male breed pig is less than 15%. The inbreeding coefficient of pigs obtained by mating with male pigs is maintained below a certain value, so that inbred degeneration does not occur, and healthy and well-developed pigs are obtained.

It is a flowchart which shows roughly the production method of the pig which has the desired character which concerns on embodiment of this invention. It is explanatory drawing which shows the relationship between the hair color of a pig, and the genotype of a KIT gene. It is explanatory drawing which shows the KIT genotype of each original pig, various pigs, and the target pig (commercial pig) which can be selected when a desired character is “white hair color”. It is explanatory drawing which shows SLA type | mold of each original breed pig, various pigs, and a commercial pig which can be selected when a desired character is "SLA haplotype is homozygous type". It is a graph which shows distribution of the SLA type haplotype which makes a population the 73 super small pigs in Example 3. FIG. It is explanatory drawing which shows the KIT genotype and SLA type | mold of each original breed pig, various pigs, and a commercial pig in Examples 6-8. It is a graph which shows transition of the body weight which shows the growth condition of the commercial pig produced in Example 8 in Example 9, and the growth situation of the micro pig produced by (b) natural selection of the non-selection.

  As shown in FIG. 1, the method for producing a pig having a desired trait according to an embodiment of the present invention includes a step S0 for determining a desired trait, and analysis of a gene related to the desired trait in an arbitrary pig population. Step S1 to perform, Step S2a for selecting female breeder pigs from the pig population, Step S2b for selecting male breeder pigs from the pig population, Step S3a for mating the selected female breeder pigs to produce female pigs, Step S3b for producing a male offspring by mating the selected male breed pig, Step S4 for selecting a male pig from this male offspring, and mating the produced female pig and male pig And a step S5 for producing a target pig (commercial pig).

(Determining the desired trait)
First, step S0 for determining a desired character will be described. The desired character is a character possessed by the target pig (commercial pig) produced by the present invention. In the present invention, traits include various phenotypes and genotypes. Therefore, traits that do not appear in the appearance are also included in the traits of the present invention. The desired character is not particularly limited, but in the case of a laboratory pig used as a medical laboratory animal, the hair color is white and the haplotype of swine leukocyte antigen (hereinafter referred to as SLA) is homozygous. Examples thereof include a junction type, a small physique, a pathological model having an etiological gene, and no specific gene (gene knockout). At least one desired trait is determined, and a plurality of desired traits may be determined as necessary. If analysis of genes related to desired traits in any swine population has already been completed and a library of each individual constituting the swine population has been created, the analyzed gene analysis process Subsequent to S1, a desired character determination step S0 may be performed.

(Analysis of genes related to desired traits in any pig population)
Next, step S1 for analyzing a gene related to a desired trait in an arbitrary pig population will be described. Here, genetic analysis of each individual of an arbitrary pig population is performed. The gene analysis is performed on the genotype of the gene related to the desired character determined in the above-described step S1. Analysis of the genotype in each individual can be performed by a known method. Specifically, for example, when it is selected that the haplotype of SLA is a homozygous type as a desired trait, an analysis of the SLA type of each individual in the swine population is performed. The analysis of pig SLA type is performed, for example, by extracting DNA from pig tissue and analyzing DNA fragment analysis to analyze SLA type composed of a plurality of genes (for example, Tanaka et al, Immunogenetics). 57, 2005, p. 690-696). In this method, the DNA extracted from each pig individual is subjected to PCR using a plurality of microsatellite markers covering the entire SLA region, and the haplotype type and genotype of each individual are examined by examining the fragment length of the PCR product. Are analyzed.

  In addition, when it is selected that the hair color is white as a desired trait, the genotype analysis of the KIT gene, which is a hair color-related gene, is performed for each individual in the pig population. Methods for extracting DNA from pig tissues and analyzing the genotype of the KIT gene have already been established (Reiko Shiotani, five others, “Mini Pig Hair Color Control”, Shizuoka Prefectural Livestock Research Institute) Report, No. 3, 2010, p.9-16). This KIT gene is located on the short arm of pig chromosome 8 and is known to be duplicated in white hair individuals. Furthermore, in one gene of this duplicated gene, a mutation occurs at the boundary between exon 17 and intron 17, and the first base of intron 17 changes from G to A, so that the transcript may lack exon 17. Are known. As a specific genotype analysis method, for a DNA extracted from each pig individual, after obtaining a PCR product of a region containing the first base of intron 17 of the KIT gene, the first base of intron 17 is examined. Is mentioned. FIG. 2 shows the relationship between the hair color of an ultra-small pig and the genotype of the KIT gene and the first base of intron 17 of the KIT gene. As shown in FIG. 2, this I locus is responsible for the dominant white of the pig's hair color, and white hair individuals are homozygous (I / I) or heterozygous (I / i) of the dominant allele I. It is. On the other hand, the hair color of the homozygous (i / i) individual of the recessive allele i is gray-black in ultra-small pigs, and other colored pigs exhibit colored hair other than white. In the present specification, the gene analysis includes determining the genotype from the appearance and form of each individual. For example, if a recessive trait appears in the appearance of an individual, the genotype is a homozygous version of the recessive allele.

