JP2006101871A - Dna marker for determining potential for increasing number of vertebrae of swine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining the number of vertebrae of a swine by using the transcription suppressing action of NR6A1 on the 1st chromosome of swine, the bindability of NR6A1 to corepressor, the mutation of one or more amino acids in the amino acid sequence of NR6A1, the mutation of one or more bases in the base sequence of NR6A1 and a microsatellite sequence on the 1st chromosome of swine as indices. <P>SOLUTION: A BAC alignment map is prepared by using a range from SJ819 to SJ273 as a QLT candidate region, a genome region having a chain unbalance to the vertebral number is searched by using a microsatellite marker arranged in high density, the base sequence near (650 kb) of the found region is determined, and all VNTR-type polymorphic markers are extracted. It has been found by reanalysis that the size of the region having the chain unbalance is 250 kb, the microsatellite marker in the region has remarkably small polymorphism in an individual having an allele to increase the number of vertebrae, and the difference in the vertebral number is caused by the single amino acid substitution of NR6A1 in the region. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the transcriptional repression action of NR6A1 on porcine chromosome 1, the binding ability of NR6A1 to the corepressor, the mutation of one or more amino acids in the amino acid sequence of NR6A1, the one or more bases in the nucleotide sequence of NR6A1 The present invention relates to a mutation, a method for identifying the number of porcine vertebrae using the microsatellite sequence on the first chromosome of pig as an index, and a mutant NR6A1 possessed by a pig having a large number of vertebrae.

  Pigs are said to have been domesticated in several parts of the Eurasian continent, with their ancestors as their ancestors. In Europe from the middle of the 19th century, selection and breeding of pigs with good growth and physique began to take place, and the current commercial pigs were formed based on them. Up until now, the main goal was to improve meat production, but recently, breeding improvements that require better meat quality are also desired. Asian native pigs and Japanese carp are not bred in terms of growth and physique, and they are expected to be genetic resources for new breeding, although their physique is small and their meat quality is low.

  In recent years, DNA polymorphic markers have also been developed in pigs, and linkage maps using microsatellite markers have been prepared. In the group of the present inventors, F2 family (MG family) was produced using different varieties of Umeyama pig and Göttingen minipig, and a linkage map of microsatellite markers was produced (Non-patent Document 1). Most of the economic traits of livestock are quantitative traits, but their loci (quantitative trait locus; QTL) have also been isolated by linkage analysis with DNA markers. Currently, there are 350 reports on various QTLs for pigs (NCBI, LocusLink; Pig & QTL), and our group also analyzed the number of vertebrae, nipples, birth weight, QTL involved in back fat thickness, daily gain, etc. is detected (Non-patent Document 2). Quantitative traits are dominated by multiple loci and are also greatly influenced by environmental factors. Therefore, accurate mapping of QTL is difficult, and it is very difficult to identify responsible genes and polymorphisms that cause diversity. In the QTL elucidated at the gene level in pigs, there are the PRKAG3 gene (non-patent document 3) involved in meat quality (glycogen content) and the IGF2 gene (non-patent document 4) involved in meat content.

  The present inventors made a plurality of F2 experimental families and conducted QTL analysis (Non-Patent Documents 5 to 11). As a result, various QTLs were detected. Among them, QTLs involved in the number of vertebrae were detected in two genomic regions (multiple chromosomes q arm end, chromosome 7 q arms center) through multiple families, Its existence was judged to be certain (Table 1, FIG. 1). Porcine cervical vertebrae are seven like other mammals, but the number of thoracic vertebrae and lumbar vertebrae has been known for a long time, varying from 14 to 16 for thoracic vertebrae and 5 to 7 for lumbar vertebrae. (Non-patent document 12). The total number of these (vertebral vertebrae) is 19 for the ancestor of pigs, but it is 21 to 23 for current meat varieties. It is considered that the number of vertebrae has been increased in the process. In fact, an increase in the number of vertebrae has been shown to increase the average length by 1.5 cm (a population with an average length of 80 cm). Further, an analysis of the heritability of the number of vertebrae has reported a high value of 0.74 (Non-patent Document 13). The two vertebra number QTLs detected by the present inventors have alleles that increase the number of vertebrae, and their effects are almost equal, and the average of the results of each family is about 0.5 to 0.6 per allele. The two QTLs acted independently of each other. When all the alleles were increased in number of vertebrae, the average number of vertebrae increased by about 2.3 (Table 2, Fig. 2). In the analysis of experimental families so far, the number of vertebrae in the q-arm end region of chromosome 1 has been confirmed to increase the number of vertebrae in Western varieties such as Landrace, Large White, Duroc and Berkshire. No increase effect was observed. In the QTL on chromosome 7, the effect of increasing the number of vertebrae was observed in some alleles of Western varieties (Table 1).

  Of these two, the present inventors have advanced fine mapping of the number of vertebrae QTL on the first chromosome. Vertebral number QTL is peaked at SW373 (AF225095) to SW705 (AF235342) in the region of about 25 cM between microsatellite markers SW1311 (AF253583) and SW1301 (AF225115) at the end of chromosome 1 q arm. Since it was detected (FIG. 3 (A), SW series is a microsatellite marker developed by the US Department of Agriculture), a comparative gene map with humans was prepared for this region (FIG. 4). As a result, the porcine chromosome 1 q-arm terminal region corresponded to the human chromosome 9 q-arm terminal region, and the gene sequence was highly conserved. We have also developed a new microsatellite marker based on comparative genetic map information. In QTL interval mapping (linkage analysis) using them, a result having a peak at SJ641 (AB167457) was obtained (FIG. 3 (B), SJ series is a marker developed by the present inventors). In addition, by analyzing the recombination of chromosomes of F2 individuals with 19 (minimum) vertebrae in the experimental family, the range where QTL is located is about 2 cM between microsatellite markers SJ819 (unregistered) and SJ273 (AB167432). Narrow down to. (FIG. 3 (C), FIG. 5, Non-Patent Document 14). SJ819 was present in the same BAC clone as KIAA1608 and SJ273 as PBX3 porcine homologue.

