JP5736655B2 - Pig vertebral number gene diagnostic kit - Google Patents

Description

  The present invention relates to a reagent and a kit for a method for determining the presence or absence of an inherited trait with increased number of vertebrae in pigs using as a marker a polymorphic marker present on or near the Vertnin gene on chromosome 7 of pigs.

  Pigs are said to have been domesticated in several parts of the Eurasian continent, with wild boars as their ancestors. In Europe from the middle of the 19th century, pigs with good growth and physique were selected and bred, and the current commercial varieties were formed based on them.

  The present inventors have been proceeding with the isolation of loci involved in economic traits for the purpose of improving pigs. Many of the economic traits such as meat quality and growth are quantitative traits, and the locus that controls them is called the quantitative trait locus (QTL). Because quantitative traits are dominated by multiple loci and are also greatly influenced by environmental factors, it is difficult to map QTL accurately, identify the responsible gene, and identify polymorphisms that cause diversity It is very difficult to do. In the QTL elucidated at the gene level in pigs, there are a PRKAG3 gene (non-patent document 1) involved in meat quality (glycogen content) and an IGF2 gene (non-patent document 2) involved in meat content.

  The present inventors created a plurality of F2 experimental families and conducted QTL analysis on various economic traits such as growth, meat production, and meat quality (Non-Patent Documents 3 to 10). As a result, various QTLs were detected. Among them, the QTLs involved in the number of vertebrae shown by the sum of the number of thoracic vertebrae and the number of lumbar vertebrae (Fig. 1) are distributed in two genomic regions (first chromosome q It was detected at the end of the arm, chromosome 7 q center of the arm (Table 1 (F2 experimental family and number of vertebrae QTL), FIG. 2).

  In Table 1 above, the increased allele Q had an effect of increasing the number of vertebrae by about +0.5 in the first chromosome, and the increased allele Q had an effect of increasing the number of vertebrae by about +0.6 in the seventh chromosome.

  The number of cervical vertebrae in pigs is 7 as in other mammals, but the number of thoracic vertebrae and lumbar vertebrae varies, with 13 to 16 thoracic vertebrae and 5 to 7 lumbar vertebrae (non-patent literature). 11). The total number of these (vertebral vertebrae) is 19 in the boar ancestor wild boar, but in the current meat variety, it is 20 to 23. In pigs only (Figure 1). Pigs were selected to increase their body size to increase meat volume and improve fertility, and it was thought that the number of vertebrae increased in the process, and in fact, the increase in the number of vertebrae increased the body length by an average of about 1.5 cm (Non-Patent Document 12).

  Each of the two vertebra counts QTL has an allele that increases the number of vertebrae (hereinafter referred to as an augmented allele), the effects of which are almost equal, and the average result of each family is about 0.5 to 0.6 vertebrae per allele Increased the number. In addition, the two QTLs worked independently of each other, and on average all 2.3 vertebral numbers increased when alleles increased (Table 2 (effects of two QTLs on porcine vertebral number)).

  In Table 2 above, the JTL family was used and the QTL type was estimated by the microsatellite marker.

  In the previous F2 experimental family analysis, the vertebral number QTL in the chromosome 1 q-terminal region showed no effect of increasing the vertebral number in alleles of Asian native species such as Umeyama pig and Kinka pig, and Japanese wild boar. The alleles of Western varieties such as Landrace, Large Yorkshire, Duroc, and Berkshire were all wild-type, and the effect of increasing the number of vertebrae was observed (Table 1, Table 3 (vertebral number QTL genotype)). As for the number of vertebrae QTL on the first chromosome, a region of about 300 kb fixed in a western variety with an increased number of vertebrae was found, and the responsible gene NR6A1 located there was identified (Patent Document 1, Non-Patent Document 13). ).

  In the QTL at the center of chromosome 7 q vertebrae number QTL, some alleles derived from Western varieties used in F2 experimental families were found to have an effect of increasing the number of vertebrae (Tables 1 and 3), Patent Document 14). In addition, using a large Yorkshire breed pig in Tokushima Prefecture, we conducted a half sibling analysis of the number of vertebrae QTL on chromosome 7 in the pig, and we detected individuals with heterozygous QTL (Fig. 3) (non-patented) Reference 14). In other words, the number of vertebrae QTL at the center of chromosome 7 q is the cause of diversity in the number of vertebrae in the varieties currently used for pork production. It can greatly contribute to the improvement of meat quality such as meat production, reproduction and muscle fat content.

  Prior art document information related to the invention of the present application is shown below.

JP2006-101871

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. 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. 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 “Meat production, linkage of meat quality and DNA markers in the Kinhua pork / Yorkshire breeding family” ”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 “QTL analysis of economic traits in Jinhua pork / Duroc crossbreds 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 Kei Kanaya, Yoko Uchiguchi, Tomoko Yamaguchi, Yoshinori Nakazawa, Takeshi Hayashi, Takashi Hamada , Volume 40, No.4, P213, October 2003 King, J. W. B., and R. C. 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 Mikawa S., Morozumi T., Shimanuki S., Hayashi T., Uenishi H., Domukai M., Okumura N., Awata T. 2007 Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1). Genome Research 17: 586-593 Mikawa S., Hayashi T., Nii M., Shimanuki S., Morozumi T., Awata T., 2005 Two quantitative trait loci on Sus scrofa chromosomes 1 and 7 affecting the number of vertebrae. J Anim Sci. 83: 2247- 2254.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a reagent for efficiently judging a pig having an increased vertebral number inheritance. More specifically, an object of the present invention is to provide a reagent for genetic diagnosis using a polymorphism on or near the Vertnin (VRTN) gene on the seventh chromosome of pig.