  As described above, a genotype analysis of a gene related to a desired trait in an arbitrary pig population is performed, but the analyzed gene is not limited to the above. In addition, the data obtained by the above-described genetic analysis, the date of birth, sex, birth history, number of offspring per birth and related data, etc. It is preferable to create a library and create a library for the entire pig population. Thereby, in the next process S2a and S2b, selection of the original breed pig from the pig population can be easily performed. In addition, by storing the somatic cells of each individual, it is possible to create a cloned pig from an individual that is not alive and apply it to the breeding pig. Can be included in the pig population.

  The arbitrary swine population may be a population composed of a plurality of species or a population composed of a specific species, but a population composed of small pigs having a body weight of less than 20 kg is preferable, and disclosed in Patent Document 1 and the like in particular. A population consisting of very small pigs weighing less than 10 kg is preferred. Thereby, since the trait that a physique is small is also provided for the target pig (commercial pig), a pig with a physique suitable as a laboratory animal can be obtained.

(Selection of female native pigs from the pig population)
Next, process S2a which selects a female system breed pig from a pig group is explained. In this step, a female genital pig and a female genital pig are selected from an arbitrary pig population. In the selection in this step, according to the result of the analysis step S1, when the desired trait is a dominant trait, the genotype of the gene related to the desired trait is arbitrary, and (b) the desired trait is In the case of a haplotype homozygous type, the haplotype of the locus associated with the desired trait is homozygous, and (c) when the desired trait is a recessive trait, the gene of the gene associated with the desired trait Individuals whose genotype is recessive homozygous are selected from the pig population as female breeders.

  Among the selections described above, (a) selection in the case where the desired character is a dominant character will be specifically described with reference to FIG. For example, when “the hair color is white” is selected as the desired trait, as described above, the white hair color is due to the dominant allele I of the KIT gene and is a dominant trait. Therefore, as shown in FIG. 3, the genotype of the KIT gene of an individual selected as a female progenitor pig and a female male progenitor pig, which are female progeny pigs, is arbitrary, and a dominant homozygous type (I / I ), Heterozygous type (I / i) and recessive homozygous type (i / i).

  Further, (b) the case where the desired character is a haplotype homozygous type will be specifically described with reference to FIG. For example, if “the SLA haplotype is homozygous” is selected as the desired trait, the female progenitor pig and female male progenitor pig will be assigned to the SLA type locus. Individuals that are homozygous for a set of allele haplotypes are selected. The present inventors have found that there are nine types of SLA haplotypes from A to I as described in the following examples. FIG. 4 shows an example of an A-type homozygote (AA) as an SLA haplotype, but the desired trait is not limited to the A-type homozygote, and other types of homozygotes are used. Also good. A haplotype refers to any one combination of alleles at each locus, and basically one haplotype is inherited from a parent to a child. Therefore, if the genotype is heterozygous, it is unclear which haplotype is inherited from the parent to the child, but if the genotype is homozygous, the same haplotype is inherited from the parent to the child.

  Furthermore, (c) selection when the desired character is a recessive character will be specifically described. For example, when “the color of the hair is a color other than white” is selected as the desired character, the color of the color other than white is inferior because the KIT gene is the recessive allele i as described above. It is a trait. Therefore, individuals having a homozygous type (i / i) of the recessive allele i of the KIT gene are selected as a female genital pig and a female genital pig.

  The selection according to the above (a) to (c) is performed using the gene analysis result of the swine population in step S1. That is, with reference to the genotype related to the desired traits of each individual constituting the pig population, those corresponding to (a) to (c) are selected as female female progenitor pigs or female male progenitor pigs. At this time, when there are a plurality of desired traits, the selection according to (a) to (c) described above is performed for each of the genotypes related to the desired traits, and all the individuals corresponding to the genotypes are female progenitors. Selected as a pig. For example, when “the SLA haplotype is homozygous” and “the hair color is white” is selected as a desired trait, the SLA haplotype set is homozygous (eg, AA type). And an individual whose KIT gene genotype is dominant homozygous (I / I), heterozygous (I / i) or recessive homozygous (i / i) is selected.