Prior art document information related to the invention of the present application is shown below.
Mikawa, S., T. Akita, N. Hisamatsu, Y. Inage, Y. Ito et al., 1999 A linkage map of 243 DNA markers in an intercross of Gottingen miniature and Meishan pigs. Anim. Genet. 30: 407- 417. Wada, Y., T. Akita, T. Awata, T. Furukawa, N. Sugai et al., 2000 Quantitative trait loci (QTL) analysis in a Meishan × Gottingen cross population. Anim. Genet. 31: 376-384. Milan D., Jeon JT., Looft C., Amarger V., Robic A. et al., 2000 A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle.Science. 288: 1248-1251. Van Laere AS., Nguyen M., Braunschweig M., Nezer C., Collette C. et al., 2003 A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig.Nature. 425: 832-836. Eiji Matsubara, Kazuyuki Tsuji, Yuko Inage, Koji Enomoto, Satoshi Mikawa, Yasuhiko Wada, Eiji Kobayashi, Mitsuru Minezawa, Hiroshi Yasue “Linkage Analysis of Economic Traits and Microsatellite Markers in the Innobuta Experimental Family”, Journal of Japanese Pig Research Vol.36, No.4, P189, November 1999 Manabu Naito, Satoshi Yamada, Yoko Uchida, Yuko Inage, Masashi Miyake, Satoshi Mikawa, Yasuhiko Wada, Eiji Kobayashi, Mitsuru Minezawa, Takashi Hamada, Hiroshi Yasue “Umeyama Pig x Large Yorkshire Breed Experiment Family DNA Markers and Economic Traits Linkage Analysis ”Journal of Japanese Society of Pig Farming, Vol. 37, No. 4, P182, October 2000 Junichi Murobushi, Tatsuo Kawarazaki, Atsushi Horiuchi, Noriko Hisamatsu, Koji Enomoto, Satoshi Mikawa, Yasuhiko Wada, Mitsuru Minezawa, Hiroshi Yasue ”Journal of Japanese Society of Pig Farming, Vol. 35, No. 2, P66, June 1998 Kazusa Oji, Masaaki Indo, Masashi Miyake, Eiji Kobayashi, Yasuhiko Wada, Satoshi Mikawa, Mitsuru Minezawa, Hiroshi Yasue, "Linkage Analysis Using Berkshire and Crown Minipig Hybrid DNA Markers," Japanese Society for Pig Research, Vol. 35, 2 No., P66, June 1998 Masahiro Nii, Fumio Tani, Akito Niki, Takeshi Hayashi, Hirohide Kaminishi, Satoshi Mikawa, Eiji Kobayashi, Yoko Uchida, Takashi Hamada, Hiroshi Yasue "QTL analysis in Japanese wild boar and large Yorkshire breeding family" The 98th Annual Meeting of the Japanese Society of Animal Science Meeting Abstracts, P19, March 2001 Ryoko Yamaguchi, Satoshi Emori, Koji Osawa, Masao Naito, Yoshizo Kamiyama, Naoe Kanaya, Yoko Uchida, Atsushi Horiuchi, Yoshinori Nakazawa, Takeshi Hayashi, Takashi Hamada Marker-based selection for intramuscular fat content "Journal of Japanese Society of Pig Farming, Vol. 40, No. 4, P212, October 2003 Atsushi Horiuchi, Mikio Tomohisa, Hanako Ide, Naoe Kaneda, Yoko Uchida, Ryoko Yamaguchi, Yoshinori Nakazawa, Takeshi Hayashi, Takashi Hamada “Analysis of QTLs related to meat quality in crossbred families of Kinka pork and Duroc” , Volume 40, No.4, P213, October 2003 King, JWB, and RC Roberts, 1960 Carcass length in the bacon pig: its association with vertebrae numbers and prediction from radiographs of the young pig.Anim.Prod. 2: 59-65 Berge, S., 1948 Genetical researches on the number of vertebrae in the pigs. J. Anim. Sci. 7: 233-238 Shimanuki S., Morozumi T., Domukai M., Shinkai H., Mikawa A., Uchida Y., Uenishi H., Hayashi T., Mikawa S., Awata T., 2004 Fine mapping of QTL affecting the number of vertebrae on SSC1. Plant & Animal Genome XII, P241 Chung AC, Katz D, Pereira FA, Jackson KJ, DeMayo FJ, Cooney AJ, O'Malley BW., 2001 Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation. Biol. 21 (2): 663-77. David R, Joos TO, Dreyer C. Mech Dev., 1998 Anteroposterior patterning and organogenesis of Xenopus laevis require a correct dose of germ cell nuclear factor (xGCNF). 79 (1-2): 137-52. Yan Z, Jetten AM., 2000 Characterization of the repressor function of the nuclear orphan receptor retinoid receptor-related testis-associated receptor / germ cell nuclear factor.J Biol Chem. 275 (45): 35077-85. Seol W, Mahon MJ, Lee YK, Moore DD., 1996 Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13 / N-CoR. Mol Endocrinol. 10 (12): 1646-55. Yan Z, Kim YS, Jetten AM., 2002 RAP80, a novel nuclear protein that interacts with the retinoid-related testis-associated receptor.J Biol Chem. 277 (35): 32379-88.

  The present invention has been made in view of such circumstances, and an object thereof is to provide a method for identifying a pig having a large number of vertebrae. More specifically, the present invention relates to the transcriptional repressive action of NR6A1 on porcine chromosome 1, the binding ability of NR6A1 to the corepressor, the mutation of one or more amino acids in the amino acid sequence of NR6A1, the 1 or 2 in the base sequence of NR6A1 It is an object of the present invention to provide a method for discriminating the number of porcine vertebrae using a plurality of base mutations and a microsatellite sequence on the porcine first chromosome as an index.

  Linkage disequilibrium analysis is generally used to further identify the location of the causative gene from the range narrowed down by linkage analysis using an experimental family. Since livestock have a short generation interval and related and tabular values are recorded, a method for mapping common chromosomal regions from the same ancestor related to the difference in traits using existing animal trait data (identical by decent analysis) ; IBD analysis) is commonly used. However, the number of vertebrae QTL targeted by the inventors of the present invention includes alleles that have an effect of increasing the number of vertebrae in a specific variety and are fixed. Therefore, there is no group that has diversity in this QTL. Therefore, in order to map in detail by this method, it is necessary to construct a new large-scale family starting from a crossbred with a pig with a small number of vertebrae, which is not realistic.

  Since a normal method cannot be taken, the present inventors considered linkage disequilibrium analysis using unrelated samples. In pigs, generational renewal is as fast as about one year, and the region where linkage disequilibrium is observed is smaller than in humans. Furthermore, since each variety was bred genetically, there were some polymorphisms that were biased depending on the variety, and it was considered difficult to analyze. However, the present inventors have selected a specific allele of the gene involved in the trait to be bred and rapidly spread it to the population, so that genetic diversity is reduced to a greater extent than usual. I expected. Therefore, genetic diversity was analyzed using microsatellite markers with a large number of alleles, not SNPs that could be arranged at high density.

  Specifically, the present inventors made a BAC alignment map of this region, and used a microsatellite marker arranged at a high density to identify a genomic region that has linkage disequilibrium with the number of vertebrae (in varieties with a large number of vertebrae). We searched for areas in which genetic diversity declined and genetic diversity was maintained in other varieties. Further, in the vicinity of the found region (650 kb), the base sequence was determined, and all VNTR (variable number of tandem repeats) type polymorphic markers were extracted. Reanalysis revealed that the region with linkage disequilibrium was 250 kb, and the microsatellite marker contained therein was significantly less diverse in individuals with alleles that increased the number of vertebrae.

  In addition, since there are two genes, NR5A1 and NR6A1, in the 250 kb region with linkage disequilibrium, polymorphism analysis was performed on F2 experimental family parent generation pigs (Table 1). As a result, it was found that NR6A1, which has a transcriptional inhibitory effect, has a mutation between pigs with a large number of vertebrae and pigs with a small number of vertebrae. Specifically, it was found that the 192nd amino acid of the amino acid sequence of NR6A1 was fixed with proline in pigs with a small number of vertebrae and with leucine in pigs with a large number of vertebrae.

  The transcription repressing action of NR6A1 is exhibited by binding to a corepressor. Therefore, when the binding ability of NR6A1 in which the 192nd amino acid is proline (natural type) and NR6A1 in which the 192nd amino acid is leucine (mutant) and the corepressor NCOR1 were examined, the binding ability of the mutant NR6A1 and NCOR1 was It was found to be less than half of the binding ability of natural NR6A1 and NCOR1. From this result, it can be determined that the mutant NR6A1 possessed by pigs with a large number of vertebrae has a low binding activity with a corepressor, and therefore has a low transcription repressing action.