  For isolation of genes associated with increased vertebral numbers, the QTL region was narrowed down to a 45 kb region by first using a microsatellite marker placed in detail in the QTL region and performing haplotype analysis. It was also elucidated that the Vertnin (VRTN) gene is located in this region. In the large Yorkshire pigs used for haplotype analysis, there were 42 polymorphic sites in the increased and wild types (FIG. 4), and haplotypes corresponding to each of the increased and wild types were formed (Table 4 ( Haplotypes due to polymorphic sites near the VRTN gene in large Yorkshire pigs)).

  In Table 4 above, “del” represents a deletion mutation and “ins” represents an insertion mutation.

  Currently, in addition to the large Yorkshire variety, Landrace and Duroc are widely used for pork production in Japan. Berkshire breeds are also produced as black pigs. There are a small number of Hampshire and medium Yorkshire species. Overseas, Chester White, Spotted and Pietren are also used, and some overseas companies produce hybrid pigs from these breeds.

  In order to collect polymorphism information for domestic pigs (produced from Landrace and large Yorkshire F1 females and Duroc males), we sampled pigs from the field and located in the promoter region. The relationship between the SNP (NV027-1) and the number of vertebrae is shown in Table 5 (and the relationship between the promoter region SNP (NV027-1) and the number of vertebrae in the field sample). The effect of increasing the number was observed. The other 41 polymorphic sites were also in linkage disequilibrium with NV027-1 and were related to the number of vertebrae, but the results of polymorphism analysis also suggested the presence of haplotypes other than the above two.

  Since there are haplotypes other than the two haplotypes shown in Table 4, it is important to select and use only polymorphisms highly related to augmented alleles for genetic diagnosis. For this purpose, it is necessary to collect pig samples widely from inside and outside the country and to analyze their polymorphisms in detail.

  The number of pigs shipped (16,596 thousand) is higher than that of cattle (Wagyu: 460,000 pigs, others: 770,000), and the carcass price per cow is low (pig: 28,000 yen, Wagyu: 810 Thousand yen). Therefore, it is essential to reduce the cost of genetic diagnosis.

  In pigs, the number of vertebrae is greatly related to meat production and reproduction, and it has been pointed out that the number of vertebrae is related to meat quality such as intramuscular fat content. Therefore, the spread of genetic diagnosis regarding the vertebral number gene can greatly contribute to the efficiency of today's pork production.

  In the 45 kb region containing the Vertnin (VRTN) gene, the large Yorkshire pig had 42 polymorphic sites that matched the QTL type, and there were two haplotypes composed of these polymorphisms. However, haplotype analysis using field samples (Landrace, large Yorkshire, and Duroc hybrids) suggested the presence of minor haplotypes in addition to these two haplotypes. For accurate genetic diagnosis, we collected pig breeds widely used for meat from home and abroad, accumulated information on their polymorphisms, and clarified the polymorphism characteristic of the vertebral number increase type.

  Methods for determining polymorphism in genetic diagnosis include amplification of the region and decoding of the nucleotide sequence, RFLP (or PCR-RFLP) using restriction enzymes, mass spectrometry, and hybridization to the polymorphic site. DNA chip etc. These have differences in simplicity, cost, and suitability for mass analysis.

  In order to widely spread genetic diagnosis for pigs, it is important that the cost is low and the operation is easy. In the present invention, the PCR-SSP (sequence specific primers) method using allele-specific primers was used. In order to improve the accuracy of diagnosis, multiple polymorphic sites were used. Multiple polymorphic sites were analyzed by multiplex PCR for the purpose of reducing costs and improving convenience.

  Specifically, 487 meat breeding pigs were collected from Japan and overseas, and 7 polymorphic sites highly related to the increased number of vertebrae were selected by analyzing their polymorphisms. These 7 polymorphic sites are capable of allele-specific amplification, have amplified DNA fragment lengths that can be distinguished and distinguished by agarose gel electrophoresis, and can be amplified simultaneously by multiplex PCR. PCR primers satisfying all were searched, and their proper mixing ratio was determined. As a result, a reagent for judging 7 polymorphic sites was completed using 15 types of primers.

That is, the present invention relates to a reagent capable of efficiently determining a pig having an increased vertebral number inheritance, more specifically,
[1] A polynucleotide that hybridizes under stringent conditions to the nucleotide sequence according to any one of SEQ ID NOs: 1 to 7 present on pig chromosome 7 or a complementary strand thereof, A polynucleotide for PCR primer for amplifying a region containing the polymorphic site of any of the following (1) to (7):
(1) Position 68 of the base sequence described in SEQ ID NO: 1 (2) Position 141 of the base sequence described in SEQ ID NO: 2 (3) Position 355 of the base sequence described in SEQ ID NO: 3 (4) SEQ ID NO: : Position 207 of the base sequence described in 4 (5) Position 152 of the base sequence described in SEQ ID NO: 5 (6) Positions 155 to 445 of the base sequence described in SEQ ID NO: 6 (7) 320-322 positions of the described nucleotide sequence [2] A porcine vertebral number gene diagnostic agent comprising the polynucleotide according to [1] as an active ingredient,
[3] A porcine vertebral number gene diagnostic agent comprising the primer set of any one of (1) to (7) below as an active ingredient,
(1) Primer set consisting of the polynucleotide described in SEQ ID NO: 8 and the polynucleotide described in SEQ ID NO: 9 (2) The polynucleotide described in SEQ ID NO: 10 and described in SEQ ID NO: 11 Primer set (3) consisting of the prepared polynucleotide (3) Polynucleotide described in SEQ ID NO: 12 and primer set consisting of the polynucleotide described in SEQ ID NO: 13 (4) Polynucleotide described in SEQ ID NO: 14 And a primer set consisting of the polynucleotide described in SEQ ID NO: 15 (5) a polynucleotide described in SEQ ID NO: 16 and a primer set consisting of the polynucleotide described in SEQ ID NO: 17 (6) SEQ ID NO: And the polynucleotide described in SEQ ID NO: 19 or 20. Primer set consisting of the prepared polynucleotide (7) Polynucleotide set forth in SEQ ID NO: 21 and primer set consisting of the polynucleotide set forth in SEQ ID NO: 22 [4] All of SEQ ID NOS: 8-22 Porcine vertebral number gene diagnostic agent, comprising a polynucleotide of
[5] A polynucleotide that hybridizes with a nucleotide sequence on pig chromosome 7 under stringent conditions, and is described in any one of (1) to (7) below: Porcine vertebral number gene diagnostic agent comprising, as an active ingredient, a probe polynucleotide capable of distinguishing each polymorphic variation of
(1) In the base sequence described in SEQ ID NO: 1, (A) the base species at position 68 is A, and (B) the base species at position 68 is G.
(2) In the base sequence shown in SEQ ID NO: 2, (A) the base type at position 141 is T, and (B) the base type at position 141 is A.
(3) In the base sequence set forth in SEQ ID NO: 3, the base type at position 355 of (A) is T, and the base type at position 355 is C.
(4) In the base sequence set forth in SEQ ID NO: 4, the base type at position 207 in (A) is A, and the base type at position 207 is (B) T.
(5) In the base sequence set forth in SEQ ID NO: 5, the base type at position 152 in (A) is C, and the base type at position 152 (A) is A.
(6) In the base sequence described in SEQ ID NO: 6, (A) an insertion mutation having a base sequence at positions 155 to 445, (B) a deletion mutation having no base sequence at positions 155 to 445 (7) sequence In the base sequence of No. 7, (A) the base sequence at positions 320 to 322 is AAA, and (B) the base sequence at positions 320 to 322 is AAAAA.
[6] A diagnostic kit comprising the porcine vertebra number gene diagnostic agent according to any one of [2] to [5],
I will provide a.