  Furthermore, in this selection, a multi-productive individual with a higher average number of pups per birth than the average value of the number of pups produced per sow in the pig population is It is preferable to be selected as a pig. As a result, the fecundity traits can be inherited by the female pigs that are the female offspring of the female primiparous pigs, so the number of offspring per birth of the female pigs increases, and the desired trait A large number of the desired pigs (commercial pigs) can be obtained in a single delivery. Furthermore, it is preferable to select individuals that have an inbred coefficient of less than 15% for commercial pigs obtained after two generations as female female breed pigs and female male breed pigs. As a result, the inbreeding coefficient of a female pig produced by mating with a female female genital pig and female male genital pig and a target pig produced by mating with a male pig described below is less than a certain value. Since it is maintained, inbred degeneration does not occur, and a healthy and well-developed pig can be obtained. As an example, inbred coefficient calculation software (CoeFR ver 3.7 (Satoh, M., Jpn. J. Sine Science, 2000, Vol. 37, No. 3, p. 122-126) ) And the like. The selection of the female original breed pigs may be performed by selecting at least one male and one female, but a plurality of female pigs may be selected.

(Production of female pigs by mating female breed pigs)
Next, step S3a for producing female pigs by mating male and female female breed pigs will be described. In this step, the female genital pig and the male genital pig selected in the previous female genital pig selection process S2a are crossed to produce a female bred. Examples of mating methods include natural mating and artificial insemination. The female offspring thus obtained can be used as female pigs without any genetic analysis or selection. At this time, it is preferable that the female genital pigs and / or female genus pigs used for mating are a plurality of cloned pigs produced using somatic cells of individuals selected from the pig population. As a result, a large number of pigs having the same genotype as the selected female breeder pig can be obtained, so even if the selected female breeder pig is not alive or difficult to mate or give birth, the same gene Individuals having a pattern can be obtained, and commercial pigs having a desired character can be stably produced.

  A method for creating clones from porcine somatic cells by somatic cell cloning technology has already been established (Tatsuo Kawarazaki, 16 others, J Biomed Opt, 2009, Vol. 14, No. 5, 054017), which was described above. If somatic cells are stored in a library of pig populations, cloned pigs can be produced by conventional methods. Examples of methods for producing cloned pigs include the following methods. The ovary of immature pigs is collected at the slaughterhouse, the follicle with a diameter of 3 to 5 mm is crushed with a small scalpel, the immature ovum is collected, and the cumulus cell complex (COCs) with several layers of cumulus cells attached is collected. Mature culture. In mature culture, 10 IU / mL eCG, 10 IU / mL hCG, and 1 mM dbcAMP are added to the basal culture solution to prepare a culture solution, which is then cultured for 20 hours with COCs, and then transferred to a culture solution that does not contain eCG, hCG, and dbcAMP. . Around 38 hours after the start of the culture, cumulus cells are mechanically removed by pipetting after hyaluronidase treatment, and somatic cell nuclei are transplanted to those in which the first polar body has been confirmed by confirming the presence or absence of polar body release. It is activated by applying a direct current voltage of 150 kv / cm and 99 μs for 1 to 2 hours after nuclear transfer. Subsequent culture is performed in PZM. Nuclear transplanted embryos 2 to 5 days after the activation treatment are transplanted to the oviduct or uterus of recipient pigs whose estrus expression is adjusted 0 to 2 days later than the collected pigs by laparotomy. Thereby, a cloned pig having the same genotype as the selected individual is obtained. In particular, in the present invention, female pigs produced from female breeder pigs do not require genetic analysis and selection, and all female offspring of female breeder pigs can be used as female pigs. By obtaining the original pig as a cloned pig, it is possible to stably produce a large number of female pigs, thereby obtaining the desired number of female pigs.

(Selection of male native pigs from pig population)
Next, the process S2b for selecting male original breed pigs from the pig population will be described. In this step, selection is made from male hog breed pigs and male hog breed pigs, which are male breed pigs, from an arbitrary pig population. In the selection in this step, according to the result of the analysis step S1, (d) when the desired trait is a dominant trait, the genotype of the gene related to the desired trait is a dominant homozygous type or heterozygous type, (E) When the desired trait is haplotype homozygous, the haplotype of the locus related to the desired trait is homozygous or heterozygous, and (f) When the desired trait is a recessive trait The individual whose genotype of the gene relating to the desired trait is recessive homozygous or heterozygous is selected from the pig population as a male progenitor pig.