That is, the present invention relates to the transcriptional repression action of NR6A1 on porcine chromosome 1, the binding ability of NR6A1 to the corepressor, the mutation of one or more amino acids in the amino acid sequence of NR6A1, and one or more of the nucleotide sequence of NR6A1. More specifically, a method for identifying the number of porcine vertebrae using a base mutation and a microsatellite sequence on pig chromosome 1 as an index, and a mutant NR6A1 possessed by a swine having a large number of vertebrae, include the following [1 ] To [16] are provided.
[1] A method for identifying the number of porcine vertebrae using NR6A1 on the first chromosome of pig as an index.
[2] The method for identifying the number of porcine vertebrae according to [1], wherein the transcription repressing action of NR6A1 on porcine chromosome 1 is used as an index.
[3] The method for identifying the number of porcine vertebrae according to [1], wherein the binding ability of NR6A1 on the porcine chromosome 1 to the corepressor is used as an index.
[4] The method for identifying the number of porcine vertebrae according to [3], wherein the corepressor is NCOR1.
[5] The method for identifying the number of porcine vertebrae according to [1], wherein a mutation of one or more amino acids is used as an index in the amino acid sequence of SEQ ID NO: 110.
[6] The method for identifying the number of porcine vertebrae according to [5], wherein the amino acid mutation includes a mutation at the 192nd amino acid of the amino acid sequence represented by SEQ ID NO: 110.
[7] The method for identifying the number of porcine vertebrae according to [6], wherein when the 192nd amino acid of the amino acid sequence represented by SEQ ID NO: 110 is leucine, the pig is judged to have a large number of vertebrae.
[8] The method for identifying the number of porcine vertebrae according to [1], wherein a mutation of one or a plurality of bases is used as an index in the base sequence described in SEQ ID NO: 109.
[9] The method for identifying the number of porcine vertebrae according to [8], wherein the base mutation includes a mutation in the 574th to 576th bases of the base sequence described in SEQ ID NO: 109.
[10] The method for identifying the number of porcine vertebrae according to [9], wherein when the 575th base of the base sequence described in SEQ ID NO: 109 is thymine, the pig is judged to have a large number of vertebrae.
[11] The DNA according to the following (a) or (b).
(A) DNA comprising the base sequence set forth in SEQ ID NO: 111
(B) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 112
[12] A protein encoded by the DNA of [11].
[13] A probe or primer for use in the method for identifying the number of porcine vertebrae according to [1] to [10].
[14] A method for identifying a pig having a large number of vertebrae, comprising detecting the microsatellite sequence according to any one of the following (a) to (n) on the first chromosome of the pig.
(a) a microsatellite sequence at positions 1913 to 1932 in the base sequence set forth in SEQ ID NO: 1
(b) a microsatellite sequence at positions 1957 to 1970 in the base sequence set forth in SEQ ID NO: 1
(c) a microsatellite sequence at positions 1419 to 1442 in the base sequence described in SEQ ID NO: 2
(d) a microsatellite sequence at positions 396 to 439 in the base sequence described in SEQ ID NO: 3
(e) a microsatellite sequence at positions 1938 to 1961 in the base sequence described in SEQ ID NO: 4
(f) a microsatellite sequence at positions 2057 to 2064 in the nucleotide sequence set forth in SEQ ID NO: 5
(g) a microsatellite sequence at positions 2067 to 2076 in the base sequence described in SEQ ID NO: 5
(h) a microsatellite sequence at positions 2079 to 2084 in the base sequence set forth in SEQ ID NO: 5
(i) a microsatellite sequence at positions 1227 to 1263 in the base sequence described in SEQ ID NO: 6
(j) a microsatellite sequence at positions 1967 to 1998 in the nucleotide sequence set forth in SEQ ID NO: 7
(k) a microsatellite sequence at positions 2003 to 2018 in the base sequence set forth in SEQ ID NO: 7
(l) a microsatellite sequence at positions 1195 to 1256 in the nucleotide sequence set forth in SEQ ID NO: 8
(m) a microsatellite sequence at positions 1427 to 1448 in the base sequence described in SEQ ID NO: 9
(n) An oligo that hybridizes under stringent conditions with the base sequence described in any one of the microsatellite sequences at positions 1046 to 1067 in the base sequence described in SEQ ID NO: 10 [15] SEQ ID NOs: 1 to 10 A reagent for identifying a pig having a large number of vertebrae, which contains a nucleotide as an active ingredient.
[16] The reagent according to [15], which contains, as an active ingredient, an oligonucleotide having the base sequence according to any one of SEQ ID NOs: 11 to 30.

  Asian varieties such as Japanese rice bran and Chinese pigs are expected as breeding resources for improving meat quality, but they are not generally used except for very small groups. The reason is low productivity due to small physique. Since the present invention enables efficient selection of genetically large pigs, the production of new lines using Western and Asian breeds becomes realistic. As a result, various characteristics of Asian varieties can be imparted to Western varieties with excellent growth and physique, and new varieties with added value can be produced.

The present invention provides the mutant NR6A1 DNA described in any of the following (a) to (e), and a protein encoded by the DNA.
(A) DNA comprising the base sequence set forth in SEQ ID NO: 111
(B) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 112
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 112
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 111
(E) DNA comprising a base sequence having at least 70% identity with the base sequence set forth in SEQ ID NO: 111

  The porcine NR6A1 has a natural form (base sequence shown in SEQ ID NO: 109, amino acid sequence shown in SEQ ID NO: 110) observed in pigs with a small number of vertebrae, and a mutant form observed in pigs with a large number of vertebrae. (The base sequence is shown in SEQ ID NO: 111, and the amino acid sequence is shown in SEQ ID NO: 112). The difference in amino acid sequence between the natural type and the mutant type is in the 192nd amino acid, the natural type is proline (corresponding codon is ccg), and the mutant type is leucine (corresponding codon is ctg).

  The DNA of the present invention can be isolated by those skilled in the art by generally known methods. For example, hybridization techniques (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) techniques (Saiki, RK. Et al., Science, 1985, 230, 1350., Saiki, RK. Et al., Science 1988, 239, 487.). The DNA of the present invention isolated using a hybridization technique or the like is at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably, with the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 111. Refers to 95% or more, more preferably 97% or more (eg, 98 to 99%) sequence identity.

  The identity of the amino acid sequence and nucleotide sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF., Proc. Natl by Carlin and Arthur. Acad. Sei. USA, 1993, 90, 5873.). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov/).

  Well-known to those skilled in the art for preparing DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 112 Examples of such methods include the above-described hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Saiki, RK. Et al., Science, 1985, 230, 1350). , Saiki, RK. Et al., Science, 1988, 239, 487.), for example, the site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Methods Enzymol, 1987) , 154, 350.). The number of amino acids to be modified is generally within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids). The amino acid modification is preferably a conservative substitution. The hydropathic index (Kyte and Doolitte, (1982) J Mol Biol. 1982 May 5; 157 (1): 105-32) and Hydrophilicity value (US Pat. No. 4,554,101) for each amino acid before and after modification are as follows: , Preferably within ± 2, more preferably within ± 1, and most preferably within ± 0.5. Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. In addition, even if the base sequence is mutated, there are cases where the mutation does not accompany amino acid mutation (degenerate mutation), and such degenerate mutated DNA is also included in the present invention.

  The DNA of the present invention includes genomic DNA, cDNA, and chemically synthesized DNA. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means.

  The present invention relates to the transcriptional repression action of NR6A1 on porcine chromosome 1, the binding ability of NR6A1 to the corepressor, the mutation of one or more amino acids in the amino acid sequence of NR6A1, the one or more bases in the nucleotide sequence of NR6A1 A method for identifying the number of porcine vertebrae as indexed by mutations and microsatellite sequences on porcine chromosome 1 is provided.

  The transcriptional repressive action of NR6A1 on porcine chromosome 1 can be determined by detecting the expression level of a gene that NR6A1 represses transcription. Here, gene expression includes transcription and translation.

  A test at the transcription level of a gene for which NR6A1 represses transcription is performed by comparing the amount of RNA transcribed from the gene for which NR6A1 represses transcription with a control. Such methods include Northern blotting using a probe that hybridizes to a polynucleotide encoding a gene that NR6A1 represses transcription, or a primer that hybridizes to a polynucleotide encoding a gene that NR6A1 represses transcription. The RT-PCR method used can be exemplified. In addition, DNA arrays (new genetic engineering handbook, Masaaki Muramatsu, Masaru Yamamoto, Yodosha, p280-284) can also be used for testing at the transcription level of genes that NR6A1 represses transcription.

  On the other hand, a test at the translation level of a gene for which NR6A1 represses transcription is performed by comparing the amount of the polypeptide transcribed and translated from the gene for which NR6A1 represses transcription with a control. Such methods include SDS polyacrylamide electrophoresis and Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunoassay (ELISA) using an antibody that binds to a gene that NR6A1 represses transcription. And immunofluorescence.

  In the above method, when the expression level of the RNA or polypeptide of the gene that NR6A1 represses transcription is increased compared to the control, it is assumed that NR6A1 itself is mutated and the NR6A1 is derived from It is predicted that the number of vertebrae will increase for those individuals.

  The binding ability of NR6A1 on porcine chromosome 1 to the corepressor can be detected using various methods known to those skilled in the art. An example is the Two-Hybrid analysis described in Example 3. Other methods include immunoprecipitation method, pull-down method, far western method, and crosslinker method. The NR6A1 corepressor used in the present invention is preferably NCOR1.

  In the above method, when the binding ability of NR6A1 and the corepressor is reduced as compared to the control, it is assumed that NR6A1 is mutated, and the number of vertebrae increases for the individual from which NR6A1 is derived. It is predicted that

  The mutation of one or more amino acids in the amino acid sequence of NR6A1 means that one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 110. The region where the amino acid mutation is observed is preferably within the hinge region of NR6A1 (regions 133 to 291 of SEQ ID NO: 110). More preferably, the mutation is a mutation at the 192nd amino acid of the amino acid sequence set forth in SEQ ID NO: 110, and even more preferably, the mutation of the 192nd amino acid in the amino acid sequence set forth in SEQ ID NO: 110 Mutation from proline to leucine. When the 192nd amino acid is leucine, it can be judged that the number of porcine vertebrae is large.