  By providing a reagent that can easily determine the vertebral number genotype, the genetic ability of the breeding pig can be accurately determined, and the pig's meat productivity, fertility, and meat quality can be improved efficiently. In addition, individuals with the necessary genotype can be supplied by genetic diagnosis while maintaining the genetic diversity of the basic population.

It is a figure which shows the skeleton of a pig and the number of vertebrae of mammals. The number of vertebrae is the sum of the number of thoracic vertebrae and the number of lumbar vertebrae. In pigs, the number of vertebrae varies. It is a figure which shows the QTL analysis (JD family line) result regarding the number of vertebrae. Two QTLs were detected for the number of vertebrae in the first arm and the middle arm of chromosome 7. It is a figure which shows the half sibling analysis result of the vertebra number QTL on the 7th chromosome using the pig of the Tokushima prefecture large Yorkshire breed pig. The transmission of the QTL region on the homologous chromosome of the pig to piglets was analyzed using microsatellite markers. Divided into two groups (arbitrarily shown in black and white) according to the QTL region of the breeding pigs from which they were derived, the average value of the number of vertebrae was plotted. The number of piglets and the standard error were also shown. In the t test, the QTL of the sow was judged to be heterozygous at P <0.01 (**). The Z test was performed according to the method of Nezer et al. With an effect of increasing the number of vertebrae of 0.43, and Z <-2.0 was made homozygous. In addition, the number of vertebrae in each pig of homozygous pigs was multiple-tested with the number of vertebrae in piglets (Q: 371, q: 383) that were transmitted by heterozygous pigs with increased vertebral number and wild type alleles. (Steel test) and vertebral number increased type or wild type was determined. It is a figure which shows the genome structure analysis result of the vertebra number QTL vicinity region. Among the polymorphic sites in and near the VRTN gene, polymorphic sites consistent with the QTL type were shown in large Yorkshire pigs. Boxes indicate exons and shaded areas indicate translation regions. It is a photograph which shows the result of vertebra number genetic diagnosis. In wild type homozygous individuals (wt / wt), four bands of 297 bp, 213 bp, 202 bp and 115 bp were confirmed. In homozygous individuals with increased vertebral number (Q / Q), four bands of 242 bp, 165 bp, 146 bp, and 94 bp were confirmed. In the heterozygous form, 8 bands were confirmed. M is molecular weight marker (50 bp ladder) It is a figure which shows the haplotype which consists of a polymorphism in the VRTN gene and its vicinity. The polymorphism pattern and the number detected for each haplotype are shown. In NV123, "i" is 291 bp insertion, "d" is 291 bp deletion, NV107 is "3" is AAA, "5" is AAAAA, NV055-2 and NV067 are "d" is deletion type. Show. Q (increased type) for which the effect of QTL has been judged and 7 polymorphic sites separated in q1 and L1 (both wild type) are shown in circles. M2 and J1 are among haplotypes detected in Asian pigs. It is a figure which shows the determination method of NV123. The insertion / deletion mutation of the PRE1 sequence indicated by the diagonal lines is determined using three types of primers (123f1, 123r1, 123r2). In the insertion type, a DNA fragment of 504 bases by 123f1 and 123r2 is not detected due to low amplification efficiency, and only a band of 242 bases by 123f1 and 123r1 is detected. A 213 base band of 123f1 and 123r2 is detected from the deletion type. From the heterozygous form, a band of 504 bases is not detected, and two bands of 242 bases and 213 bases are generated. It is a figure which shows the sequence of NV123 insertion type and NV123 deletion type. The PRE1 sequence is double underlined, the overlapping sequence is boxed, and the border sequence (characteristic in the insertion type) is italicized.

  The present invention relates to a method for determining the presence or absence of a porcine vertebral number-increasing genetic trait using as an index a polymorphic marker present on or in the vicinity of the Vertnin (VRTN) gene on the seventh chromosome of pig (in the present specification, “ In some cases, the determination method of the present invention may be described), and a reagent usable for the method is provided.

  The nucleotide sequence of the Vertnin gene and the amino acid sequence of the protein encoded by the gene are described in SEQ ID NOs: 23 and 24, respectively.

  In the present invention, “determination” usually means that a test pig (pig subjected to the determination method of the present invention) has or does not have a vertebral number wild-type genetic trait. Further, it refers to discriminating between swine having an increased vertebral number-inherited genetic trait and other pigs. It may be expressed as “inspection” or the like.