  Among the selections described above, (d) selection in the case where the desired character is a dominant character will be specifically described with reference to FIG. For example, when “the hair color is white” is selected as the desired trait, as described above, the white hair color is due to the dominant allele I of the KIT gene and is a dominant trait. Therefore, as shown in FIG. 3, male genital pigs and male genital pigs, which are male prototypical pigs, have a KIT gene genotype of dominant homozygous (I / I) or heterozygous (I / I) individuals are selected.

  (E) The case where the desired character is a haplotype homozygous type will be specifically described with reference to FIG. For example, if “the SLA haplotype is homozygous” is selected as the desired trait, the male progenitor pig and the male male progenitor pig, which are male progenitor pigs, are assigned to the SLA type locus. Individuals whose homozygous or heterozygous set of haplotypes of an allele are selected. FIG. 4 shows an example in which the desired trait is a haplotype A homozygote (AA) of SLA, and in this case, it has a haplotype A homozygote (AA) or a haplotype A type. Individuals of heterozygotes (A ○, ○ are arbitrary meanings) are selected. When the desired character determined in step S0 is another type of homozygote, an individual having a homozygote of another type of haplotype or a heterozygote having the type of haplotype is selected.

  Further, (f) selection when the desired character is a recessive character will be specifically described. For example, when “the color of the hair is a color other than white” is selected as the desired character, the color of the color other than white is inferior because the KIT gene is the recessive allele i as described above. It is a trait. For this reason, male genital pigs and male genital pigs, which are male genital pigs, have individuals with homozygous (i / i) or heterozygous (I / i) recessive alleles of the KIT gene. Selected.

  The selection according to the above (d) to (f) is performed using the gene analysis result of the swine population in step S1. That is, with reference to the genotype related to the desired traits of each individual constituting the pig population, those corresponding to (d) to (f) are selected as male genital breed pigs or male genital breed pigs. At this time, if there are a plurality of desired traits, the selection according to (d) to (f) described above is performed for each of the genotypes related to the desired traits, and all the individuals corresponding to the genotypes are male progenitors. Selected as a pig. For example, when “the SLA haplotype is homozygous” and “the hair color is white” is selected as a desired trait, the SLA haplotype set is homozygous (eg, AA type). Alternatively, individuals that are heterozygous (eg A type) and whose KIT gene genotype is dominant homozygous (I / I) or heterozygous (I / i) are selected.

  Furthermore, in this selection, it is preferable to select individuals such that the inbred coefficient of commercial pigs obtained after two generations is less than 15% as male genital pigs and male genital pigs. As a result, the inbreeding coefficient of a male pig produced by mating with a male genital pig and a male genital breed pig and a target pig produced by mating with the female pig described above is less than a certain value. Since it is maintained, inbred degeneration does not occur, and commercial pigs that are healthy and in good growth can be obtained.

(Production of male offspring by crossbreeding of male breeding pigs)
Next, the process S3b for producing male offspring by mating male and female of the male breeder pig will be described. In this step, the male genital pig selected in the previous male protospecies pig selection step S2b and the male genital breed pig are crossed to produce a male offspring that is a male breed candidate. Examples of mating methods include natural mating and artificial insemination. At least one of the obtained male offspring is selected as a male pig in the selection step S4 described later. The male genital breed pig and / or male genital breed pig used for mating may be a cloned pig produced using somatic cells of individuals selected from a pig population in the same manner as the female genital breed pig.

(Selection of male pigs from male offspring of male breeding pigs)
Next, process S4 which selects a male pig from the male offspring of a male original breed pig is explained. In this step, among the offspring obtained in the above-described step S3b, a male pig that will be a parent pig of a commercial pig is selected from the male offspring. In the present invention, the selection conditions for the genotype are set so that the selection process of the breeding pig is performed only for the male male pig. That is, boars can generally breed throughout the year, and semen can be collected twice / week from about 6 months to 5 years of age, and two females can be collected in one semen collection. In addition to being able to be used for breeding of individuals, semen can be stored. Therefore, if there is at least one individual selected as a male pig, a large number of commercial pigs can be produced. On the other hand, sows generally require about 114 days of gestation and breed at most twice a year, so that a large number of female pigs are required to produce a large number of commercial pigs. Therefore, in the present invention, by selecting only male pigs and using all female pigs as female pigs without selecting female offspring produced from female breed pigs, a large number of commercial pigs can be efficiently produced. Is possible.