  In the present invention, in order to detect a mutation of one or more amino acids in the amino acid sequence of NR6A1, the above-described methods such as detection of the binding ability of NR6A1 and a corepressor, detection of the transcriptional repressive action of NR6A1 can be used. .

  The mutation of one or more bases in the base sequence of NR6A1 means that one or more bases are substituted, deleted, inserted, and / or added in the base sequence set forth in SEQ ID NO: 111. At this time, the base sequence is preferably mutated so as not to cause a polymorphism that does not change the amino acid sequence even if the base sequence is changed. The region where the base mutation is observed is preferably within the hinge region of NR6A1 (regions 397 to 873 of SEQ ID NO: 109). More preferably, the mutation is a mutation at the 574th to 576th bases of the base sequence shown in SEQ ID NO: 109, and even more preferably, the 575th base in the base sequence shown in SEQ ID NO: 109. Mutation of the base cytosine to thymine. When the 575th base is thymine, it can be determined that the number of porcine vertebrae is large.

The `` microsatellite sequence '' of the present invention is a microsatellite sequence represented by a microsatellite marker, SJ641, SJ878, SW705, SJ932, SJ885, SJ930, SJ884, SJ929, SJ928 or SJ820, and specifically, a microsatellite array according to any one of a) to (n);
(a) a microsatellite sequence at positions 1913 to 1932 in the base sequence set forth in SEQ ID NO: 1
(b) a microsatellite sequence at positions 1957 to 1970 in the base sequence set forth in SEQ ID NO: 1
(c) a microsatellite sequence at positions 1419 to 1442 in the base sequence described in SEQ ID NO: 2
(d) a microsatellite sequence at positions 396 to 439 in the base sequence described in SEQ ID NO: 3
(e) a microsatellite sequence at positions 1938 to 1961 in the base sequence described in SEQ ID NO: 4
(f) a microsatellite sequence at positions 2057 to 2064 in the nucleotide sequence set forth in SEQ ID NO: 5
(g) a microsatellite sequence at positions 2067 to 2076 in the base sequence described in SEQ ID NO: 5
(h) a microsatellite sequence at positions 2079 to 2084 in the base sequence set forth in SEQ ID NO: 5
(i) a microsatellite sequence at positions 1227 to 1263 in the base sequence described in SEQ ID NO: 6
(j) a microsatellite sequence at positions 1967 to 1998 in the nucleotide sequence set forth in SEQ ID NO: 7
(k) a microsatellite sequence at positions 2003 to 2018 in the base sequence set forth in SEQ ID NO: 7
(l) a microsatellite sequence at positions 1195 to 1256 in the nucleotide sequence set forth in SEQ ID NO: 8
(m) a microsatellite sequence at positions 1427 to 1448 in the base sequence described in SEQ ID NO: 9
(n) a microsatellite sequence at positions 1046 to 1067 in the base sequence set forth in SEQ ID NO: 10

  The base sequence described in SEQ ID NO: 1 indicates the peripheral sequence of microsatellite marker SJ641, the base sequence described in SEQ ID NO: 2 indicates the peripheral sequence of microsatellite marker SJ878, and the base sequence described in SEQ ID NO: 3 The sequence indicates the peripheral sequence of microsatellite marker SW705, the base sequence described in SEQ ID NO: 4 indicates the peripheral sequence of microsatellite marker SJ932, and the base sequence described in SEQ ID NO: 5 indicates the peripheral sequence of microsatellite marker SJ885. The base sequence described in SEQ ID NO: 6 indicates the peripheral sequence of microsatellite marker SJ930, the base sequence described in SEQ ID NO: 7 indicates the peripheral sequence of microsatellite marker SJ884, and the base sequence described in SEQ ID NO: 8 The sequence shows the peripheral sequence of the microsatellite marker SJ929, and the base sequence described in SEQ ID NO: 9 is the microsatellite. The peripheral sequence of light marker SJ928 is shown, and the base sequence described in SEQ ID NO: 10 is the peripheral sequence of microsatellite marker SJ820.

  Those skilled in the art can detect microsatellite sequences and mutations of one or more bases by various methods. The most reliable technique for detecting a microsatellite sequence and one or more base mutations is to directly determine the base sequence of the DNA containing the microsatellite sequence and one or more base mutations. In this method, a DNA sample is first prepared from a pig. The DNA sample can be prepared from, for example, various porcine tissues, blood, and semen samples, but is not particularly limited thereto. These samples may be commercially available or may be obtained by extraction themselves. Extraction of DNA from pigs can be performed by those skilled in the art using generally known methods such as the phenol / chloroform method or a commercially available genomic DNA extraction kit.

  In this method, a DNA containing a microsatellite site and one or more base mutations is then isolated. The isolation of the DNA can be performed, for example, by performing PCR using a DNA sample as a template, using a primer that hybridizes to the DNA containing the microsatellite site of the present invention and one or more base mutations. Is possible. For those skilled in the art, PCR can be performed by appropriately selecting optimal conditions for the reaction conditions and the like based on experiments or experience. Usually, PCR can be easily carried out using a commercially available reagent kit containing a reaction solution and a heat-resistant polymerase, a commercially available PCR device, and the like. The DNA region amplified by PCR is not particularly limited as long as it is a DNA region containing a microsatellite sequence and one or more base mutations. For example, in the case of a microsatellite sequence, any one of SEQ ID NOs: 1 to 10 is used. A DNA region containing the above microsatellite sequence in the base sequence described above is preferred. In this method, the base sequence of the isolated DNA is then determined. Those skilled in the art can easily determine the base sequence of the isolated DNA using a DNA sequencer or the like.

  In the detection of the microsatellite sequence, a method of detecting indirectly without determining the base sequence can be used in addition to the method of detecting directly by determining the base sequence as described above. A typical example of the indirect method is SSLP (Simple Sequence Length Polymorphism) method (Nucleic Acids Res. 1989; 17: 6463, Genomics. 1994; 19: 137). This method uses the fact that the length of microsatellite present in the genome varies depending on the pig type, and sets the primer that sandwiches the microsatellite to perform PCR, and the length of the amplified DNA fragment This is a method for detecting the difference between the two.

  In this method, first, a DNA sample is prepared from pigs, and then PCR using the DNA sample as a template is performed using primers that hybridize to DNA containing the microsatellite site of the present invention. Next, the DNA amplified by PCR is separated on a gel, and the chain length of the separated DNA is analyzed. The difference in the chain length of the DNA separated on the gel can be detected as a difference in band pattern after electrophoresis of the DNA fragment. If one of the primers used for PCR is fluorescently labeled, the DNA fragment after electrophoresis can be analyzed using software (eg, GeneScan software, Genotyper software (Applied Biosystems)).

  In the present invention, when the length of the detected microsatellite array is the same as the microsatellite array of (a) to (n) above, it is determined that the pig has a large number of vertebrae. For example, when the identification method of the present invention is carried out using a forward primer consisting of the base sequence set forth in SEQ ID NO: 13 and a reverse primer consisting of the base sequence set forth in SEQ ID NO: 14, When a microsatellite array identical to the described microsatellite array is detected, the test pig is determined to be a pig with a large number of vertebrae.

  In detecting one or a plurality of base mutations, since the base mutation site in the base sequence of NR6A1 is known, an allele specific oligonucleotide (ASO) hybridization method can also be used. An allele-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where the SNPs to be detected are present. When ASO is hybridized to sample DNA, the efficiency of hybridization decreases if a mismatch occurs in the SNPs site due to polymorphism. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.

  Other methods for detecting mutations in one or more bases in the nucleotide sequence of NR6A1 include LAMP (Loop-mediated isothermal amplification) method, primer extension method, invader method, and OLA (Oligo ligation assay) method.

  The present invention also provides a reagent for identifying a pig having a large number of vertebrae, which contains, as an active ingredient, an oligonucleotide that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NOs: 1 to 10. provide. The oligonucleotide is, for example, a PCR primer used in a method for identifying a pig having a large number of vertebrae, and amplifies a DNA region containing the microsatellite sequence according to any one of (a) to (n) above For synthetic oligonucleotides.

