JP2009219451A - Method for individual identification and method for parentage test of cattle using single nucleotide polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a marker and a method for accurately, inexpensively, and rapidly carrying out individual identification and parentage test of cattle. <P>SOLUTION: Provided is the method for individual identification and the method for parentage test, a primer set useful for the determination and a reagent kit including the primer set, wherein the method includes a step of finding out 44 kinds of gene polymorphisms having a specific sequence, in particular a single nucleotide polymorphism and an indel polymorphism, a step of using these single nucleotide polymorphism as a marker for identification and parentage test of cattle, analyzing DNA of the subject cattle using these single nucleotide polymorphism as an index, and a step of determining the index polymorphism. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention includes a step of analyzing the DNA of a test cow using a gene polymorphism, specifically, a single nucleotide polymorphism and an insertion deletion (indel) polymorphism as an index, and determining the polymorphism as the index. The present invention relates to an individual identification method and a parent-child discrimination method of cattle. The present invention also relates to the use of gene polymorphisms in bovine genes, specifically single nucleotide polymorphisms and insertion deletion (indel) polymorphisms, as markers for individual identification of cattle and parentage discrimination. Furthermore, the present invention relates to a primer used for detecting the gene polymorphism and a reagent kit containing the primer.

  Currently, there are four varieties of Japanese beef that are bred in Japan: Japanese Black, Brown Japanese, Hornless Japanese, and Japanese Shorthorn. Among them, Japanese black cattle have excellent genetic characteristics in carcass traits such as fat crossing, meat color selection, and tightening, are treated as high-quality beef in the domestic market, and are attracting international attention. However, in recent years, in relation to the BSE problem, cheap foreign beef and domestic first-generation hybrids (F1) of Holstein and Japanese Black are illegally labeled as Japanese Black and are sold. Food impersonation problems are occurring, consumers are worried about food, and their safety is growing. For this reason, traceability using genetic analysis techniques has recently attracted attention as a means of ensuring food safety and restoring consumer confidence. Traceability is a system that can keep track of records from the time a calf is born until it is put on the market, such as the production area and distribution channels, and now it is obliged to make the production history of domestic beef public. In addition, with recent developments in molecular biology and genetic engineering, a method for analyzing polymorphisms of a DNA body has attracted attention. In livestock, DNA polymorphism markers have been developed for the purpose of “selection of livestock based on DNA polymorphism” (Non-patent Document 1). At present, DNA markers are widely used for parentage determination and individual identification of livestock as a useful genetic marker in order to complement traceability.

Currently, the most commonly used DNA marker in parentage determination and individual identification of Japanese black hair is a microsatellite (MS) marker. MS is a repetitive sequence of a simple sequence of 2 to several bp found in a non-coding region, and is widely distributed in the genome. The difference in the number of repeats in MS causes polymorphism, and the alleles are rich in diversity and are used as useful markers with a large amount of genetic information. However, in the case of inheritance from parent to child, the number of repeats may change due to slip at the time of DNA replication, and the mutation rate is 10 ⁻² to 10 ⁻⁵ per generation (non-patent document). 2). Therefore, in the parent-child determination, there is a difficulty that the reliability as a marker is lacking.

For this reason, single nucleotide polymorphism (SNPs) markers have attracted attention as new DNA markers. SNPs are polymorphisms caused by single base substitution, and are estimated to exist at a rate of one place in several hundred to 1,000 p. It is said that there are 3 to 10 million SNPs per genome. . Most of the SNPs consist of two alleles, have a lower polymorphism per marker than MS, and have less genetic information. Therefore, when performing parent-child determination and individual identification using SNPs, it is necessary to develop more markers than MS. On the other hand, SNPs have a mutation rate between parents and children of ˜2.5 × 10 ⁻⁸ per generation (Non-patent Document 2), and the mutation rate is 1000 to 1 million times lower than MS. have. Therefore, a stable discrimination rate can be expected for parent-child discrimination using SNP markers. Furthermore, since automatic analysis is possible and the cost can be reduced, the SNP marker is expected to be a useful marker in place of the MS marker. In humans, the construction of an individual identification system using SNPs is in progress (Non-patent Document 3). In addition, there is a report that evaluates the individual identification of pigs using the SNPs and the appraisal ability of parent-child determination (Non-Patent Document 4). However, bovine SNP markers have not yet been developed to a practical number in parentage determination and individual identification. This is because it took a great deal of labor and cost to identify and marker the identified SNPs on the chromosome. On the other hand, the entire base sequence of the bovine genome has been clarified as a result of genome projects being promoted around the world, and the bovine genome database can be used accordingly.

Hideyuki Mannen, “Funded Research Report on Meat in Fiscal 1997”, 1998, Vol. 16, p. 145-148 Agrafioti I. Et al., “Nucleic Acids Research”, 2006, Vol. 35, Database Issue, p. D71-75 Paktis A. J. et al. Et al., “Human Genetics”, 2007, Vol. 121, No. 3-4, p. 305-317 Rohrer G. A. Et al., “Animal Genetics”, 2007, Vol. 38, p. 253-258

  In parent-child determination and individual identification using SNPs, it is necessary to develop a marker with a higher amount of genetic information in order to obtain a marker with high appraisal ability. In order to maximize the amount of genetic information, it is essential to develop markers suitable for each group and variety.

  The present invention has been made in view of the problems to be solved, and an object of the present invention is to provide a marker and a method capable of accurately, inexpensively and rapidly implementing individual identification and parent-child differentiation of cattle.

  As a means of searching for information-rich SNPs, the present inventors have used the Amplified Fragment Length Polymorphism (AFLP) method (Vos P. et al., “Nucleic Acids Research” ”, 1995. 23, No. 21, p.4407-4414). This method is described in terms of genetic affinities (Moreno Y. et al., “FEMS Microbiology Letters”, 2002, Vol. 211, No. 1, p. 97-103), QTL (quantitative trait loci). ) Analysis (Otsen M. et al., “Genomics”, 1996, Vol. 37, No. 3, p. 289-294), linkage map creation (Lee EJ et al., “Animal Genetics (Animal) Genetics) ", 2002, Vol. 33, No. 1, pp. 42-48). By using this AFLP method, it becomes possible to predict the allele frequency in the population of each SNPs, and it can be considered that a useful marker with a high amount of genetic information can be searched more efficiently. .

  The present inventors have used the AFLP method to search for SNPs for application to parent-child determination and individual identification in Japanese black hair breeds, and further to make them markers. Moreover, the effectiveness of the developed marker was evaluated by calculating the appraisal ability based on the estimated gene frequency. Furthermore, the effectiveness of the AFLP method in developing SNPs was verified by comparing the allele frequency predicted from the analysis result by the AFLP method with the allele frequency of the developed marker. And from these verification results, we found a marker capable of accurately, inexpensively and quickly performing cow individual identification and parent-child differentiation, and completed the present invention.