  In addition, the determination method of the present invention is not necessarily limited to the method for determining a pig individual, for example, meat or processed meat (for example, ham), or a biological sample derived from a test pig. And a method for determining the presence or absence of an inherited trait with increased vertebral number.

  In the present invention, “polymorphism” refers to, for example, single nucleotide polymorphism (SNP), deletion mutation, insertion mutation, microsatellite and the like.

A preferred embodiment of the determination method of the present invention is a method characterized by using a polymorphic marker present in the pig genome.
The polymorphic marker in the present invention refers to a DNA site (polymorphic site) capable of determining the presence or absence of an inherited trait with increased number of vertebrae in pigs. Therefore, using a polymorphic marker in the method of the present invention usually means determining the presence or absence of an inherited trait of increased swine vertebral number based on the difference in DNA sequence (base species) at the polymorphic site of the present invention. Say.

Specifically, the polymorphic marker used in the present invention is a polymorphic marker present on porcine chromosome 7, which is a polymorphic marker at the following site (in the present specification, “polymorphic marker of the present invention”). May be indicated).
(1) Position 68 of the base sequence described in SEQ ID NO: 1 (2) Position 141 of the base sequence described in SEQ ID NO: 2 (3) Position 355 of the base sequence described in SEQ ID NO: 3 (4) SEQ ID NO: : Position 207 of the base sequence described in 4 (5) Position 152 of the base sequence described in SEQ ID NO: 5 (6) Positions 155 to 445 of the base sequence described in SEQ ID NO: 6 (7) 320-322 positions of the described base sequence

As a preferred embodiment of the above method, the presence or absence of an inherited trait of increased swine vertebral number by evaluating a polymorphic site in the polymorphic marker (for example, evaluating a difference in base type (DNA sequence) in the polymorphic site) This is a method of determining.
More specifically, the method uses the one or a plurality of polymorphic markers according to any one of (1) to (7) present in the porcine genome, and the base species in each of the polymorphic markers. This is a method for determining the presence or absence of an inherited trait with increased vertebral number of pigs based on the following determination table in which (DNA sequence) is described.

  In Table 6 above, “del” represents a deletion mutation and “ins” represents an insertion mutation.

  The polymorphic markers (1) to (7) of the present invention are shown in Tables 7-1 and 7-2 below. In the following Tables 7-1 and 7-2, the letters enclosed by squares indicate the polymorphic sites. The underline indicates the position of the primer, and the italic letters in SEQ ID NOs: 8 and 13 indicate mismatch bases. The insertion of tctgtt (61..66) of SEQ ID NO: 5 is found in some of the wild type alleles of Landrace, Berkshire, and Middle Yorkshire.

As a preferred embodiment of the reagent of the present invention, a test reagent for determination (reagent) for use in the above method is provided.
As a preferred embodiment of the test agent of the present invention, one or a plurality of polymorphic sites described in the following (1) to (7) existing on the seventh chromosome of pig (in the present specification, “polymorphic site of the present invention”). And a PCR primer polynucleotide for amplifying a DNA region containing the DNA region.

More specifically, a polynucleotide that hybridizes under stringent conditions to the nucleotide sequence of any one of SEQ ID NOs: 1 to 7 present on swine chromosome 7 or a complementary strand thereof, wherein the nucleotide Examples of the polynucleotide for PCR primer for amplifying a region containing the polymorphic site of any one of the following (1) to (7) present in the sequence.
(1) Position 68 of the base sequence described in SEQ ID NO: 1 (2) Position 141 of the base sequence described in SEQ ID NO: 2 (3) Position 355 of the base sequence described in SEQ ID NO: 3 (4) SEQ ID NO: : Position 207 of the base sequence described in 4 (5) Position 152 of the base sequence described in SEQ ID NO: 5 (6) Positions 155 to 445 of the base sequence described in SEQ ID NO: 6 (7) 320-322 positions of the described base sequence

  The polynucleotide is useful as a diagnostic agent for porcine vertebral number gene. Accordingly, the present invention provides a porcine vertebral number gene diagnostic agent comprising the above polynucleotide as an active ingredient.

The method for determining the presence or absence of an inherited trait with increased number of vertebrae in the present invention can be preferably performed using a PCR-SSP (sequence specific primers) method. The PCR-SSP method is a method in which a DNA site having a specific sequence is selectively amplified by PCR.
For example, a primer capable of selectively amplifying the “enhanced Q” base species (DNA sequence) in the polymorphic marker of the present invention is set and PCR is performed. Based on the presence or absence of the PCR amplification product, it is possible to determine the presence or absence of an inherited trait with an increased number of vertebrae in pigs. For example, when an amplification product of the PCR is detected, it is determined that the test pig has an increased number of vertebrae. On the other hand, when the PCR amplification product is not detected, it is determined that the test pig does not have an increased vertebral number inheritance.

Examples of the primer set that can be used in the above method include the following (1) to (7). That is, this invention provides the porcine vertebra number gene diagnostic agent which contains the primer set in any one of the following (1)-(7) as an active ingredient.
(1) Primer set consisting of the polynucleotide described in SEQ ID NO: 8 and the polynucleotide described in SEQ ID NO: 9 (2) The polynucleotide described in SEQ ID NO: 10 and described in SEQ ID NO: 11 Primer set (3) consisting of the prepared polynucleotide (3) Polynucleotide described in SEQ ID NO: 12 and primer set consisting of the polynucleotide described in SEQ ID NO: 13 (4) Polynucleotide described in SEQ ID NO: 14 And a primer set consisting of the polynucleotide described in SEQ ID NO: 15 (5) a polynucleotide described in SEQ ID NO: 16 and a primer set consisting of the polynucleotide described in SEQ ID NO: 17 (6) SEQ ID NO: And the polynucleotide described in SEQ ID NO: 19 or 20. It has been made of the polynucleotide primer sets (7) SEQ ID NO: 21 and a polynucleotide described in SEQ ID NO: primer set consisting of polynucleotides described 22

  As a preferred embodiment of the reagent of the present invention, preferably, at least one set from the primer sets (1) to (7) above, more preferably two sets, more preferably 3 or 4 sets, and further preferably 5 or 6 sets. Most preferably, it is a reagent (diagnostic agent) containing all sets (7 sets) as active ingredients.