In the present invention, “stringent conditions” means that 0.1 to 1 μM PCR primer is hybridized at 55 ° C. in a reaction solution of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl _{2 and} 0.001% gelatin. This refers to the conditions under which soybeans are made, or hybridization conditions with stringency equivalent thereto. A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  A primer oligonucleotide sequence capable of amplifying the microsatellite sequence used in the present invention can be appropriately designed by those skilled in the art based on the sequence information of DNA serving as a template. Preferably, it is an oligonucleotide comprising 15 consecutive bases complementary to DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 1 to 10 or DNA which is a complementary strand thereof. In PCR, when a DNA region containing the microsatellite sequence is amplified, a pair of oligonucleotides set so as to sandwich the microsatellite sequence is usually used. The oligonucleotide consisting of the base sequence shown in the following SEQ ID NOs: 11 to 30 is an example of the most preferred oligonucleotide of the present invention.

SJ641 amplification primer set SEQ ID NO: 11 (forward primer), SEQ ID NO: 12 (reverse primer)
SJ878 amplification primer set SEQ ID NO: 13 (forward primer), SEQ ID NO: 14 (reverse primer)
SW705 amplification primer set SEQ ID NO: 15 (forward primer), SEQ ID NO: 16 (reverse primer)
SJ932 amplification primer set SEQ ID NO: 17 (forward primer), SEQ ID NO: 18 (reverse primer)
SJ885 amplification primer set SEQ ID NO: 19 (forward primer), SEQ ID NO: 20 (reverse primer)
SJ930 amplification primer set SEQ ID NO: 21 (forward primer), SEQ ID NO: 22 (reverse primer)
SJ884 amplification primer set SEQ ID NO: 23 (forward primer), SEQ ID NO: 24 (reverse primer)
SJ929 amplification primer set SEQ ID NO: 25 (forward primer), SEQ ID NO: 26 (reverse primer)
SJ928 amplification primer set SEQ ID NO: 27 (forward primer), SEQ ID NO: 28 (reverse primer)
SJ820 amplification primer set SEQ ID NO: 29 (forward primer), SEQ ID NO: 30 (reverse primer)

  The oligonucleotide of the present invention does not need to be completely complementary to the DNA region as long as the DNA region containing the microsatellite sequence can be amplified. For example, even an oligonucleotide having a substitution mutation to other bases on the 5 'end side or about several bases, or an arbitrary base added to the 5' end side, can be used as the oligonucleotide of the present invention. Is considered possible.

  The above-mentioned oligonucleotide of the present invention can be easily obtained by those skilled in the art, usually using a commercially available oligonucleotide synthesizer or a synthetic oligonucleotide contract service.

When the oligonucleotide of the present invention is used as a reagent, in addition to the above oligonucleotide, Tris-HCl, EDTA, KCl, MgCl ₂ , gelatin, Tween-20, NP-40, nucleotides (dATP, dCTP, dGTP, dTTP), nucleotide derivatives (7 deaza-dGTP, etc.) and the like.

  The present invention also provides a reagent for discriminating the number of porcine vertebrae using as an index the mutation of one or more bases in the nucleotide sequence of NR6A1 or the mutation of one or more amino acids in the amino acid sequence of NR6A1.

  The reagent for discriminating the number of porcine vertebrae using the mutation of one or more bases in the base sequence of NR6A1 of the present invention as an index includes the following primers and / or probes.

  In the present invention, a primer for amplifying a region containing a mutation site refers to a primer that can initiate complementary strand synthesis toward the mutation site using a DNA containing the mutation site as a template. The primer of the present invention can also be expressed as a primer for providing a replication origin on the 3 ′ side of a mutation site in DNA containing the mutation site. The interval between the region where the primer hybridizes and the mutation site is arbitrary. For the interval between the two, a suitable number of bases can be selected according to the analysis method of the base at the mutation site. The primer of the present invention can be modified. For example, a primer labeled with a fluorescent substance or a binding affinity substance such as biotin or digoxin is also included in the present invention.

  On the other hand, in the present invention, a probe that hybridizes to a region containing a mutation site refers to a probe that can hybridize to a polynucleotide having the base sequence of the region containing the mutation site. More specifically, a probe containing a mutation site in the probe base sequence is preferred as the probe of the present invention. Alternatively, depending on the base analysis method at the mutation site, the probe may be designed so that the end of the probe corresponds to the base adjacent to the polymorphic site. Accordingly, a probe containing a base sequence complementary to a region adjacent to the mutation site can be shown as a desirable probe in the present invention, although the base sequence of the probe itself does not contain the mutation site.

  The primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it. The length of the base sequence complementary to DNA of the primer or probe of the present invention is usually 15 to 100, generally 15 to 50, usually 15 to 30. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.

  Various reagents, enzyme substrates, buffers and the like can be combined with the reagent of the present invention according to the method. As the enzyme, an enzyme necessary for various analysis methods such as DNA polymerase, DNA ligase, or IIs restriction enzyme can be shown. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.

  The reagent for discriminating the number of porcine vertebrae using the mutation of one or more amino acids in the amino acid sequence of NR6A1 as an index of the present invention includes an antibody that binds to NR6A1. Antibodies include polyclonal antibodies and monoclonal antibodies. The antibody may be labeled as necessary.

  The present invention also provides a reagent for discriminating the number of porcine vertebrae using the binding ability of NR6A1 on the porcine chromosome 1 and a corepressor as an index. These reagents include a NR6A1 corepressor, preferably NCOR1.

  In the above-mentioned reagents, in addition to oligonucleotides and antibodies that are active ingredients, for example, sterilized water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protein stabilizers (BSA, gelatin, etc.) In addition, a preservative or the like may be mixed as necessary.

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

(I) Preparation of BAC alignment map and development of microsatellite marker PCR primers were prepared from the gene located between the KIAA1608 gene and PBX3 gene in the human chromosome 9 q-arm end region and the sequence of the porcine homologue. Using them, a BAC library (pig genome library, Suzuki, K., S. Asakawa, M. Iida, S. Shimanuki, N. Fujishima et al., 2000 Anim. Genet. 31: 8-12), Screened by a two-step PCR system. The resulting BAC clones and the BAC clones isolated by walking with their end sequences were used to generate an alignment map. Microsatellite marker is a method using direct sequencing of BAC DNA with modified (CA) 10 or (GT) 10 as a primer (Fujishima-Kanaya, N., D. Toki, K. Suzuki, T. Sawazaki, H. Hiraiwa et al., 2003 Anim. Genet. 34: 135-141). The sequencing reaction was performed on 20 μl of the reaction solution using ABI PRISM BigDye Terminator Kit (Applied Biosystems) using 10 pmol of primer and analyzed using ABI3700 sequencer.

(Ii) BAC clone sequencing
BAC clones were sequenced by the shotgun method. The purified BAC DNA was fragmented with a nebulizer (CIS-US, Inc., Bedford, Mass.), Treated with T4 DNA polymerase and Klenow, and then introduced into a plasmid vector (pBluescript II, STRATAGENE). Randomly selected subclones were sequenced for 10x redundancy. The sequencing reaction was performed using ABI PRISM BigDye Terminator Kit (Applied Biosystems) and analyzed using an ABI3700 sequencer. Individual sequences were assembled by Phred / Phrap / Consed.

(Iii) Analysis of microsatellite markers
One of the PCR primers was fluorescently labeled (6-FAM, HEX, NED), and PCR was performed using AmpliTaqGold DNA polymerase. For 15 μl of the reaction solution, 20 ng of genomic DNA and 10 pmol of primer were used. Electrophoresis was performed using an ABI3700 sequencer, and fragment analysis was performed using GeneScan software and Genotyper software (Applied Biosystems). The length of each allele PCR fragment was corrected using the value determined by sequencing.

(Iv) Porcine genomic DNA panel For Berkshire, DNA was extracted from edible meat produced in Japan (14), the United States (54), and the United Kingdom (8). Duroc (51), Landrace (15), Large White (20), and Naka Yorkshire (7) extracted DNA from semen for artificial insemination sold in Japan. For the semen, we selected the breeding boar so that the same ancestor would not exist within 3 generations. Hampshire (19) prepared DNA from blood samples of Kumamoto and Okinawa prefectures, Umeyama pig (14) livestock improvement center, and Kinka pig (6) from basic pigs kept in Shizuoka prefecture. Japanese rabbits (6) prepared DNA from wild animals captured in six prefectures.