  That is, this invention consists of the following.

(1). Analyzing the DNA of a test cow using at least 10 polymorphisms of the following 44 gene polymorphisms as an index, and determining the polymorphism as an index, comprising the step of identifying a bovine individual or parent and child Identification method:
1. A single nucleotide polymorphism at the 291st base of the base sequence shown in SEQ ID NO: 1,
2. A single nucleotide polymorphism at the 505th base of the base sequence shown in SEQ ID NO: 2,
3. A single nucleotide polymorphism at the 64th base of the base sequence shown in SEQ ID NO: 3,
4). A single nucleotide polymorphism at the 93rd base of the base sequence shown in SEQ ID NO: 4,
5. A single nucleotide polymorphism at the 27th base of the base sequence shown in SEQ ID NO: 5,
6). A single nucleotide polymorphism at the 147th base of the base sequence shown in SEQ ID NO: 6,
7). A single nucleotide polymorphism at the 113th base of the base sequence shown in SEQ ID NO: 7,
8). A single nucleotide polymorphism at the 594th base of the base sequence shown in SEQ ID NO: 8,
9. An insertion deletion (indel) polymorphism at the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9;
10. A single nucleotide polymorphism at the 446th base of the base sequence shown in SEQ ID NO: 10,
11. A single nucleotide polymorphism at the 185th base of the base sequence shown in SEQ ID NO: 11,
12 A single nucleotide polymorphism at the 194th base of the base sequence shown in SEQ ID NO: 12,
13. A single nucleotide polymorphism at the 143rd base of the base sequence shown in SEQ ID NO: 13,
14 A single nucleotide polymorphism at the 206th base of the base sequence shown in SEQ ID NO: 14,
15. A single nucleotide polymorphism at the 217th base of the base sequence shown in SEQ ID NO: 15,
16. A single nucleotide polymorphism at the 130th base of the base sequence shown in SEQ ID NO: 16,
17. A single nucleotide polymorphism at the 221nd base of the base sequence shown in SEQ ID NO: 17,
18. A single nucleotide polymorphism at the 382nd base of the base sequence shown in SEQ ID NO: 18,
19. A single nucleotide polymorphism at the 148th base of the base sequence shown in SEQ ID NO: 19,
20. A single nucleotide polymorphism at the 179th base of the base sequence shown in SEQ ID NO: 20,
21. A single nucleotide polymorphism at the 129th base of the base sequence shown in SEQ ID NO: 21,
22. A single nucleotide polymorphism at position 151 of the base sequence shown in SEQ ID NO: 22,
23. A single nucleotide polymorphism at the 252nd base of the base sequence shown in SEQ ID NO: 23,
24. A single nucleotide polymorphism at the 271st base of the base sequence shown in SEQ ID NO: 24,
25. A single nucleotide polymorphism at the 141st base of the base sequence shown in SEQ ID NO: 25,
26. A single nucleotide polymorphism at the 137th base of the base sequence shown in SEQ ID NO: 26,
27. A single nucleotide polymorphism at the 344th base of the base sequence shown in SEQ ID NO: 27,
28. A single nucleotide polymorphism at the 246th base of the base sequence shown in SEQ ID NO: 28,
29. A single nucleotide polymorphism at the 108th base of the base sequence shown in SEQ ID NO: 29,
30. An insertion deletion (indel) polymorphism at the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30;
31. A single nucleotide polymorphism at the 62nd base of the base sequence shown in SEQ ID NO: 31,
32. A single nucleotide polymorphism at the 156th base of the base sequence shown in SEQ ID NO: 32,
33. A single nucleotide polymorphism at the 669th base of the base sequence shown in SEQ ID NO: 33,
34. A single nucleotide polymorphism at the 183rd base of the base sequence shown in SEQ ID NO: 34,
35. An insertion deletion (indel) polymorphism at the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35,
36. An insertion deletion (indel) polymorphism in the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36;
37. A single nucleotide polymorphism at the 361st base of the base sequence shown in SEQ ID NO: 37,
38. A single nucleotide polymorphism at the 139th base of the base sequence shown in SEQ ID NO: 38,
39. A single nucleotide polymorphism at the 320th base of the base sequence shown in SEQ ID NO: 39,
40. A single nucleotide polymorphism at the 265th base of the base sequence shown in SEQ ID NO: 40,
41. A single nucleotide polymorphism at the 337th base of the base sequence shown in SEQ ID NO: 41,
42. A single nucleotide polymorphism at the 312th base of the base sequence shown in SEQ ID NO: 42,
43. A single nucleotide polymorphism at the 160th base of the base sequence shown in SEQ ID NO: 43,
44. A single nucleotide polymorphism at the 314th base of the base sequence shown in SEQ ID NO: 44.