  For example, the present invention provides a porcine vertebra number gene diagnostic agent containing 15 primers shown in Table 8 below (details of vertebra number gene diagnosis kit) in the concentrations or mixing ratios described.

  As a preferred embodiment of the present invention, there is provided a porcine vertebral number gene diagnostic agent comprising all of the above 15 kinds of primer polynucleotides (polynucleotides described in SEQ ID NOs: 8 to 22). With the above 15 types of primers, it is possible to simultaneously determine 7 polymorphic sites.

The gene diagnostic agent of the present invention is preferably provided as a mixed primer and used in addition to PCR using ordinary genomic DNA as a template. If it is the density | concentration described in Table 8, 3 microliters will be usually added to a PCR reaction liquid of 15 microliters by 5-fold dilution, for example. The composition of the reaction solution is x1 PCR buffer (including Applied Biosystems, 1.5 mM MgCl ₂ ), 0.2 mM dNTP, 0.375 units AmpliTaq Gold DNA polymerase (Applied Biosystems), 20-40 ng genome in addition to the above mixed primers. DNA. The PCR cycle is [94 ° C: 9 minutes], [94 ° C: 30 seconds, 55 ° C: 30 seconds, 72 ° C: 30 seconds] x 40 times, [72 ° C: 5 minutes]. PCR products are electrophoresed in TBE buffer or TAE buffer using an agarose gel with high resolution, such as a 3% agarose gel from Agarose 1000 (Invitrogen).

  As a result of agarose gel electrophoresis, when four bands corresponding to 297 bp, 213 bp, 202 bp, and 115 bp are confirmed, it is determined that the sample is derived from a wild-type homozygous individual. When four bands corresponding to 242 bp, 165 bp, 146 bp, and 94 bp are confirmed, it is determined that the sample is derived from an increased homozygous individual. If 8 bands corresponding to 297 bp, 242 bp, 213 bp, 202 bp, 165 bp, 146 bp, 115 bp, 94 bp are confirmed, samples from wild-type and augmented heterozygous individuals (FIG. 5, Table 9 (judgment table for gene diagnosis)).

  As a minor band pattern other than the above, when five bands of 297 bp, 213 bp, 202 bp, 146 bp, and 115 bp were generated, a wild type allele derived from Landrace species (L3 (FIG. 6)) ) Or a wild type homozygous individual containing a Berkshire species-derived wild type allele (B1 (FIG. 6)). When five bands of 242 bp, 165 bp, 146 bp, 115 bp, and 94 bp are generated, an increased homozygous individual containing an increased allele (H1 (FIG. 6)) derived from a Hampshire species (Table 9).

  In Table 9 above, * 1 indicates that when the Landrace species-derived wild type allele (L3) or Berkshire species-derived wild type allele (B1) is included, amplification occurs. * 2 indicates that amplification occurs when an increased allele (H1) derived from a Hampshire species is included.

The present invention also relates to a polynucleotide that hybridizes under stringent conditions with a nucleotide sequence on the chromosome 7 of pig, and is described in any one of the following (1) to (7): Provided is a porcine vertebral number gene diagnostic agent (reagent) comprising, as an active ingredient, a probe polynucleotide capable of distinguishing each polymorphic variation of B). In addition, (A) represents “enhanced type (Q)” and (B) represents “wild type (wt)”.
(1) In the base sequence described in SEQ ID NO: 1, (A) the base species at position 68 is A, and (B) the base species at position 68 is G.
(2) In the base sequence shown in SEQ ID NO: 2, (A) the base type at position 141 is T, and (B) the base type at position 141 is A.
(3) In the base sequence set forth in SEQ ID NO: 3, the base type at position 355 of (A) is T, and the base type at position 355 is C.
(4) In the base sequence set forth in SEQ ID NO: 4, the base type at position 207 in (A) is A, and the base type at position 207 is (B) T.
(5) In the base sequence set forth in SEQ ID NO: 5, the base type at position 152 in (A) is C, and the base type at position 152 (A) is A.
(6) In the base sequence described in SEQ ID NO: 6, (A) an insertion mutation having a base sequence at positions 155 to 445, (B) a deletion mutation having no base sequence at positions 155 to 445 (7) sequence In the base sequence of No. 7, (A) the base sequence at positions 320 to 322 is AAA, and (B) the base sequence at positions 320 to 322 is AAAAA.

  For example, when the base species (or base sequence, insertion mutation) described in (A) is detected using the above reagent, the test pig is determined to have an “increased” genetic trait for the number of vertebrae. The On the other hand, when the base species (or base sequence, deletion mutation) described in (B) is detected, the genetic character of the number of vertebrae in the test pig is determined to be “wild type”.

  In addition, it would be particularly difficult for those skilled in the art to prepare a probe for detecting an arbitrary polymorphic mutation if the peripheral sequence of the polymorphic site and DNA information on the polymorphic mutation are given. It is possible to implement without. Therefore, those skilled in the art can easily prepare the above-described probe polynucleotide based on the information on the polymorphism described in the present specification (peripheral sequence of the polymorphic site and the type of mutation of the polymorphism, etc.) Can do.

  For example, when preparing a probe polynucleotide in (1) above, in the sequence described in SEQ ID NO: 1 consisting of a DNA region containing a polymorphism to be detected, position 68 (polymorphic site to be detected) A DNA sequence that specifically hybridizes with a DNA sequence containing a base species can be used as a probe polynucleotide. The polynucleotide is usually composed of a DNA sequence fragment containing a base species at position 68 (polymorphic site to be detected) in the sequence shown in SEQ ID NO: 1, or a DNA sequence fragment complementary to the sequence.