(V) Correspondence between the microsatellite markers used in the examples and the primer sets for amplification thereof and the sequences in the sequence listing
Since the peripheral sequences and primer sets for SJ641, SJ878, SW705, SJ932, SJ885, SJ930, SJ884, SJ929, SJ928, and SJ820 have been described above, they are omitted.

SJ819
The peripheral sequence is shown in SEQ ID NO: 31, the forward primer is shown in SEQ ID NO: 32, and the reverse primer is shown in SEQ ID NO: 33.
SJ847
The peripheral sequence is shown in SEQ ID NO: 34, the forward primer is shown in SEQ ID NO: 35, and the reverse primer is shown in SEQ ID NO: 36.
SJ852
The peripheral sequence is shown in SEQ ID NO: 37, the forward primer is shown in SEQ ID NO: 38, and the reverse primer is shown in SEQ ID NO: 39.
SJ853
The peripheral sequence is shown in SEQ ID NO: 40, the forward primer is shown in SEQ ID NO: 41, and the reverse primer is shown in SEQ ID NO: 42.
SJ854
The peripheral sequence is shown in SEQ ID NO: 43, the forward primer is shown in SEQ ID NO: 44, and the reverse primer is shown in SEQ ID NO: 45.
SJ856
The peripheral sequence is shown in SEQ ID NO: 46, the forward primer is shown in SEQ ID NO: 47, and the reverse primer is shown in SEQ ID NO: 48.
SJ859
The peripheral sequence is shown in SEQ ID NO: 49, the forward primer is shown in SEQ ID NO: 50, and the reverse primer is shown in SEQ ID NO: 51.
SJ872
The peripheral sequence is shown in SEQ ID NO: 52, the forward primer is shown in SEQ ID NO: 53, and the reverse primer is shown in SEQ ID NO: 54.
SJ881
The peripheral sequence is shown in SEQ ID NO: 55, the forward primer is shown in SEQ ID NO: 56, and the reverse primer is shown in SEQ ID NO: 57.
SJ883
The peripheral sequence is shown in SEQ ID NO: 58, the forward primer is shown in SEQ ID NO: 59, and the reverse primer is shown in SEQ ID NO: 60.
SJ887
The peripheral sequence is shown in SEQ ID NO: 61, the forward primer is shown in SEQ ID NO: 62, and the reverse primer is shown in SEQ ID NO: 63.
SJ891
The peripheral sequence is shown in SEQ ID NO: 64, the forward primer is shown in SEQ ID NO: 65, and the reverse primer is shown in SEQ ID NO: 66.
SJ892
The peripheral sequence is shown in SEQ ID NO: 67, the forward primer is shown in SEQ ID NO: 68, and the reverse primer is shown in SEQ ID NO: 69.
SJ898
The peripheral sequence is shown in SEQ ID NO: 70, the forward primer is shown in SEQ ID NO: 71, and the reverse primer is shown in SEQ ID NO: 72.
SJ909
The peripheral sequence is shown in SEQ ID NO: 73, the forward primer is shown in SEQ ID NO: 74, and the reverse primer is shown in SEQ ID NO: 75.
SJ910
The peripheral sequence is shown in SEQ ID NO: 76, the forward primer is shown in SEQ ID NO: 77, and the reverse primer is shown in SEQ ID NO: 78.
SJ913
The peripheral sequence is shown in SEQ ID NO: 79, the forward primer is shown in SEQ ID NO: 80, and the reverse primer is shown in SEQ ID NO: 81.
SJ914
The peripheral sequence is shown in SEQ ID NO: 82, the forward primer is shown in SEQ ID NO: 83, and the reverse primer is shown in SEQ ID NO: 84.
SJ915
The peripheral sequence is shown in SEQ ID NO: 85, the forward primer is shown in SEQ ID NO: 86, and the reverse primer is shown in SEQ ID NO: 87.
SJ916
The peripheral sequence is shown in SEQ ID NO: 88, the forward primer is shown in SEQ ID NO: 89, and the reverse primer is shown in SEQ ID NO: 90.
SJ917
The peripheral sequence is shown in SEQ ID NO: 91, the forward primer is shown in SEQ ID NO: 92, and the reverse primer is shown in SEQ ID NO: 93.
SJ918
The peripheral sequence is shown in SEQ ID NO: 94, the forward primer is shown in SEQ ID NO: 95, and the reverse primer is shown in SEQ ID NO: 96.
SJ924
The peripheral sequence is shown in SEQ ID NO: 97, the forward primer is shown in SEQ ID NO: 98, and the reverse primer is shown in SEQ ID NO: 99.
SJ925
The peripheral sequence is shown in SEQ ID NO: 100, the forward primer is shown in SEQ ID NO: 101, and the reverse primer is shown in SEQ ID NO: 102.
SJ926
The peripheral sequence is shown in SEQ ID NO: 103, the forward primer is shown in SEQ ID NO: 104, and the reverse primer is shown in SEQ ID NO: 105.
SJ927
The peripheral sequence is shown in SEQ ID NO: 106, the forward primer is shown in SEQ ID NO: 107, and the reverse primer is shown in SEQ ID NO: 108.

[Example 1] Detection of microsatellite markers fixed in pigs with a large number of vertebrae
A range from SJ819 to SJ273 was used as a QTL candidate region, and a BAC alignment map was prepared in this region (FIG. 6). SJ819 and SJ273 are contained in the same BAC clone as KIAA1608 and PBX3, respectively. PCR primers were prepared from the gene between human chromosome 9 q arm KIAA1608 and PBX3, LHX2, NEK6, PSMB7, NR5A1, NR6A1, ARPC5L, GOLGA1, RAB9P40, HSPA5, and MAPKAP1.

  Alignment was performed with isolated BAC clones and BAC clones obtained by walking from the end sequence. MS sequences were developed from clones on the BAC alignment map (SJ898, SJ853, SJ856, SJ909, SJ910, SJ852, SJ847, SJ854, SJ878, SJ885, SJ884, SJ820, SJ872, SJ859, SJ887, SJ891, SJ881, SJ892, SJ3 ). Using these markers and existing markers located in this area (SJ819, SJ641, SW705, SJ344 (AB167439), SJ273), 188 pigs with a large number of vertebrae (Berkshire (76), Duroc (51) Hampshire (19), Landrace (15), Large White (20), Middle Yorkshire (7)) were used to search for regions with linkage disequilibrium. As a reference, 26 pigs with a small number of vertebrae (Umeyama pig (14), Jinhua pig (6), Japanese carp (6)) were used.

  As a result, SJ641, SJ878, SW705, SJ885, SJ884, and SJ820 had significantly less allele in pigs with a large number of vertebrae, and were fixed in SJ878 and SJ885 (Table 3). In SJ641, SJ884, and SJ820, alleles that appear to have been mutated have been present at a frequency of about 1% in recent years. SW705 had 92% of alleles with the highest frequency and 5% of allyl at the second position. Since 10 alleles were detected even in 26 heads with few vertebrae, it is considered to be a marker with a high mutation rate.

Underlined markers have lost diversity in swine with a high number of vertebrae

  In order to determine in detail the area where the microsatellite marker is fixed in this swine with a large number of vertebrae, the base sequence of the non-fixed marker, the area from SJ854 to SJ872, is determined, and the VNTR type such as the microsatellite marker is determined. All polymorphic markers were extracted (SJ918, SJ917, SJ916, SJ914, SJ915, SJ913, SJ932, SJ930, SJ929, SJ928, SJ925, SJ924, SJ927, SJ926). As a result of the analysis again, pigs with a large number of vertebrae showed a very small diversity and the region where linkage disequilibrium was confirmed was in the range from SJ641 to SJ820 (FIG. 6, Table 3). The newly obtained markers, SJ932, SJ930, SJ929, and SJ928, were all fixed in pigs with a large number of vertebrae.

  From these results, it is considered that the QTL involved in the number of vertebrae is located in the range of about 250 kb from SJ641 to SJ820, a microsatellite marker in the first arm q-arm end region. Microsatellite markers located in this region are highly fixed in pigs with a large number of vertebrae, and are therefore highly useful for breeding. By using a marker in this region, individuals having an ability to increase the number of vertebrae can be selected, and the effect of increasing the number of vertebrae in the offspring is about 0.6 per allele on average.

  In addition, what was expected as a result of this analysis at first was that a region with low genetic diversity was detected statistically in pigs with a large number of vertebrae, and the candidate regions could be further narrowed down It was that. However, as a result, there was no polymorphism in the microsatellite marker in the 250 kb range, and it was almost certain that the allele that increased the number of vertebrae was probably derived from one mutated pig. Thus, it was unexpected that a microsatellite marker having a qualitative relationship far beyond statistical relevance was detected.