  For each male offspring produced from a male breeder pig, the genotype analysis of the gene associated with the desired trait is performed in the same manner as in the gene analysis step S1 described above. According to the results of gene analysis of the male offspring, (A) when the desired trait is a dominant trait, the genotype of the gene related to the desired trait is a dominant homozygous type, and (B) the desired trait is In the case of a haplotype homozygous type, the haplotype of the locus related to the desired trait is homozygous, and (C) when the desired trait is a recessive trait, the gene of the gene related to the desired trait Individuals whose genotype is recessive homozygous are selected from male offspring as male pigs.

  Among the selections described above, (A) selection in the case where the desired character is a dominant character will be specifically described with reference to FIG. For example, when “the hair color is white” is selected as the desired trait, the white hair color is due to the dominant allele I of the KIT gene, so as shown in FIG. Individuals with a dominant homozygous type (I / I) are selected. Thus, if individuals with duplicated dominant alleles in homozygous form are selected as male pigs and used for mating, a dominant trait will always be produced regardless of the genotype of the female pig. Since it is transmitted to the offspring, it becomes possible to efficiently produce an individual having a dominant character.

  Further, (B) the case where the desired character is a haplotype homozygous type will be specifically described with reference to FIG. For example, if “the SLA haplotype is homozygous” is selected as the desired trait, an individual with a homozygous haplotype set of alleles at the SLA type locus is selected as a male pig. Selected. In FIG. 4, the desired trait shows an example of a haplotype A homozygote (AA) of SLA. In this case, an individual of a haplotype A homozygote (AA) is selected. .

  Furthermore, (C) selection when the desired character is a recessive character will be specifically described. For example, when “the hair color is a color other than white” is selected as the desired trait, as described above, the colored hair color other than white is a recessive trait and the KIT gene is a recessive allele i Therefore, individuals whose homozygous (i / i) recessive allele i of the KIT gene is selected.

  The selection according to the above (A) to (C) is performed using the gene analysis result of the male offspring produced in step S3b. That is, with reference to the genotype relevant to the desired traits of each male offspring, those corresponding to (A) to (C) are selected as male pigs. At this time, when there are a plurality of desired traits, the selection according to (A) to (C) described above is performed for each of the genotypes related to the desired traits, and all the corresponding individuals are male pigs. As selected. For example, when “the SLA haplotype is homozygous” and “the hair color is white” is selected as a desired trait, the SLA haplotype set is homozygous (eg, AA type). And an individual whose KIT gene genotype is dominant homozygous (I / I) is selected.

(Production of commercial pigs by mating female and male pigs)
Next, the production process S5 of a commercial pig by crossing a female pig and a male pig will be described. In this process, the target pig (commercial pig) is produced by crossing the female pig obtained in the above-described process S3a with the male pig selected in the process S4. Examples of mating methods include natural mating and artificial insemination. All of the resulting litters have the desired traits. For example, as shown in FIG. 3, when “the hair color is white” is selected as the desired trait, the genotype of the resulting commercial pig KIT gene is dominant homozygous (I / I) or heterozygous. Only the junction type (I / i) is used, and the baby hair color is all white. In addition, as shown in FIG. 4, when “the SLA haplotype is homozygous” is selected as a desired trait, all the commercial pig haplotype sets obtained are homozygous (eg, AA type). Thus, since the obtained offspring are all provided with the desired character, it is not necessary to select the offspring, and all offspring can be used as commercial pigs.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not particularly limited to these examples.

[Example 1]
1. Determination of desired traits of commercial pigs The characteristics required for experimental pigs used as medical laboratory animals are as follows: (1) Small physique, (2) To improve the reproducibility of safety tests and transplantation experiments, SLA It is mentioned that the hair color is “white” because the genotype of (swine leukocyte antigen) is complete, and (3) it is easy to observe the state of health and skin (Reiko Shiotani, three others, “Questionnaire survey on experimental pigs”, Shizuoka Prefectural Livestock Research Institute, Small and Medium Livestock Research Center Research Report, No. 5, 2012, p. 1-9). In this example, for ultra-small pigs with a small physique, select “the SLA genotype is homozygous” and “the hair color is white” as the desired traits. An attempt was made to produce a very small pig.

[Example 2]
2. Characteristic survey About the pig population of 73 ultra-small pigs described in Patent Document 1, (1) Date of birth, (2) Gender, (3) Hair color, (4) Production history, and (5) Production per birth We investigated the number of offspring. In addition, about the hair color, it investigated also about the hair color of the parent pig of an investigation object pig. The survey results were as follows.
(1) Birth date: February 2001 to March 2009.
(2) Gender: 37 males and 36 females.
(3) Hair color: 26 white, 18 black, and 29 gray. Individuals whose parent generation was white and whose child generation was colored (black or gray) were confirmed.
(4) Birth history: Of 36 females, 21 females had a history of birth.
(5) Number of offspring per birth: 13 were the largest and 0 in the few. The average number of offspring per birth was 4-5, and individuals with an average number of offspring per birth of 5 or more were considered prolific.