  In the polymorphism (6), there are an “insertion type” (enhancement type) and a “deletion type” (wild type) as shown in FIG. Examples of probes that can distinguish the difference between the polymorphic mutations include probes consisting of the following DNA sequences.

(Insert type)
The boundary sequence (italic part) between the DNA sequence part (indicated by the box) duplicated in the insertion type and the PRE1 sequence (double underline) may be described as a characteristic sequence in the insertion type (hereinafter referred to as “characteristic sequence”) ). Therefore, examples of the probe of the present invention include a partial sequence of the DNA sequence set forth in SEQ ID NO: 6 and a DNA sequence containing the characteristic sequence (italic part).

[Deletion type]
As a probe that specifically recognizes the deletion type, a sequence that recognizes a DNA region that extends from the box portion of FIG. 8 to its front and back, for example, a partial sequence of the DNA sequence described in SEQ ID NO: 6, A DNA sequence containing the sequence of the box part can be mentioned.

  In the present invention, “hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, No. 2). In the condition described in the edition 1989), it means that cross-hybridization with DNA other than the target does not occur significantly. If the specific hybridization is possible, the polynucleotide does not need to be completely complementary to the base sequence to be detected.

  Hybridization conditions can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. Low stringent conditions are, for example, conditions of 42 ° C., 0.1 × SSC, and 0.1% SDS, and preferably 50 ° C., 0.1 × SSC, and 0.1% SDS in washing after hybridization. More preferable hybridization conditions include highly stringent conditions. Highly stringent conditions are, for example, conditions of 65 ° C., 5 × SSC and 0.1% SDS. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, 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 the same stringency by appropriately selecting these factors. .

  The polynucleotide can be used as a probe or primer in the determination method of the present invention. When the polynucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify DNA containing the polymorphic site of the present invention. Examples of the primer include a polynucleotide having a sequence described in Table 11 to be described later. However, the primer is not particularly limited to this polynucleotide.

  In addition, when the polynucleotide of the present invention is used as a probe, the probe is not particularly limited as long as it specifically hybridizes to a DNA region containing the polymorphic marker of the present invention. The probe may be a synthetic polynucleotide and usually has a chain length of at least 15 bp.

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

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

  In the present invention, a primer for amplifying a region containing a mutation site refers to a primer capable of initiating 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 usually 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.

  In the synthesis of the oligonucleotide to be the primer or probe of the present invention, 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.

  In the above identification or determination reagent (inspection drug of the present invention), in addition to the active ingredient polynucleotide, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, Protein stabilizers (such as BSA and gelatin), preservatives, and the like may be mixed as necessary.

  Also included in the present invention is a kit for determining the presence or absence of a porcine vertebral number-increasing genetic trait (pig vertebral number gene diagnostic kit) containing the reagent (diagnostic agent) of the present invention as a constituent. In addition to the reagent of the present invention, the kit can contain, for example, a reaction solution, an enzyme (polymerase, etc.), a control sample, a reaction vessel, an operating instrument, instructions, etc. for use in the method of the present invention. .

  It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

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

[Example 1] Selection of polymorphic site used for gene diagnosis
(1) Collection of Western breed pig samples used for meats On condition that no blood relatives are recognized by the grandparents generation, 48 Landrace species, 48 large Yorkshire species, 48 Duroc species, 48 Berkshire species Collected. In addition, 48 Hampshire species, 13 Chester White species, 10 medium Yorkshire species, 3 spotted species, 2 Pietren species, and 1 LWD ternary hybrid were collected. We also collected samples of 48 Duroc breeds and 170 LWD ternary pigs produced as beef pigs that were bred at the Central Research Institute for Agricultural Feed Livestock. A total of 487 (number of chromosomes: 974).

(2) Analysis of 42 polymorphic sites in and near the VRTN gene 42 polymorphic sites in and near the VRTN gene isolated in large and wild types in large Yorkshire pigs (Fig. 4, Table 4) The polymorphism was determined using MALDI-TOF-MS or by direct sequencing of PCR amplified fragments.

By MALDI-TOF-MS, 35 polymorphic sites were analyzed by 3 sets of iPlex systems.
Group 1: 14 locations (NV025, NV028, NV030-4, NV040-1, NV040-6, NV048, NV049-2, NV055-2, NV058-3, NV058-4, NV062-2, NV064, NV067, NV071) ,
Group 2: 14 locations (NV015-2, NV024, NV027-2, NV030-1, NV030-2, NV035, NV040-2, NV040-5, NV040-7, NV049-1, NV058-1, NV090, NV107, NV149),
Group 3: 7 locations (NV027-1, NV030-3, NV040-4, NV062-1, NV065, NV083, NV104)
By direct sequence, polymorphism was determined at 7 locations (NV004, NV007, NV015-1, NV123, NV40-3, NV055-1, NV058-2).

(3) Reconfiguration of haplotypes
Using the determination results of 42 polymorphic sites, haplotypes in 487 samples (974 locus) were reconstructed. Downstream from NV064, there was a sample in which no haplotype block was retained, and discrimination was performed based on haplotypes from NV004 to NV062 (FIG. 6).

  If the haplotype corresponding to the increased allele that has been confirmed so far in large Yorkshire species is Q type, and the haplotype corresponding to the wild type allele wt is q1 type, Duroc bred at the National Institute of Agricultural Research In species, only 3 species were detected: Q / Q: 11, q1 / q1: 10, and Q / q1: 27.

  In LWD ternary hybrids (170 heads) produced at the Central Research Institute for Agricultural Animal Feed Livestock, Q / Q: 59 heads, q1 / q1: 7 heads, Q / q1: 47 heads, only NV028 is newly different Nine q1 / q2 including haplotype q2 and 19 Q / q2 were observed. In addition, other haplotypes were detected, Q / L1: 20 and q1 / L1: 9.