[Example 2] Polymorphism analysis of genes present in a region with linkage disequilibrium In the region of about 250 kb from SJ641 to SJ820, where microsatellite markers are fixed in swine with a large number of vertebrae, NR5A1 (nuclear receptor subfamily 5, group A, member 1: Other Aliases: AD4BP, ELP, FTZ1, FTZF1, SF-1, SF1) and NR6A1 (nuclear receptor subfamily 6, group A, member 1: Other Aliases: GCNF, RTR) The gene is located (Figure 6).

NR5A1 is a transcription factor of ftz (Fushitarazu gene) in Drosophila. However, no homolog of ftz has been confirmed in mammals, and NR5A1 is involved in sex differentiation.
NR6A1 is a nuclear receptor expressed in the embryo, adult testis, and ovary. The ligand is unknown and is an orphan receptor. Knockout mice are embryonic lethal and dysplastic in the posterior body (Chung et al., 2001). In Xenopus, it is involved in longitudinal axis formation (David et al., 1998).

Therefore, the present inventors performed polymorphism analysis in the F2 experimental family parent generation pigs (Table 1) constructed so far for these NR5A1 and NR6A1. Polymorphism analysis was also performed using a porcine genomic DNA panel.
As a result of the polymorphism analysis, a substitution in which the proline at the 192nd amino acid in the amino acid sequence of NR6A1 (sequence of SEQ ID NO: 110) was leucine was detected, which completely matched the QTL of the F2 experimental family (Table 4). In the porcine genomic DNA panel, 200 Western varieties (pigs with a large number of vertebrae) were fixed with leucine, and 30 Oriental varieties (pigs with a small number of vertebrae) were fixed with proline. (FIG. 7). Analysis of this amino acid substitution at the DNA level revealed that the NR6A1 cDNA (SEQ ID NO: 109) of the natural form (pig with few vertebrae, proline form) 575th base was C, and the mutant type (pig with many vertebrae, leucine form). ) Of NR6A1 cDNA (SEQ ID NO: 111) of) was T.
From this result, it is predicted that the amino acid substitution from proline to leucine at the 192nd amino acid in the amino acid sequence of NR6A1 is responsible for the increase in the number of vertebrae in pigs.

[Example 3] Analysis of functional change by amino acid substitution of NR6A1 (Two-Hybrid analysis)
It has been reported that NR6A1 binds to a corepressor (NCOR1, RAP80) like other nuclear receptors and exerts a transcription repressing action, and that the hinge region of NR6A1 is necessary for this binding. Since the amino acid substitution detected this time is located in the hinge region (FIG. 7), a two-hybrid analysis was performed to analyze the change in the binding ability to the corepressor due to the amino acid substitution.

  NCOR1 has two receptor binding domains (ID-I, ID-II) in the C-terminal region (FIG. 8, Seol et al., 1996, Yan et al., 2000, Yan et al., 2002). The present inventors first prepared a porcine NCOR1 fragment comprising the region containing these ID-I and ID-II (SEQ ID NO: 113). The region of this fragment corresponds to 6205-7587 of mouse Ncor1 (NM_011308, CDS: 256..7587) and has 75.3% homology. ID-II is 217-472 of the fragment (SEQ ID NO: 113) and corresponds to 6442-6681 (2063-2142 aa) of NM_011308. ID-I is 760-945 of the fragment (SEQ ID NO: 113) and corresponds to 6970-7155 (2239-2300 aa) of NM_011308. It corresponds to human NCOR1 (NM_006311, CDS: 241-7563) 6172-7562 and has 79.9% homology.

Next, a plasmid having a region containing NCOR1 ID-I and ID-II (FIG. 8) and a plasmid having NR6A1 proline type or leucine type were constructed (FIG. 9). For construction of the plasmid, CheckMate ™ mammalian Two-Hybrid System (Promega) was used. The constructed plasmid is as follows.
pBINDpigNCORI ・ ・ ・ pBIND inserts porcine NCORI fragment (1386 bp, SEQ ID NO: 113)
pACTpigNR6A1 (P) ・ ・ ・ inserts proline porcine NR6A1 (1440 bp, SEQ ID NO: 109) into pACT
pACTpigNR6A1 (L) ・ ・ ・ insert leucine porcine NR6A1 (1440 bp, SEQ ID NO: 111) into pACT

Next, 2 × 10 ⁵ CHO-K1 cells were seeded in a 6 well plate (9.6 cm ² ) and cultured at 37 ° C. for 20 hours with 5% CO ₂ . 3 μl of FuGENE6 (Loche) and plasmid DNA were mixed and transfected. pG5luc was used at 0.1 μg, and pBIND, pBINDpigNCORI, pACT, pACTpigNR6A1P, and pACTpigNR6A1L were used at 0.5 μg. After culturing at 37 ° C. and 5% CO ₂ for 48 hours, the cells were washed twice with PBS (−), 500 μl of Glolysis buffer (Promega) was added, and the cells were allowed to stand at room temperature for 15 minutes to lyse the cells. 200 μl of the cell lysate was transferred to a 96 well plate, centrifuged at 5000 rpm for 5 minutes, and the supernatant (10 μl) was used for measurement. Firefly luciferase and Renilla luciferase activities were measured using Dual-Luciferase ™ Reporter Assay System.

  As a result, it was clarified that the mutant type had half or less binding ability with the corepressor compared to the natural type (FIG. 10). That is, it is predicted that the transcriptional repression action of NR6A1 will not be exhibited due to the amino acid mutation from proline to leucine.

[Example 4] Expression analysis and in situ hybridization in mouse embryo Using mouse 10.5 day embryos, expression of Ncor1 mRNA and Nr6a1 mRNA was analyzed by in situ hybridization. The experiment was performed as follows.

<Preparation of mouse>
C57BL / 6 mouse embryos (10.5 days old) were removed from pregnant mice under Nembutal anesthesia, fixed with formaldehyde, embedded in paraffin, and sliced to a thickness of 6 μm.

<Preparation of probe>
The following DNA fragment obtained by RT-PCR using total RNA prepared from testis was cloned into a plasmid, and a digoxigenin-labeled RNA probe was prepared by in vitro transcription method (DIG RNA labeling Mix, Roche).
Probe for Ncor1
NM_011308 2319-2739, NM_011308 (1..7780, CDS: 117..7478) → Ncor1 mRNA (mouse)
Probe for Nr6a1
NM_010264 1586-2084, NM_010264 (1..6360, CDS: 390..1877) → Nr6a1 mRNA (mouse)

<Hybridization>
After dehydration by xylene treatment and ethanol treatment, the cells were washed twice with PBS and fixed with 4% paraformardehyde for 15 minutes. After treatment with 7.5 μg / ml proteinase K at 37 ° C. for 1 hour, it was re-fixed with 4% paraformardehyde, washed with PBS for 2 minutes, 0.2N HCl for 10 minutes, and PBS for 1 minute. The mixture was acetylated with 0.1M triethanolamine-HCl, pH 8.0 for 1 minute, further treated with 0.1M triethanolamine-HCl, 0.25% acetic anhydride for 10 minutes, washed with PBS, and dehydrated with ethanol. Hybridization was carried out at 55 ° C. for 16 hours using 200-500 ng / ml probe in 50% formamide, 5X SSC, 1% SDS, 50 μg / ml tRNA, 50 μg / ml heparin. Wash the specimens after hybridization at 55 ° C for 15X with 5X SSC, and 50% formamide, 55 ° C with 2X SSC, then 50 μg / ml RNase A in 10 mM Tris-HCl, pH 8.0, 1 M NaCl, Treated with 1 mM EDTA for 15 minutes. Washing at 2X SSC at 50 ° C for 15 minutes was performed twice, washing at 0.2X SSC at 50 ° C for 15 minutes was performed twice, and washing with TBST (0.1% Tween20 in TBS) was performed once for 5 minutes. After treatment with 0.5% Blocking reagent (Roche) in TBST for 1 hour, it was incubated with anti-DIG AP conjugate (Roshe, diluted 1: 2000 with TBST) for 1 hour. Washed twice with TBST containing 2 mM levamisole, then incubated with 100 mM NaCl, 50 mM MgCl ₂ , 0.1% Tween 20, 100 mM Tris-HCl pH 9.5, 2 mM levamisole. NBT / BCIP was used as a chromogenic substrate, and after staining, counterstaining was performed with a Kernechtrot Strain Sol. (Muto Chemical).