(2) The 44 individual genetic polymorphisms are the following genetic polymorphisms, the cow individual identification method or parent-child differentiation method of (1):
1. A single nucleotide polymorphism represented by whether the 291st base of the base sequence shown in SEQ ID NO: 1 is "G" or "A";
2. A single nucleotide polymorphism represented by whether the 505th base of the base sequence shown in SEQ ID NO: 2 is “G” or “A”;
3. A single nucleotide polymorphism represented by whether the 64th base of the base sequence shown in SEQ ID NO: 3 is “C” or “G”;
4). A single nucleotide polymorphism represented by whether the 93rd base of the base sequence shown in SEQ ID NO: 4 is “C” or “T”;
5. A single nucleotide polymorphism represented by whether the 27th base of the base sequence shown in SEQ ID NO: 5 is "T" or "C";
6). A single nucleotide polymorphism represented by whether the 147th base of the base sequence shown in SEQ ID NO: 6 is “G” or “A”;
7). A single nucleotide polymorphism represented by whether the 113th base of the base sequence shown in SEQ ID NO: 7 is "A" or "T";
8). A single nucleotide polymorphism represented by whether the 594th base of the base sequence shown in SEQ ID NO: 8 is “C” or “T”;
9. An insertion deletion (indel) polymorphism represented by whether the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9 are “GGAC” or “deletion”;
10. A single nucleotide polymorphism represented by whether the 446th base of the base sequence shown in SEQ ID NO: 10 is “G” or “C”;
11. A single nucleotide polymorphism represented by whether the 185th base of the base sequence shown in SEQ ID NO: 11 is “C” or “T”;
12 A single nucleotide polymorphism represented by whether the 194th base of the base sequence shown in SEQ ID NO: 12 is “G” or “C”;
13. A single nucleotide polymorphism represented by whether the 143rd base of the base sequence shown in SEQ ID NO: 13 is "G" or "A";
14 A single nucleotide polymorphism represented by whether the 206th base of the base sequence shown in SEQ ID NO: 14 is “G” or “T”;
15. A single nucleotide polymorphism represented by whether the 217th base of the base sequence shown in SEQ ID NO: 15 is "G" or "A";
16. A single nucleotide polymorphism represented by whether the 130th base of the base sequence shown in SEQ ID NO: 16 is "G" or "A";
17. A single nucleotide polymorphism represented by whether the 221nd base of the base sequence shown in SEQ ID NO: 17 is “C” or “T”;
18. A single nucleotide polymorphism represented by whether the 382nd base of the base sequence shown in SEQ ID NO: 18 is "G" or "A";
19. A single nucleotide polymorphism represented by whether the 148th base of the base sequence shown in SEQ ID NO: 19 is “C” or “T”;
20. A single nucleotide polymorphism represented by whether the 179th base of the base sequence shown in SEQ ID NO: 20 is "T" or "C";
21. A single nucleotide polymorphism represented by whether the 129th base of the base sequence shown in SEQ ID NO: 21 is "A" or "G";
22. A single nucleotide polymorphism represented by whether the 151st base of the base sequence shown in SEQ ID NO: 22 is "G" or "A";
23. A single nucleotide polymorphism represented by whether the 252nd base of the base sequence shown in SEQ ID NO: 23 is “G” or “A”;
24. A single nucleotide polymorphism represented by whether the 271st base of the base sequence shown in SEQ ID NO: 24 is "C" or "T";
25. A single nucleotide polymorphism represented by whether the 141st base of the base sequence shown in SEQ ID NO: 25 is "G" or "A";
26. A single nucleotide polymorphism represented by whether the 137th base of the base sequence shown in SEQ ID NO: 26 is “G” or “A”;
27. A single nucleotide polymorphism represented by whether the 344th base of the base sequence shown in SEQ ID NO: 27 is “G” or “A”;
28. A single nucleotide polymorphism represented by whether the 246th base of the base sequence shown in SEQ ID NO: 28 is “C” or “T”;
29. A single nucleotide polymorphism represented by whether the 108th base of the nucleotide sequence shown in SEQ ID NO: 29 is “C” or “T”;
30. An insertion deletion (indel) polymorphism represented by whether the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30 are “TAAT” or “deletion”;
31. A single nucleotide polymorphism represented by whether the 62nd base of the base sequence shown in SEQ ID NO: 31 is "T" or "C";
32. A single nucleotide polymorphism represented by whether the 156th base of the base sequence shown in SEQ ID NO: 32 is “G” or “T”;
33. A single nucleotide polymorphism represented by whether the 669th base of the base sequence shown in SEQ ID NO: 33 is “G” or “A”;
34. A single nucleotide polymorphism represented by whether the 183rd base of the base sequence shown in SEQ ID NO: 34 is "G" or "C";
35. An insertion deletion (indel) polymorphism represented by whether the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35 are “GA” or “deletion”;
36. An insertion deletion (indel) polymorphism represented by whether the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36 are “AG” or “deletion”;
37. A single nucleotide polymorphism represented by whether the 361st base of the base sequence shown in SEQ ID NO: 37 is “C” or “T”;
38. A single nucleotide polymorphism represented by whether the 139th base of the base sequence shown in SEQ ID NO: 38 is "G" or "A";
39. A single nucleotide polymorphism represented by whether the 320th base of the base sequence shown in SEQ ID NO: 39 is “C” or “T”;
40. A single nucleotide polymorphism represented by whether the 265th base of the base sequence shown in SEQ ID NO: 40 is "G" or "A";
41. A single nucleotide polymorphism represented by whether the 337th base of the base sequence shown in SEQ ID NO: 41 is "A" or "C";
42. A single nucleotide polymorphism represented by whether the 312th base of the base sequence shown in SEQ ID NO: 42 is "A" or "C";
43. A single nucleotide polymorphism represented by whether the 160th base of the base sequence shown in SEQ ID NO: 43 is “G” or “A”;
44. A single nucleotide polymorphism represented by whether the 314th base of the nucleotide sequence shown in SEQ ID NO: 44 is "T" or "C".

  (3) Analyzing the DNA of the test cow using the gene polymorphism as an index, and determining the polymorphism as the index, the step of amplifying a DNA fragment containing each polymorphism by a gene amplification method, The individual identification method or parent-child differentiation method of the cow according to (1) or (2), which is a determination step.