[Example 3]
3. Genetic analysis (SLA type)
The swine leukocyte antigen (SLA) genotype was examined for 73 ultra-small pigs whose traits were examined in Example 2. Genetic analysis on the SLA type of each individual was performed as follows. Tissue pieces (ear pieces, tail pieces, etc.) of each individual were collected, and the tissue pieces were washed with PBS (−). The tissue piece was dissolved in a DNA extraction buffer (0.2 mg / mL Proteinase K, 10 mM EDTA, 120 mM NaCl, 1% SDS, 10 mM Tris-HCl [pH 8.0]) containing Proteinase K, and then treated with phenol / chloroform. The genomic DNA was extracted from the ethanol precipitate. This genomic DNA was subjected to PCR targeting eleven types of microsatellite markers located in the SLA class I to III region (about 2.4 Mb). The target markers are shown in Table 1. For PCR, a final concentration of 0.025 U / μL of AmpliTaq (registered trademark) Gold DNA polymerase (Applied Biosystems Japan Co., Ltd.), 1 × PCR buffer (attached to the above-mentioned DNA polymerase. The final concentration of MgCl is 1.5 mM), 0.2 mM dNTPs, 0.25 μM of each primer, and 20 ng of each DNA were added to make a total volume of 15 μL. In the PCR reaction, DNA polymerase was activated by heating at 94 ° C. for 9 minutes, followed by denaturation reaction at 94 ° C. for 30 seconds, annealing reaction at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 30 seconds. These reactions were performed by repeating 40 cycles.

  As a result of fragment analysis of the obtained PCR product, as shown in Table 2, nine haplotypes A to I were confirmed in the population consisting of 73 ultra-small pigs in this example. The numerical value in the table | surface has shown the length of the DNA fragment of each marker in various haplotypes. In addition, the haplotype distribution of the population is shown in FIG. As shown in FIG. 5, among the nine types of haplotypes, the A type accounted for 39.7% and the C type accounted for 25.3%, and it was confirmed that the frequency of these two haplotypes was high.

  Table 3 shows the results of genotype analysis in this population. A to I indicate the type of each confirmed haplotype, and X indicates the type that could not be determined. Among the numerical values in the table, the upper row shows the number of corresponding individuals, and the lower row shows the abundance ratio in the population (n = 73). In the population of 73 ultra-small pigs in this example, individuals with homozygous haploid set were 10 AA (13.7%), 1 CC (1.4%), and DD One (1.4%) was confirmed, and it was found that the heterozygous type (38 heads; 52.1%) genotype having type A accounted for more than half of the population.

[Example 4]
4). Genetic analysis (KIT gene)
The 73 tiny pigs examined in Examples 2 and 3 were examined for the genotype of the KIT gene, which is a coat color-related gene. Specifically, gene analysis regarding the KIT gene of each individual was performed as follows. The genomic DNA of each individual obtained in Example 3 was subjected to PCR targeting the region containing the first base of intron 17 of the KIT gene. The primers and amplification regions used in PCR are shown in Table 4 below. Primers were designed and produced with reference to a report by Okumura et al. (Naoko Okumura, 5 others, 2000, Journal of the Japanese Society of Animal Science, No. 7, J222-J234).

  For PCR, a final concentration of 0.025 U / μL of AmpliTaq (registered trademark) Gold DNA polymerase (Applied Biosystems Japan Co., Ltd.), 1 × PCR buffer (attached to the above-mentioned DNA polymerase. The final concentration of MgCl is 1.5 mM), 0.2 mM dNTPs, 0.25 μM of each primer, and 20 ng of each DNA were added to make a total volume of 15 μL. In the PCR reaction, DNA polymerase was activated by heating at 94 ° C. for 9 minutes, followed by denaturation reaction at 94 ° C. for 30 seconds, annealing reaction at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 30 seconds. These reactions were performed by repeating 40 cycles.

  About the obtained PCR product, the base sequence was analyzed by the dideoxy method using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Japan Co., Ltd.). For 26 white hair individuals, in the sequence data of the first base of intron 17, two homozygous (I / I) dominant white alleles I with G: A of 1: 1, and G: A 24 heterozygous types (I / i) of 2: 1 were confirmed. For 47 colored hair individuals (black: 18; gray: 29), the sequence data of the first base of intron 17 is only G, and all are homozygous for recessive allele i (i / i) Met.