  As a result, it was confirmed that both Ncor1 mRNA and Nr6a1 mRNA were expressed in the arch, lung bud, and dorsal root ganglion. Nr6a1 mRNA expression was weak (FIGS. 11 and 12).

[Example 5] Expression analysis and immunostaining of NR6A1 in mouse embryo Using mouse 10.5 day embryo, expression of Nr6a1 was analyzed by immunostaining. The experiment was performed as follows.
Rabbits were immunized 6 times with a synthetic partial peptide of NR6A1 (CQDELAELDPSTISV (KLH conjugate), SEQ ID NO: 114). After whole blood collection, serum was 0.22 μm filtered. As the mouse 10.5 day embryo, a C57BL / 6 mouse embryo (10.5 day old) was taken out from a pregnant mouse under Nembutal anesthesia and fixed with ethanol.
As a result, it was confirmed that the protein was expressed in both sides of the neural tube and in the body segment (FIG. 13).

  From the above, the responsible gene of QTL that fluctuates the number of vertebrae is NR6A1, and the cause of the increase in the number of vertebrae is that the ability to bind to the corepressor is reduced due to the mutation from proline to leucine. Therefore, the SNP that is the basis of this amino acid substitution can be used as an index of the ability to increase the number of vertebrae.

It is a graph of the QTL analysis result about the number of vertebrae (Hokkaido 1 family). Two QTLs were detected with almost the same statistics in the q-arm terminal region of chromosome 1 (SSC1) and the center of chromosome 7 (SSC7) q-arm. White circles indicate the positions of the microsatellite markers used in the analysis. It is a figure which shows the effect of two vertebra number QTL in a three prefecture joint family. The average number of vertebrae of F2 individuals with the respective genotypes of the two QTLs is shown. Each QTL has mainly additive effects, and the two QTLs work independently. It is a figure of the fine mapping of the vertebra number QTL in the 1st chromosome q arm end. (A) Analysis results by Wada et al. (B) Results of interval mapping using a new microsatellite marker. (C) Mapping by chromosome recombination of F2 individuals with 19 vertebrae from three prefectures. (D) A linkage map of microsatellite markers. It is a figure which shows preparation of the comparative gene map with a human in the QTL area | region of the 1st chromosome q arm terminal, and development of a microsatellite marker. A comparative gene map between pig and human was prepared using Radiation Hybrid (RH) Panel. A microsatellite marker was developed from a BAC clone isolated using the base sequence of the porcine gene located in the QTL region. It is a figure which shows the recombination in the 1st chromosome q arm terminal area | region of the individual | organism | solid of 19 vertebrae of a 3 prefecture joint family. Each marker was shown to be Jinhua pig type (J, q / q), Duroc type (D, Q / Q), and Heterotype (H, q / Q). Nineteen without recombination are shown together. It is a figure which shows development of the BAC alignment map and microsatellite marker of the area | region from SJ819 to SJ273. Dotted clones indicate those obtained by walking. Long arrows indicate existing markers and short arrows indicate new markers. The nucleotide sequences of SJ854 to SJ872 were determined. After determination of the base sequence, the marker obtained from the sequence is marked with *. Markers surrounded by squares are markers with small vertebrae and large numbers of vertebrae. It is a figure of NR6A1. The position of the amino acid substitution detected in the pig and the region binding to the corepressor are shown. It is a figure which shows a corepressor NCOR1 gene and two receptor binding domains which this gene has. It is a figure which shows the principle of the two hybrid analysis using the receptor binding domain of NCOR1. It is a graph which shows the result of the two hybrid analysis using the receptor binding domain of NCOR1. It is the photograph of the expression analysis (In situ hybridization) in the mouse embryo of NR6A1 and NCOR1. 1: olfactory plate, 2: forebrain, 3: 4th ventricle, 4: arch / mandibular progenitor tissue, 5: ball-stem junction, 6: hindlimb bud, 7: pericardium, 8: lung Buds, 9: neural tube, 10: dorsal root ganglion. It is an enlarged photograph of expression analysis (In situ hybridization) in mouse embryos of NR6A1 and NCOR1. It is a photograph of expression analysis (immunostaining) in mouse embryo of NR6A1.

Claims (16)

A method for identifying the number of porcine vertebrae using NR6A1 on porcine chromosome 1 as an index. The method for identifying the number of porcine vertebrae according to claim 1, wherein the transcription repressing action of NR6A1 on porcine chromosome 1 is used as an index. The method for discriminating the number of porcine vertebrae according to claim 1, wherein the binding ability of NR6A1 on the porcine chromosome 1 to the corepressor is used as an index. The method for identifying the number of porcine vertebrae according to claim 3, wherein the corepressor is NCOR1. The method for discriminating the number of porcine vertebrae according to claim 1, wherein the amino acid sequence of SEQ ID NO: 110 uses a mutation of one or more amino acids as an index. The method for identifying the number of porcine vertebrae according to claim 5, wherein the amino acid mutation comprises a mutation at the 192nd amino acid of the amino acid sequence represented by SEQ ID NO: 110. The method for identifying the number of porcine vertebrae according to claim 6, wherein when the 192nd amino acid of the amino acid sequence shown in SEQ ID NO: 110 is leucine, it is determined that the pig has a large number of vertebrae. The method for identifying the number of porcine vertebrae according to claim 1, wherein a mutation of one or a plurality of bases is used as an index in the base sequence set forth in SEQ ID NO: 109. The method for identifying the number of porcine vertebrae according to claim 8, wherein the base mutation includes a mutation at the 574th to 576th bases of the base sequence shown in SEQ ID NO: 109. The method for identifying the number of porcine vertebrae according to claim 9, wherein when the 575th base of the base sequence described in SEQ ID NO: 109 is thymine, it is determined that the pig has a large number of vertebrae. DNA as described in the following (a) or (b).
(A) DNA comprising the base sequence set forth in SEQ ID NO: 111
(B) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 112 A protein encoded by the DNA of claim 11. A probe or primer for use in the method for identifying the number of porcine vertebrae according to claim 1. A method for identifying a pig having a large number of vertebrae, comprising detecting the microsatellite sequence according to any one of the following (a) to (n) on the first chromosome of the pig.
(a) a microsatellite sequence at positions 1913 to 1932 in the base sequence set forth in SEQ ID NO: 1
(b) a microsatellite sequence at positions 1957 to 1970 in the base sequence set forth in SEQ ID NO: 1
(c) a microsatellite sequence at positions 1419 to 1442 in the base sequence described in SEQ ID NO: 2
(d) a microsatellite sequence at positions 396 to 439 in the base sequence described in SEQ ID NO: 3
(e) a microsatellite sequence at positions 1938 to 1961 in the base sequence described in SEQ ID NO: 4
(f) a microsatellite sequence at positions 2057 to 2064 in the nucleotide sequence set forth in SEQ ID NO: 5
(g) a microsatellite sequence at positions 2067 to 2076 in the base sequence described in SEQ ID NO: 5
(h) a microsatellite sequence at positions 2079 to 2084 in the base sequence set forth in SEQ ID NO: 5
(i) a microsatellite sequence at positions 1227 to 1263 in the base sequence described in SEQ ID NO: 6
(j) a microsatellite sequence at positions 1967 to 1998 in the nucleotide sequence set forth in SEQ ID NO: 7
(k) a microsatellite sequence at positions 2003 to 2018 in the base sequence set forth in SEQ ID NO: 7
(l) a microsatellite sequence at positions 1195 to 1256 in the nucleotide sequence set forth in SEQ ID NO: 8
(m) a microsatellite sequence at positions 1427 to 1448 in the base sequence described in SEQ ID NO: 9
(n) a microsatellite sequence at positions 1046 to 1067 in the base sequence set forth in SEQ ID NO: 10 A reagent for identifying a pig with a large number of vertebrae, which contains, as an active ingredient, an oligonucleotide that hybridizes under stringent conditions with the nucleotide sequence of any one of SEQ ID NOs: 1 to 10. The reagent of Claim 15 which contains the oligonucleotide which consists of a base sequence in any one of sequence number 11-11 as an active ingredient.