(4) The step of amplifying a DNA fragment containing each polymorphism by a gene amplification method is a step of amplifying a DNA fragment containing each polymorphism using a primer set selected from the following primer set (3) ) Cattle individual identification method or parent-child differentiation method:
1. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 1, which gives a gene amplification product capable of detecting the 291st base of the base sequence shown in SEQ ID NO: 1.
2. A primer set designed based on the base sequence shown in SEQ ID NO: 2 to give a gene amplification product capable of detecting the 505th base of the base sequence shown in SEQ ID NO: 2;
3. A primer set designed based on the base sequence shown in SEQ ID NO: 3 to give a gene amplification product capable of detecting the 64th base of the base sequence shown in SEQ ID NO: 3;
4). A primer set designed based on the base sequence shown in SEQ ID NO: 4 to give a gene amplification product capable of detecting the 93rd base of the base sequence shown in SEQ ID NO: 4;
5. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 5, which gives a gene amplification product capable of detecting the 27th base of the base sequence shown in SEQ ID NO: 5;
6). A primer set designed based on the base sequence shown in SEQ ID NO: 6 to give a gene amplification product capable of detecting the 147th base of the base sequence shown in SEQ ID NO: 6;
7). A primer set designed based on the base sequence shown in SEQ ID NO: 7 to give a gene amplification product capable of detecting the 113th base of the base sequence shown in SEQ ID NO: 7;
8). A primer set designed based on the base sequence shown in SEQ ID NO: 8, which gives a gene amplification product capable of detecting the 594th base of the base sequence shown in SEQ ID NO: 8;
9. A primer set designed based on the base sequence shown in SEQ ID NO: 9, which gives a gene amplification product capable of detecting the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9;
10. A primer set designed based on the base sequence shown in SEQ ID NO: 10, which gives a gene amplification product capable of detecting the 446th base of the base sequence shown in SEQ ID NO: 10;
11. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 11, which gives a gene amplification product capable of detecting the 185th base of the base sequence shown in SEQ ID NO: 11;
12 A primer set designed based on the base sequence shown in SEQ ID NO: 12, which gives a gene amplification product capable of detecting the 194th base of the base sequence shown in SEQ ID NO: 12;
13. A primer set designed based on the base sequence shown in SEQ ID NO: 13, which gives a gene amplification product capable of detecting the 143rd base of the base sequence shown in SEQ ID NO: 13;
14 A primer set designed based on the base sequence shown in SEQ ID NO: 14, which gives a gene amplification product capable of detecting the 206th base of the base sequence shown in SEQ ID NO: 14;
15. A primer set designed based on the base sequence shown in SEQ ID NO: 15 to give a gene amplification product capable of detecting the 217th base of the base sequence shown in SEQ ID NO: 15;
16. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 16, which gives a gene amplification product capable of detecting the 130th base of the base sequence shown in SEQ ID NO: 16;
17. A primer set designed based on the base sequence shown in SEQ ID NO: 17, which gives a gene amplification product capable of detecting the 221st base of the base sequence shown in SEQ ID NO: 17;
18. A primer set designed based on the base sequence shown in SEQ ID NO: 18, which gives a gene amplification product capable of detecting the 382nd base of the base sequence shown in SEQ ID NO: 18;
19. A primer set designed based on the base sequence shown in SEQ ID NO: 19, which gives a gene amplification product capable of detecting the 148th base of the base sequence shown in SEQ ID NO: 19;
20. A primer set designed based on the base sequence shown in SEQ ID NO: 20 to give a gene amplification product capable of detecting the 179th base of the base sequence shown in SEQ ID NO: 20;
21. A primer set designed based on the base sequence shown in SEQ ID NO: 21, which gives a gene amplification product capable of detecting the 129th base of the base sequence shown in SEQ ID NO: 21;
22. A primer set designed based on the base sequence shown in SEQ ID NO: 22, which gives a gene amplification product capable of detecting the 151st base of the base sequence shown in SEQ ID NO: 22;
23. A primer set designed based on the base sequence shown in SEQ ID NO: 23, which gives a gene amplification product capable of detecting the 252nd base of the base sequence shown in SEQ ID NO: 23;
24. A primer set designed based on the base sequence shown in SEQ ID NO: 24, which gives a gene amplification product capable of detecting the 271st base of the base sequence shown in SEQ ID NO: 24;
25. A primer set designed based on the base sequence shown in SEQ ID NO: 25, which gives a gene amplification product capable of detecting the 141st base of the base sequence shown in SEQ ID NO: 25;
26. A primer set designed based on the base sequence shown in SEQ ID NO: 26, which gives a gene amplification product capable of detecting the 137th base of the base sequence shown in SEQ ID NO: 26;
27. A primer set designed based on the base sequence shown in SEQ ID NO: 27, which gives a gene amplification product capable of detecting the 344th base of the base sequence shown in SEQ ID NO: 27;
28. A primer set designed based on the base sequence shown in SEQ ID NO: 28, which gives a gene amplification product capable of detecting the 246th base of the base sequence shown in SEQ ID NO: 28;
29. A primer set designed based on the base sequence shown in SEQ ID NO: 29, which gives a gene amplification product capable of detecting the 108th base of the base sequence shown in SEQ ID NO: 29;
30. A primer set designed based on the base sequence shown in SEQ ID NO: 30 to give a gene amplification product capable of detecting the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30;
31. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 31, which gives a gene amplification product capable of detecting the 62nd base of the base sequence shown in SEQ ID NO: 31;
32. A primer set designed based on the base sequence shown in SEQ ID NO: 32, which gives a gene amplification product capable of detecting the 156th base of the base sequence shown in SEQ ID NO: 32;
33. A primer set designed based on the base sequence shown in SEQ ID NO: 33, which gives a gene amplification product capable of detecting the 669th base of the base sequence shown in SEQ ID NO: 33;
34. A primer set designed based on the base sequence shown in SEQ ID NO: 34, which gives a gene amplification product capable of detecting the 183rd base of the base sequence shown in SEQ ID NO: 34;
35. A primer set designed based on the base sequence shown in SEQ ID NO: 35, which gives a gene amplification product capable of detecting the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35;
36. A primer set designed based on the base sequence shown in SEQ ID NO: 36, which gives a gene amplification product capable of detecting the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36;
37. A primer set designed based on the base sequence shown in SEQ ID NO: 37, which gives a gene amplification product capable of detecting the 361st base of the base sequence shown in SEQ ID NO: 37;
38. A primer set designed based on the base sequence shown in SEQ ID NO: 38, which gives a gene amplification product capable of detecting the 139th base of the base sequence shown in SEQ ID NO: 38;
39. A primer set designed based on the base sequence shown in SEQ ID NO: 39, which gives a gene amplification product capable of detecting the 320th base of the base sequence shown in SEQ ID NO: 39;
40. A primer set designed based on the base sequence shown in SEQ ID NO: 40, which gives a gene amplification product capable of detecting the 265th base of the base sequence shown in SEQ ID NO: 40;
41. A primer set designed based on the base sequence shown in SEQ ID NO: 41, which gives a gene amplification product capable of detecting the 337th base of the base sequence shown in SEQ ID NO: 41;
42. A primer set designed based on the base sequence shown in SEQ ID NO: 42, which gives a gene amplification product capable of detecting the 312th base of the base sequence shown in SEQ ID NO: 42;
43. A primer set designed based on the base sequence shown in SEQ ID NO: 43, which gives a gene amplification product capable of detecting the 160th base of the base sequence shown in SEQ ID NO: 43;
44. A primer set designed based on the base sequence shown in SEQ ID NO: 44, which gives a gene amplification product capable of detecting the 314th base of the base sequence shown in SEQ ID NO: 44.

The step (3) wherein the step of amplifying a DNA fragment containing each polymorphism by a gene amplification method is a step of amplifying a DNA fragment containing each polymorphism using a primer set selected from a combination of the following polynucleotides: Cattle individual identification method or parent-child discrimination method:
1. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 53 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 54;
2. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 55 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 56;
3. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 57 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 58;
4). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 59 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 60;
5. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 61 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 62;
6). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 63 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 64;
7). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 65 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 66;
8). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 67 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 68;
9. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 69 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 70;
10. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 71 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 72;
11. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 73 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 74;
12 A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 75 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 76;
13. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 77 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 78;
14 A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 79 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 80;
15. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 81 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 82;
16. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 83 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 84;
17. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 85 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 86;
18. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 87 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 88;
19. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 89 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 90;
20. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 91 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 92;
21. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 93 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 94;
22. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 95 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 96;
23. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 97 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 98;
24. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 99 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 100;
25. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 101 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 102;
26. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 103 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 104;
27. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 105 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 106;
28. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 107 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 108;
29. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 109 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 110;
30. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 111 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 112;
31. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 113 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 114;
32. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 115 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 116;
33. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 117 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 118;
34. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 119 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 120;
35. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 121 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 122;
36. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 123 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 124;
37. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 125 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 126;
38. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 127 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 128;
39. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 129 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 130;
40. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 131 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 132;
41. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 133 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 134;
42. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 135 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 136;
43. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 137 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 138;
44. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 139 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 140.

  (6) The individual identification method or parent-child differentiation method of any of the above-mentioned (1) to (5), wherein the cow is a Japanese black cattle.