[Example 5]
5. Preparation of Library Somatic cells of 73 ultra-small pigs investigated in Examples 2 to 4 described above were collected (collected tissue: fibroblasts derived from ear skin) and stored frozen using a liquid nitrogen cylinder. . Also, individual information (birth date, sex, coat color, birth history, number of offspring per birth, SLA genotype and KIT genotype) and freezing obtained from Examples 2 to 4 A library of each individual containing the above-mentioned stored somatic cells was prepared.

[Example 6]
6). Selection of Progeny Pig Candidates In selecting female progeny pigs, that is, parent pigs of female pigs, from the library data shown in Table 5, individuals with SLA type homozygous AA (6 females and 4 males). ) As the primary candidate. When this primary candidate is selected, the desired trait “hair color is white” is due to the dominant gene, and the hair color of the commercial pig is determined by the genotype of the male pig described below. For this reason, the genotype of the KIT gene of the female breeder pig was arbitrary. Thus, by selecting the female breeder pig, all female offspring produced from the female breeder pig can be used for the production of commercial pigs as a female pig without selection.

  Next, among the female breeder pigs, for the female breeder pigs, the average number of pups per birth is the average number of pups per birth of the sows in the library (4 to 5) More prolific female individuals were selected. Thereby, since the produced female pig is provided with a prolific trait, the number of offspring per birth is increased, and commercial pigs can be produced efficiently. In this example, library NO. Thirty-four females were selected as female breeder pig candidates. Subsequently, a combination of males and females of a female progeny pig whose inbred coefficient in commercial pig generations was less than 15% was examined. The inbred coefficient is calculated using inbred coefficient measurement software (CoeFR ver. 3.7 (Satoh, M., Jpn. J. Sine Science, 2000, Vol. 37, No. 3, p. 122-126)). I went. As a result, library NO. 34 was selected and the library No. 63 were selected. The genotype of the selected individual is shown in FIG.

  On the other hand, when selecting a male breed pig, that is, a parent pig of a male pig, the SLA type has at least one A-type haploid from the library data shown in Table 5, and the KIT gene is dominant. The white allele I was examined as a candidate for one female (library No. 32) and one male (library NO. 54) of homozygous type (I / I). However, it was found that the inbreeding coefficient in the commercial pig generation exceeds 15% when both are male breeding pigs. For this reason, the dominant white allele I of the KIT gene is heterozygous (I / i), and the SLA type is homozygous AA, 3 females (library No. 3, 47, 53) and 1 male (library). No. 58) was also selected as a candidate. For these male breed pig candidates, we examined male and female combinations of male breed pigs that have an inbreeding coefficient of less than 15% in the commercial pig generation. As a result, library NO. 47 was selected, and as a male male genital pig, library NO. 54 were selected. The genotype of the selected individual is shown in FIG.

[Example 7]
7). Production of breeding pigs by crossbreeding of the breeding pigs In Example 6, the female breeding pigs selected from the library were designated as library NO. 34 (female) and library NO. 63 (male). In order to produce a large number of commercial pigs, it is necessary to have a large number of female pigs produced from female breeder pigs. Therefore, using the somatic cells of selected individuals stored in the library, 8 female and 8 male pigs were produced by the somatic cell cloning technique. The female breeding pigs thus obtained were naturally mated to obtain 31 female offspring.

  On the other hand, in Example 6, the male progeny pig selected from the library is the library NO. 47 (female) and library NO. 54 (male). There should be at least one male pig produced from a male breed pig because of its breeding characteristics. No. Since 47 individuals were alive and could be bred at the planned breeding place, they were used as male and female progenitor pigs as they were. No. Since 54 individuals were not alive, one was produced by somatic cell cloning technology using somatic cells stored in the library. The male breed pigs thus obtained were naturally mated to obtain male offspring.

[Example 8]
8). Production of commercial pigs by breeding of breed pigs Regarding female pigs, 5 of the female offspring (31 pigs) obtained by natural breeding of female breed pigs (8 males and 8 females) according to Example 7 Was used as a female pig for the production of commercial pigs. As shown in FIG. 6, the SLA types of these female pigs were all AA homozygous types. In this example, the KIT genotype of the female pig is a homozygous type (i / i) of the recessive gene i, but the genotype of the female pig KIT gene is not limited to this, and the dominant gene I Homozygous type or heterozygous type.