  In other Western breed samples, Q / Q: 74, Q / q1: 63, q1 / q1: 41, Q / q2: 42, q1 / q2: 11, and q2 / q2: There were 17 heads. The other diplotypes were 5 in Landrace (Q / L1: 3; q2 / L2: 1; Q / L3: 1); and 5 in Berkshire (Q / B1: 1) , Q1 / B2: 4), 8 Hampshire species (H1 / H1: 1; Q / H1: 1; q1 / H1: 2; q2 / H1: 3; q2 / H2: 1) In the Yorkshire species, there were 3 (q1 / Y1: 3).

  Overall, there were 11 types of haplotypes: Q, q1, q2, L1, L2, L3, B1, B2, H1, H2, and Y1 (Fig. 6, Table 10 (Western) Frequency of haplotypes due to polymorphic sites in and near the VRTN gene in breed pigs)). Systematically, Q type, H1 type, and H2 type are close, and q1 type and q2 type are the same except for NV028. The L1 type and L2 type are the same except for NV025. The L3, B1, B2, and Y1 types were systematically close to each other, and these were the same strains as the haplotypes of Japanese wild boar (J1 type) and Chinese native species (M2 type).

  Systematically, the H1 type and the H2 type are judged to be the increased type together with the Q type. Increased alleles are thought to have selected mutations during the breeding process and then spread to the population, with the Q, H1, and H2 types being highly conserved. The q2 type was determined to be a wild type along with the q1 type, and the L3 type, W2, B1, B2, and Y1 types were determined to be wild type along with wild boar-derived J1 type and Chinese species-derived M2 type. Allele types cannot be systematically determined for L1 and L2. In groups that include L1 and L2, it is necessary to verify the ability to increase the number of vertebrae.

(4) Presence or absence of vertebral number increasing effect of allele showing haplotype L1 As a result of searching for F2 family parent generation pigs with haplotype L1 so far, we found female land race and Yuumeyama pig in Miyagi prefecture. The haplotype L1 type was found on the chromosome of one side of the female land race of the F2 experimental family (LM family) (Table 1) constructed using the The other was type Q. Analysis of this F2 family shows that the chromosomal region where the L1 locus is located has no effect on increasing the number of vertebrae (Mikawa S. 2005 J Anim. Sci. 83: 2247). It was found to correspond to. Therefore, L1 type and L2 type were judged to be wild type.

(5) Selection of polymorphic sites for genetic diagnosis 98.3% (512/521) of the increased type is Q type, and wild type occupies 97.8% (443/453) of q1, q2 and L1 types Therefore, 7 polymorphic sites (NV083, NV149, NV090, NV024, NV027-1, NV123, NV107) separated between them were used for genetic diagnosis. NV083, NV024, NV027-1, NV123, and NV107 correspond to 100% genotype. NV149 matches 99.8% (972/974) and NV090 matches 99.2% (966/974).

[Example 2] Selection of PCR primers for judging polymorphism
(1) PCR primers for detecting polymorphic sites were searched. The conditions are that allele-specific amplification is possible, that the DNA fragment length is distinguishable and distinguishable by agarose gel electrophoresis, and that simultaneous amplification is possible by multiplex PCR. PCR uses AmpliTaq Gold DNA polymerase, and the PCR cycle is [94 ° C: 9 minutes], [94 ° C: 30 seconds, 55 ° C: 30 seconds, 72 ° C: 30 seconds] x 40 times, [72 ° C: 5 minutes. ]. The characteristics of the primers contained in the completed genetic diagnosis kit are as follows.

(2) PCR primer to detect polymorphism of NV123
NV123 is an insertion / deletion polymorphism of the 291 bp PRE1 sequence. With the PCR primer designed outside the polymorphic site, no insertion-type amplification was detected in the hetero-type sample due to the difference in amplification efficiency. Therefore, in addition to the PCR primer designed outside the polymorphic site, a reverse primer was designed in the insertion sequence. By using these three types of primers at the same time, insertion type, deletion type and hetero type were determined (FIG. 7). The primers used are upstream: 123f1, insertion sequence: 123r1, and downstream 123r2 (Table 11 (PCR primers for polymorphism determination and size of amplified DNA fragment)), and 242 bp from the insertion (enhanced) allele. A 213 bp DNA fragment is amplified from the type (wild type) allele. From heterozygous individuals, DNA fragments of 242 bp and 213 bp are amplified simultaneously. Amplification of 504 bp with 123f1 and 123r2 is not detected to be significantly less efficient than amplification of 242 bp with 123f1 and 123r1, and 213 bp with 123f1 and 123r2 (FIG. 7).

  In Table 11, the underlined base has allele specificity. Bases shown in italics are mismatched bases.

(3) In NV024, NV027-1, and NV149, the polymorphic site (SNP) is located at the 3 ′ end of the reverse primer. NV024 uses a wild-type base sequence, and NV027-1 and NV149 use an increased base sequence. Thus, NV024 amplifies a 202 bp DNA fragment from the wild type allele, NV027-1 amplifies the 165 bp DNA fragment from the increased allele, and NV149 amplifies the 146 bp DNA fragment from the increased allele. (Table 11).

(4) NV107 is a polymorphic site in which base A continues 3 times (increased) or 5 times (wild type). The determination primer is a reverse primer in which TTTTT is located at 3 to 7 bases from the 3 ′ end, and specifically amplifies the 5-fold type (wild type). The size of the amplified DNA fragment is 297 bp (Table 11).

(5) The primers for polymorphism determination of NV090 and NV083 contain a mismatch base of 1 base in addition to the polymorphic site (SNP) located at the 3 ′ end. NV090 uses a sequence-specific primer as a reverse primer to amplify a 115 bp DNA fragment from a wild type allele. NV083 uses a sequence-specific primer as a forward primer to amplify a 94 bp DNA fragment from the increased allele (Table 11).