  (7) Use of any one of the 44 gene polymorphisms as a bovine individual identification or parentage discrimination marker.

  (8) Any one combination selected from the combination of the said polynucleotide.

  (9) A reagent kit containing at least any one combination selected from the combinations of the polynucleotides.

  ADVANTAGE OF THE INVENTION According to this invention, the gene polymorphism marker useful for individual identification and parent-child differentiation of a cow, especially Japanese black cattle, specifically a single nucleotide polymorphism marker and an insertion deletion (indel) polymorphism marker can be provided. In addition, according to the present invention, it is possible to provide an individual identification method and a parent-child differentiation method for cattle, particularly Japanese black cattle, characterized by including a step of analyzing the DNA of test cattle to determine these polymorphic markers. According to the individual identification method and the parent-child discrimination method of cattle according to the present invention, individual identification and parent-child discrimination can be performed accurately, inexpensively, and quickly with respect to a sample collected from a bovine body or a sample collected from meat prepared from a cow. Thus, the present invention is extremely useful for establishing food traceability as a means for ensuring food safety and reliability.

  The present invention relates to a bovine individual identification method and a parent-child differentiation method, comprising the steps of analyzing DNA of a test cow using a gene polymorphism as an index and determining the polymorphism as the index. Individual identification refers to distinguishing individuals included in a target group. Parent-child discrimination refers to determining the biological father or mother of an individual with a deterministic probability.

  The individual identification method and parent-child discrimination method of cattle according to the present invention are preferably applied to the individual identification method and parent-child differentiation method of Japanese black cattle (hereinafter simply referred to as Japanese black cattle).

There are 44 polymorphisms of the bovine gene found in the present invention, of which 40 are single nucleotide polymorphisms and 4 are insertion deletion (indel) polymorphisms. These gene polymorphisms are useful as an index for carrying out an individual identification method and a parent-child differentiation method of cattle, that is, gene polymorphism markers. These gene polymorphisms were found by analyzing genomic DNA of 91 Japanese black hairs using the AFLP method. The AFLP method uses a genomic DNA cleaved with two kinds of restriction enzymes as a template DNA for amplification using an adapter specific to each restriction enzyme, and a selective primer with an arbitrary base added to the 3 ′ end. It is based on amplification and has the following advantages. (1) The amount of genomic DNA required is small. Theoretically, electrophoresis can be performed 10,000 times or more with 500 ng of genomic DNA. (2) Information on the base sequence of genomic DNA is not necessary. (3) Operation is simple and reproducibility is high. (4) Many polymorphic bands can be detected by one electrophoresis. (5) Many bands can be detected by changing the combination of primers. (6) By using three selective bases, 4 ⁶ = 4096 primer sets can be used for the same template DNA. Because of these advantages, the present method has been applied to a wide range of studies such as genetic relationships and QTL analysis. By using this AFLP method, it is possible to predict the allele frequency within each SNPs population, and it is possible to more efficiently search for useful markers having a high amount of genetic information.

  Genetic polymorphism refers to the diversity among individuals caused by mutation or the like in a DNA sequence constituting a gene of a certain species group. Single nucleotide polymorphism refers to the diversity of DNA sequences due to mutation of one base in a genome base sequence of a certain species population (substitution of nucleobase). The insertion deletion polymorphism refers to the diversity of DNA sequences caused by the insertion / deletion of short nucleobases in the genome base sequence of a certain species population. Determining polymorphism means determining the type of nucleobase at the polymorphic site and the presence or absence of an insertion deletion. Here, the nucleobase is indicated according to IUPAC (International Union of Pure and Applied Chemistry), “A” is adenine, “C” is cytosine, “G” is guanine, and “T” is thymine. is there.

The individual identification method and parent-child discrimination method of the cattle according to the present invention include a step of analyzing the DNA of a test cow using a polymorphism selected from the following 44 gene polymorphisms as an index and determining the polymorphism as the index. Features:
1. A single nucleotide polymorphism at the 291st base of the base sequence shown in SEQ ID NO: 1,
2. A single nucleotide polymorphism at the 505th base of the base sequence shown in SEQ ID NO: 2,
3. A single nucleotide polymorphism at the 64th base of the base sequence shown in SEQ ID NO: 3,
4). A single nucleotide polymorphism at the 93rd base of the base sequence shown in SEQ ID NO: 4,
5. A single nucleotide polymorphism at the 27th base of the base sequence shown in SEQ ID NO: 5,
6). A single nucleotide polymorphism at the 147th base of the base sequence shown in SEQ ID NO: 6,
7). A single nucleotide polymorphism at the 113th base of the base sequence shown in SEQ ID NO: 7,
8). A single nucleotide polymorphism at the 594th base of the base sequence shown in SEQ ID NO: 8,
9. An insertion deletion (indel) polymorphism at the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9;
10. A single nucleotide polymorphism at the 446th base of the base sequence shown in SEQ ID NO: 10,
11. A single nucleotide polymorphism at the 185th base of the base sequence shown in SEQ ID NO: 11,
12 A single nucleotide polymorphism at the 194th base of the base sequence shown in SEQ ID NO: 12,
13. A single nucleotide polymorphism at the 143rd base of the base sequence shown in SEQ ID NO: 13,
14 A single nucleotide polymorphism at the 206th base of the base sequence shown in SEQ ID NO: 14,
15. A single nucleotide polymorphism at the 217th base of the base sequence shown in SEQ ID NO: 15,
16. A single nucleotide polymorphism at the 130th base of the base sequence shown in SEQ ID NO: 16,
17. A single nucleotide polymorphism at the 221nd base of the base sequence shown in SEQ ID NO: 17,
18. A single nucleotide polymorphism at the 382nd base of the base sequence shown in SEQ ID NO: 18,
19. A single nucleotide polymorphism at the 148th base of the base sequence shown in SEQ ID NO: 19,
20. A single nucleotide polymorphism at the 179th base of the base sequence shown in SEQ ID NO: 20,
21. A single nucleotide polymorphism at the 129th base of the base sequence shown in SEQ ID NO: 21,
22. A single nucleotide polymorphism at position 151 of the base sequence shown in SEQ ID NO: 22,
23. A single nucleotide polymorphism at the 252nd base of the base sequence shown in SEQ ID NO: 23,
24. A single nucleotide polymorphism at the 271st base of the base sequence shown in SEQ ID NO: 24,
25. A single nucleotide polymorphism at the 141st base of the base sequence shown in SEQ ID NO: 25,
26. A single nucleotide polymorphism at the 137th base of the base sequence shown in SEQ ID NO: 26,
27. A single nucleotide polymorphism at the 344th base of the base sequence shown in SEQ ID NO: 27,
28. A single nucleotide polymorphism at the 246th base of the base sequence shown in SEQ ID NO: 28,
29. A single nucleotide polymorphism at the 108th base of the base sequence shown in SEQ ID NO: 29,
30. An insertion deletion (indel) polymorphism at the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30;
31. A single nucleotide polymorphism at the 62nd base of the base sequence shown in SEQ ID NO: 31,
32. A single nucleotide polymorphism at the 156th base of the base sequence shown in SEQ ID NO: 32,
33. A single nucleotide polymorphism at the 669th base of the base sequence shown in SEQ ID NO: 33,
34. A single nucleotide polymorphism at the 183rd base of the base sequence shown in SEQ ID NO: 34,
35. An insertion deletion (indel) polymorphism at the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35,
36. An insertion deletion (indel) polymorphism in the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36;
37. A single nucleotide polymorphism at the 361st base of the base sequence shown in SEQ ID NO: 37,
38. A single nucleotide polymorphism at the 139th base of the base sequence shown in SEQ ID NO: 38,
39. A single nucleotide polymorphism at the 320th base of the base sequence shown in SEQ ID NO: 39,
40. A single nucleotide polymorphism at the 265th base of the base sequence shown in SEQ ID NO: 40,
41. A single nucleotide polymorphism at the 337th base of the base sequence shown in SEQ ID NO: 41,
42. A single nucleotide polymorphism at the 312th base of the base sequence shown in SEQ ID NO: 42,
43. A single nucleotide polymorphism at the 160th base of the base sequence shown in SEQ ID NO: 43,
44. A single nucleotide polymorphism at the 314th base of the base sequence shown in SEQ ID NO: 44.