  On the other hand, for male pigs, genetic analysis of male offspring obtained by natural mating of male breeding pigs according to Example 7 was performed in the same manner as in Examples 3 to 4, and the dominant white allele I of the KIT gene was determined. Were homozygous (I / I) and SLA haplotype AA homozygous individuals were selected as male pigs. As a result of the natural mating of the above 5 female pigs and 1 male pig, as shown in FIG. 6, all are white hair (KIT gene heterozygous type (I / i)) and SLA type commercials with AA aligned. 63 pigs (38 males and 25 females) were produced.

[Example 9]
9. Growth status of commercial pigs The 19 commercial pigs produced in Example 8 (12 males, 7 females) were weighed at birth and at 2-6 months of age. The results are shown in FIG. The horizontal axis indicates the age, and the vertical axis indicates the body weight (kg). The dotted line indicates male data, and the solid line indicates female data. The average weight of commercial pigs at 6 months of age was 8.9 ± 1.8 kg (n = 12) for males and 9.9 ± 1.1 kg (n = 7) for females. On the other hand, as comparative examples, 34 ultra-small pigs (15 males and 19 females) born between July 2006 and December 2007 by unselected natural mating, 2-6 months old The body weight data is shown in FIG. The horizontal axis indicates the age, the vertical axis indicates the body weight (kg), the dotted line indicates the male data, and the solid line indicates the female data. The average body weight at 6 months of age of the ultra-small pigs was 9.4 ± 2.0 kg for males and 9.0 ± 2.3 kg for females. From these results, there was no significant difference in the body weight at the age of 6 months between the commercial pigs produced in this example and the ultra-small pigs of the comparative examples, and the commercial pigs produced in this study are characteristic of ultra-small pigs. It was confirmed that some very small traits were retained.

  The present invention is not limited to the above-described embodiments or examples, and various design changes within the scope not departing from the gist of the invention described in the claims are also included in the technical scope. Is.

Claims (7)

A method for producing a pig having a desired trait,
Determining the desired trait;
Analyzing the genotype of the gene associated with the determined desired trait in any pig population;
From the analysis of the genotype, from the pig population,
(A) When the desired trait is a dominant trait, the genotype of the gene associated with the desired trait is arbitrary;
(B) when the desired trait is haplotype homozygous, the haplotype of the locus associated with the desired trait is homozygous,
(C) When the desired trait is a recessive trait, the genotype of the gene associated with the desired trait is a recessive homozygous type,
Selecting a male pig and a female pig as a female genital pig and a female genital pig, respectively;
Crossing the selected female genital pig and female genital pig to obtain a female pig;
From the analysis of the genotype, from the pig population,
(D) when the desired trait is a dominant trait, the genotype of the gene associated with the desired trait is a dominant homozygous type or heterozygous type,
(E) when the desired trait is haplotype homozygous, the haplotype of the locus associated with the desired trait is homozygous or heterozygous,
(F) When the desired trait is a recessive trait, the genotype of the gene associated with the desired trait is a recessive homozygous or heterozygous type,
Selecting a male pig and a female pig as a male and male female pig respectively;
Crossing the selected male genital pig and male genital pig to obtain male offspring of male pig candidates;
For male offspring of the male pig candidate, analyze the genotype of the gene associated with the desired trait, from the male offspring of the male pig candidate,
(A) When the desired trait is a dominant trait, the genotype of the gene associated with the desired trait is a dominant homozygous type,
(B) If the desired trait is haplotype homozygous, the haplotype of the locus associated with the desired trait is homozygous,
(C) If the desired trait is a recessive trait, the genotype of the gene associated with the desired trait is a recessive homozygous type,
Selecting individuals as male pigs;
Cross-breeding the male pig and the female pig to obtain offspring, a method for producing pigs.   The female genital pig and / or female genital pig used in the step of obtaining the female pig is a cloned pig produced using somatic cells of individuals selected from the pig population. The method for producing pigs according to claim 1.   The female female progeny pig is selected as a prolific female pig having an average number of pups per birth that is greater than the average value of the pig population. How to make pigs.   The female genital pig, the female genital pig, and the male male so that the inbred coefficient of the offspring obtained by mating the male pig and the female pig is less than 15%. The method for producing a pig according to any one of claims 1 to 3, wherein the original breed pig and the male female breed pig are selected.   The male genital pig and / or male genital pig used in the process of obtaining male offspring of the male pig candidate were created using somatic cells of individuals selected from the pig population. It is a cloned pig, The pig production method of any one of Claims 1-4 characterized by the above-mentioned.   The pig production method according to any one of claims 1 to 5, wherein the desired traits include two types of traits: white hair and SLA haplotype homozygous.   The method for producing pigs according to any one of claims 1 to 6, wherein the pig population is a population of ultra-small pigs.

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