[Example 3] Mixing ratio of primers enabling multiplex PCR
The primer concentration during PCR was set between 0.05 μM and 0.4 μM. Details are shown in Table 12 (mixing ratio of PCR primers for gene diagnosis). As an example when supplying as a kit, the value of 5-fold concentration is described.

[Example 4] Utilization method of gene diagnosis kit The gene diagnosis kit is provided as a mixed primer and used in addition to the PCR reaction. If it is what is described in Table 12, 3 microliters will be added to a PCR reaction liquid of 15 microliters by 5-fold dilution, for example. The composition of the reaction mixture was x1 PCR buffer (including Applied Biosystems, 1.5 mM MgCl ₂ ), 0.2 mM dNTP, 0.375 units AmpliTaq Gold DNA polymerase (Applied Biosystems), 20-40 ng genomic DNA, various primers (Tables) 12). The PCR cycle is [94 ° C: 9 minutes], [94 ° C: 30 seconds, 55 ° C: 30 seconds, 72 ° C: 30 seconds] x 40 times, [72 ° C: 5 minutes]. PCR products are subjected to agarose gel electrophoresis. Electrophoresis is performed in TBE buffer or TAE buffer using an agarose gel having high resolution, for example, 3% gel of Agarose 1000 (Invitrogen).

  As a result of agarose gel electrophoresis, if four bands corresponding to 297 bp, 213 bp, 202 bp, and 115 bp are confirmed, it is determined that the sample is derived from a wild-type homozygous individual. When four bands corresponding to 242 bp, 165 bp, 146 bp, and 94 bp are confirmed, it is determined that the sample is derived from an increased homozygous individual. If 8 bands corresponding to 297 bp, 242 bp, 213 bp, 202 bp, 165 bp, 146 bp, 115 bp, 94 bp are confirmed, samples from wild-type and augmented heterozygous individuals (FIG. 5, Table 9).

  When 5 bands of 297 bp, 213 bp, 202 bp, 146 bp, 115 bp occur as minor band patterns other than the above, the wild type allele (L3) derived from Landrace or Berkshire It is a wild-type homozygous individual containing a wild-type allele (B1) derived therefrom. If five bands of 242 bp, 165 bp, 146 bp, 115 bp, and 94 bp are generated, it is determined that this is an increased homozygous individual containing the increased allele (H1) derived from Hampshire (Table 9).

Claims (6)

A polynucleotide that specifically hybridizes to the nucleotide sequence of any one of SEQ ID NOs: 1 to 7 or its complementary strand present on pig chromosome 7, which is present in the nucleotide sequence as shown below (1 A polynucleotide for PCR primer for amplifying a region containing the polymorphic site of any of (1) to (7).
(1) Position 68 of the base sequence described in SEQ ID NO: 1 (2) Position 141 of the base sequence described in SEQ ID NO: 2 (3) Position 355 of the base sequence described in SEQ ID NO: 3 (4) SEQ ID NO: : Position 207 of the base sequence described in 4 (5) Position 152 of the base sequence described in SEQ ID NO: 5 (6) Positions 155 to 445 of the base sequence described in SEQ ID NO: 6 (7) 320-322 positions of the described base sequence   A porcine vertebral number gene diagnostic agent comprising the polynucleotide according to claim 1 as an active ingredient. A porcine vertebral number gene diagnostic agent comprising the primer set of any one of (1) to (7) below as an active ingredient.
(1) Primer set consisting of the polynucleotide described in SEQ ID NO: 8 and the polynucleotide described in SEQ ID NO: 9 (2) The polynucleotide described in SEQ ID NO: 10 and described in SEQ ID NO: 11 Primer set (3) consisting of the prepared polynucleotide (3) Polynucleotide described in SEQ ID NO: 12 and primer set consisting of the polynucleotide described in SEQ ID NO: 13 (4) Polynucleotide described in SEQ ID NO: 14 And a primer set consisting of the polynucleotide described in SEQ ID NO: 15 (5) a polynucleotide described in SEQ ID NO: 16 and a primer set consisting of the polynucleotide described in SEQ ID NO: 17 (6) SEQ ID NO: And the polynucleotide described in SEQ ID NO: 19 or 20. It has been made of the polynucleotide primer sets (7) SEQ ID NO: 21 and a polynucleotide described in SEQ ID NO: primer set consisting of polynucleotides described 22   A porcine vertebral number gene diagnostic agent comprising all the polynucleotides described in SEQ ID NOs: 8 to 22. A polynucleotide that specifically hybridizes to the nucleotide sequence of any one of SEQ ID NOS: 1 to 7 or a complementary strand thereof having the following base type (A) or (B), which is present on pig chromosome 7: The number of porcine vertebrae comprising, as an active ingredient, a probe polynucleotide that can distinguish each of the polymorphic variations (A) and (B) described in any of (1) to (7) below Genetic diagnostic agent.
(1) In the base sequence described in SEQ ID NO: 1, (A) the base species at position 68 is A, and (B) the base species at position 68 is G.
(2) In the base sequence shown in SEQ ID NO: 2, (A) the base type at position 141 is T, and (B) the base type at position 141 is A.
(3) In the base sequence set forth in SEQ ID NO: 3, the base type at position 355 of (A) is T, and the base type at position 355 is C.
(4) In the base sequence set forth in SEQ ID NO: 4, the base type at position 207 in (A) is A, and the base type at position 207 is (B) T.
(5) In the base sequence set forth in SEQ ID NO: 5, the base type at position 152 in (A) is C, and the base type at position 152 (A) is A.
(6) In the base sequence described in SEQ ID NO: 6, (A) an insertion mutation having a base sequence at positions 155 to 445, (B) a deletion mutation having no base sequence at positions 155 to 445 (7) sequence In the base sequence of No. 7, (A) the base sequence at positions 320 to 322 is AAA, and (B) the base sequence at positions 320 to 322 is AAAAA.   A diagnostic kit comprising the porcine vertebral number gene diagnostic agent according to any one of claims 2 to 5.

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