  The single nucleotide polymorphism in the 291st base of the nucleotide sequence shown in SEQ ID NO: 1 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 505th base of the base sequence shown in SEQ ID NO: 2 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 64th base of the nucleotide sequence shown in SEQ ID NO: 3 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “G”.

  The single nucleotide polymorphism at the 93rd base in the nucleotide sequence shown in SEQ ID NO: 4 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 27th base of the nucleotide sequence shown in SEQ ID NO: 5 is preferably a single nucleotide polymorphism represented by whether the base is “T” or “C”.

  The single nucleotide polymorphism at the 147th base of the nucleotide sequence shown in SEQ ID NO: 6 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 113th base of the nucleotide sequence shown in SEQ ID NO: 7 is preferably a single nucleotide polymorphism represented by whether the base is “A” or “T”.

  The single nucleotide polymorphism at the 594th base in the base sequence shown in SEQ ID NO: 8 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism in the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9 is preferably an insertion deletion (indel) represented by whether the base is “GGAC” or “deletion”. ) Polymorphic.

  The single nucleotide polymorphism at the 446th base of the nucleotide sequence shown in SEQ ID NO: 10 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “C”.

  The single nucleotide polymorphism at the 185th base of the base sequence shown in SEQ ID NO: 11 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 194th base of the base sequence shown in SEQ ID NO: 12 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “C”.

  The single nucleotide polymorphism at the 143rd base in the nucleotide sequence shown in SEQ ID NO: 13 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 206th base in the nucleotide sequence shown in SEQ ID NO: 14 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “T”.

  The single nucleotide polymorphism at the 217th base of the nucleotide sequence shown in SEQ ID NO: 15 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 130th base of the nucleotide sequence shown in SEQ ID NO: 16 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 221nd base of the nucleotide sequence shown in SEQ ID NO: 17 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 382nd base of the nucleotide sequence shown in SEQ ID NO: 18 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 148th base of the nucleotide sequence shown in SEQ ID NO: 19 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 179th base of the nucleotide sequence shown in SEQ ID NO: 20 is preferably a single nucleotide polymorphism represented by whether the base is “T” or “C”.

  The single nucleotide polymorphism at the 129th base of the nucleotide sequence shown in SEQ ID NO: 21 is preferably a single nucleotide polymorphism represented by whether the base is “A” or “G”.

  The single nucleotide polymorphism at the 151st base of the nucleotide sequence shown in SEQ ID NO: 22 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 252nd base of the nucleotide sequence shown in SEQ ID NO: 23 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 271st base in the nucleotide sequence shown in SEQ ID NO: 24 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 141st base of the nucleotide sequence shown in SEQ ID NO: 25 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 137th base of the nucleotide sequence shown in SEQ ID NO: 26 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 344th base of the nucleotide sequence shown in SEQ ID NO: 27 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 246th base of the nucleotide sequence shown in SEQ ID NO: 28 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 108th base of the nucleotide sequence shown in SEQ ID NO: 29 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism in the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30 is preferably an insertion deletion (indel) represented by whether the base is “TAAT” or “deletion”. ) Polymorphic.

  The single nucleotide polymorphism at the 62nd base of the nucleotide sequence shown in SEQ ID NO: 31 is preferably a single nucleotide polymorphism represented by whether the base is “T” or “C”.

  The single nucleotide polymorphism at the 156th base in the base sequence shown in SEQ ID NO: 32 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “T”.

  The single nucleotide polymorphism at the 669th base of the base sequence shown in SEQ ID NO: 33 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 183rd base of the nucleotide sequence shown in SEQ ID NO: 34 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “C”.

  The single nucleotide polymorphism at the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35 is preferably an insertion deletion (indel) represented by whether the base is “GA” or “deletion”. ) Polymorphic.

  The single nucleotide polymorphism in the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36 is preferably an insertion deletion (indel) represented by whether the base is “AG” or “deletion”. ) Polymorphic.

  The single nucleotide polymorphism at the 361st base of the nucleotide sequence shown in SEQ ID NO: 37 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 139th base of the nucleotide sequence shown in SEQ ID NO: 38 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 320th base of the nucleotide sequence shown in SEQ ID NO: 39 is preferably a single nucleotide polymorphism represented by whether the base is “C” or “T”.

  The single nucleotide polymorphism at the 265th base in the nucleotide sequence shown in SEQ ID NO: 40 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 337th base of the nucleotide sequence shown in SEQ ID NO: 41 is preferably a single nucleotide polymorphism represented by whether the base is “A” or “C”.

  The single nucleotide polymorphism at the 312th base of the nucleotide sequence shown in SEQ ID NO: 42 is preferably a single nucleotide polymorphism represented by whether the base is “A” or “C”.

  The single nucleotide polymorphism at the 160th base of the nucleotide sequence shown in SEQ ID NO: 43 is preferably a single nucleotide polymorphism represented by whether the base is “G” or “A”.

  The single nucleotide polymorphism at the 314th base of the nucleotide sequence shown in SEQ ID NO: 44 is preferably a single nucleotide polymorphism represented by whether the base is “T” or “C”.

  The cattle individual identification method and parent-child discrimination method according to the present invention are at least 10 or more, preferably 12 or more, more preferably 14 or more, and more preferably 17 or more of the above 44 genetic polymorphisms. Even more preferably, 20 or more polymorphisms can be used as an indicator.

The type and number of genetic polymorphisms used as an index in individual identification is calculated by calculating the individual identification probability based on the genotype frequency of each polymorphism and selecting the type and number of polymorphisms that provide a high individual identification probability. Can be determined. The individual identification probability is defined as the probability that two unrelated individuals randomly selected have exactly the same genotype. The calculation of the individual identification probability can be performed, for example, according to the calculation formula of Holt et al. (“Forensic Science International”, 2000, Vol. 112, No. 2-3, p. 91-109) (Examples) 1). The individual identification method for cattle according to the present invention is represented by whether the 64th base of the nucleotide sequence shown in SEQ ID NO: 3 is “C” or “G” among the 44 gene polymorphisms. The individual identification probability of Japanese black breeds using 43 polymorphisms excluding single nucleotide polymorphisms as an index is 1.31 × 10 ⁻¹⁶ , and the number of Japanese black breeds reared in Japan (approximately 1.64 million heads) ) All can be identified (see Example 1). Moreover, as a result of calculating the individual identification probability in the same manner using 17 polymorphisms as an index among the 44 gene polymorphisms, the individual identification probability is 7.29 × 10 ⁻⁸ , one in 13.7 million heads. 17 types of polymorphisms coincide with each other (see Example 1). Thus, the individual identification method using 17 kinds of gene polymorphisms as an index has a sufficiently high individual identification ability in view of the individual identification probability. Furthermore, since the individual identification probability when using 17 types of genetic polymorphisms as an index is about 10 times the probability of identifying all the Japanese black breeds (about 1.64 million) in Japan, 200 It can be considered that individual identification of about 10,000 heads can be sufficiently performed by an individual identification method using as a marker a single nucleotide polymorphism of a kind less than the 17 types of gene polymorphisms. If the number of gene polymorphisms used as an indicator is too small, the individual identification probability decreases, and therefore, as described above, 10 or more, preferably 12 or more, more preferably 14 or more, and more preferably 17 or more, Even more preferably, it is appropriate to use 20 or more polymorphs. Of course, if there are many kinds of gene polymorphisms used as indices, a high individual identification probability can be obtained. In addition, the type of single nucleotide polymorphism used as an index can be increased or decreased according to the change in the number of breeding animals.

  The type and number of genetic polymorphisms used as an index in parent-child discrimination is calculated by calculating the paternal right denial probability based on the allele frequency of each polymorphism, and selecting the polymorphic type and number that provide a high probability of paternity denial. Can be determined. The paternity denial probability is defined as the probability that a bull who is not truly a paternal bull will be excluded from bulls considered as paternal candidates. The higher the paternity denial probability, the higher the accuracy of parent-child discrimination. Further, the paternity denial probability is increased by increasing the types of genetic polymorphisms used as indices, and as a result, the accuracy of parent-child discrimination is increased. For example, when the maternal genotype of the mother cow is known, Jameson et al. (“Heredity”, 1965, Vol. 20, p.419-441; “Proceedings of the 16th International Conference on Animal Blood Group and Biochemical Polymorphism, Leningrad 1978 (according to Proceedings of the XVIth International Conferencing on Animal Blood Group and Biochemical Poly. If the genotype of the mother cow is not known , Grabar & Morrice et ( 'inclusion professional Babiri Tees Inn Pearentijji testing (Inclusion Probabilities in Parentage Testing) ", 1983 years, p.277-280) (see Example 1) can be carried out according to the calculation formula of. The cattle parent-child discrimination method according to the present invention is represented by whether the 64th base of the nucleotide sequence shown in SEQ ID NO: 3 is “C” or “G” among the 44 gene polymorphisms. The probability of paternity denial of Japanese black cattle when conducted using 43 polymorphisms excluding single nucleotide polymorphism as an index is 99.96553% when the genotype of the mother cow is known, When the genotype was unknown, it was 99.0619%. From this, it was found that the parent-child discrimination method using the 43 gene polymorphisms as an index has high discrimination ability. When carrying out the parent-child discrimination of cattle, it is preferable to use the above-mentioned 43 gene polymorphisms, more preferably the above 44 gene polymorphisms as indices. For the purpose of performing parent-child discrimination with higher accuracy, parent-child discrimination can also be carried out by combining other gene polymorphisms as an index in addition to the above-mentioned 44 gene polymorphisms.

  Thus, the 44 types of genetic polymorphisms are useful as individual markers for cattle or for distinguishing parents. The individual identification marker means an index that can distinguish individuals in a group to be identified. The parent-child differentiation marker means an index that can determine the biological father or mother of a certain individual with a high probability.

  The present invention provides use of any one of the 44 gene polymorphisms as a bovine individual identification or parentage discrimination marker. Cattle individual identification or parent-child differentiation markers according to the present invention can be used in combination with each other, and other gene polymorphism markers useful for cow individual identification or parent-child differentiation, for example, other single nucleotide polymorphism markers and Can be used in combination.

  Analysis of the DNA of the test cow using the gene polymorphism as an index can be performed using a sample (test sample) collected from the test cow. The test sample is a sample collected from a bovine living body or a sample collected from meat prepared from a cow, and any sample containing DNA, preferably genomic DNA, can be used. Specific examples of the test sample include muscle tissue, visceral tissue, blood, peripheral blood leukocytes, spinal fluid, and hair root cells. Nucleic acid preparation from a test sample can be performed by a nucleic acid preparation method known per se. Genomic DNA can be preferably used as the nucleic acid sample. When the gene polymorphism is present in the protein coding region, cDNA or RNA can be used as the nucleic acid sample. When a genetic polymorphism is determined using cDNA or RNA, the base at the position in cDNA or RNA corresponding to the position where the genetic polymorphism exists in the genomic base sequence is determined. The prepared nucleic acid may be used directly, or a desired region may be amplified enzymatically by using polymerase chain reaction (hereinafter abbreviated as PCR) or other amplification method before analysis.

  In individual identification, when all of the genetic polymorphisms used as indices match in a plurality of test samples, it can be determined that the plurality of test samples are very likely to be samples from the same individual. Alternatively, in the parent-child discrimination, when all of the gene polymorphisms used as the index match, it can be determined that the cattle having the gene polymorphism used as the index is extremely likely to be a parent and child.

  The genetic polymorphism can be determined using a method known per se. For example, a method can be used in which a partial sequence including a desired polymorphic site is amplified by PCR, and the base sequence of the obtained amplification product is sequenced by a sequencing method, for example, a direct sequencing method. As a sequencing method, in addition to the sequencing method, a hybridization method, a restriction enzyme fragment length polymorphism (RFLP) analysis method and the like can be used. In addition, as a single nucleotide polymorphism analysis method, a method using fluorescence, for example, Invader assay (Lyamichev, V. et al., “Nature Biotechnology”, 1999, Vol. 17, p. 292- 296) and the Luminex method (Chen, J. et al., “Genome Research”, 2000, Vol. 10, pp. 549-557). Further, a method using a mass spectrum, for example, a method using a primer extension method can be used. Such methods include pinpoint assays (Ross, P. et al., “Nature Biotechnology”, 1998, Vol. 16, p. 1347-1351) and probe methods (Braun, A.) et al., “Clinical Chemistry”, 1997, Vol. 43, p. 1151-1158). In addition, DOL assay, Chen, X. et al., “Proceedings of the National Academy of Sciences, United States of America, The United States of Science of the United States of America. 94, 10756-10761), Sniper Assay (Piatek, AS, et al., “Nature Biotechnology”, 1998, 16, 359-363). And the tag array method (Fan, J-B. Et al., “Genome Research ch) ", 2000, Vol. 10, p. 853-860). These methods can be easily carried out by referring to the cited references in which they are reported. In addition, methods well known as single nucleotide polymorphism detection methods such as fluorescence resonance energy transfer (FRET) and Taqman method can be used.

  Of the 44 gene polymorphisms, 28 are single nucleotide polymorphisms in the restriction enzyme TaqI recognition site, 4 are single nucleotide polymorphisms in the restriction enzyme EcoRI recognition site, and 8 are in the 3 base selection site. There were single nucleotide polymorphisms, and four types were insertion deletion (indel) polymorphisms (see Example 1 and Tables 4-1 to 4-5). All of the 8 single nucleotide polymorphisms at the 3-base selection site were at the restriction enzyme recognition site. Determination of the single nucleotide polymorphism at the restriction enzyme recognition site is performed by amplifying a partial sequence containing the desired single nucleotide polymorphism site by PCR, and using the corresponding restriction enzyme for the obtained amplification product, the RFLP analysis method (PCR- (RFLP) can be easily performed. The determination of indel can be performed by detecting the fragment length of the amplified product amplified by PCR by a known method such as agarose or polyacrylamide gel electrophoresis.

  Amplification of each DNA fragment containing each of the 44 gene polymorphisms can be performed using a primer set designed from the nucleotide sequences shown in SEQ ID NOs: 1-44. The primer is not particularly limited as long as it can amplify a desired DNA fragment, preferably specifically. The nucleotide sequence of the DNA fragment, specifically each nucleotide sequence of SEQ ID NOs: 1 to 44 or a complementary nucleotide sequence thereof. And can be produced by conventional methods such as chemical synthesis. The primer generally has a base sequence length of preferably about 5 to 50 nucleotides, more preferably about 10 to 35 nucleotides, and even more preferably about 15 to 30 nucleotides.

  Primer set designed based on the base sequence shown in SEQ ID NO: 1 which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 291st base of the base sequence shown in SEQ ID NO: 1 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 53 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 54 can be preferably exemplified.

    Primer set designed based on the base sequence shown in SEQ ID NO: 2 that gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 505th base of the base sequence shown in SEQ ID NO: 2 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 55 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 56 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 3 which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 64th base of the base sequence shown in SEQ ID NO: 3 Can be mentioned. Specifically, a combination of the polynucleotide represented by the base sequence shown in SEQ ID NO: 57 and the polynucleotide represented by the base sequence shown in SEQ ID NO: 58 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 4 to give a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 93rd base of the base sequence shown in SEQ ID NO: 4 Can be mentioned. Specifically, a combination of the polynucleotide represented by the base sequence shown in SEQ ID NO: 59 and the polynucleotide represented by the base sequence shown in SEQ ID NO: 60 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 5 to give a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 27th base of the base sequence shown in SEQ ID NO: 5 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 61 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 62 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 6 as a primer set for amplifying a DNA fragment containing the 147th base of the base sequence shown in SEQ ID NO: 6 to give a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 63 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 64 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 7 that gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 113th base of the base sequence shown in SEQ ID NO: 7 Can be mentioned. Specifically, a combination of the polynucleotide represented by the base sequence represented by SEQ ID NO: 65 and the polynucleotide represented by the base sequence represented by SEQ ID NO: 66 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 8 to give a gene amplification product capable of detecting the base, as a primer set for amplifying a DNA fragment containing the 594th base of the base sequence shown in SEQ ID NO: 8 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 67 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 68 can be preferably exemplified.

  A primer set for amplifying a DNA fragment containing the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9 is designed based on the base sequence shown in SEQ ID NO: 9 to give a gene amplification product capable of detecting the base And primer sets. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 69 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 70 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 10 that gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 446th base of the base sequence shown in SEQ ID NO: 10 Can be mentioned. Specifically, a combination of the polynucleotide represented by the base sequence shown in SEQ ID NO: 71 and the polynucleotide represented by the base sequence shown in SEQ ID NO: 72 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 185th base of the base sequence shown in SEQ ID NO: 11, a primer set designed based on the base sequence shown in SEQ ID NO: 11 that gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 73 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 74 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 12 to give a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 194th base of the base sequence shown in SEQ ID NO: 12 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 75 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 76 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 143rd base of the base sequence shown in SEQ ID NO: 13, a primer set designed based on the base sequence shown in SEQ ID NO: 13 that gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 77 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 78 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 14 to give a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 206th base of the base sequence shown in SEQ ID NO: 14 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 79 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 80 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 217th base of the base sequence shown in SEQ ID NO: 15, a primer set designed based on the base sequence shown in SEQ ID NO: 15 that gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 81 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 82 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 16 as a primer set for amplifying a DNA fragment containing the 130th base of the base sequence shown in SEQ ID NO: 16 to give a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 83 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 84 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 17 to give a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 221st base of the base sequence shown in SEQ ID NO: 17 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 85 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 86 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 18 to give a gene amplification product capable of detecting the base, as a primer set for amplifying a DNA fragment containing the 382nd base of the base sequence shown in SEQ ID NO: 18 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 87 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 88 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 148th base of the base sequence shown in SEQ ID NO: 19, a primer set designed based on the base sequence shown in SEQ ID NO: 19 that gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 89 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 90 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 20 as a primer set for amplifying a DNA fragment containing the 179th base of the base sequence shown in SEQ ID NO: 20 to give a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 91 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 92 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 21 that gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 129th base of the base sequence shown in SEQ ID NO: 21 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 93 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 94 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 151st base of the base sequence shown in SEQ ID NO: 22, a primer set designed based on the base sequence shown in SEQ ID NO: 22 which gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 95 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 96 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 23, which gives a gene amplification product capable of detecting the base, as a primer set for amplifying a DNA fragment containing the 252nd base of the base sequence shown in SEQ ID NO: 23 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 97 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 98 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 24, which gives a gene amplification product capable of detecting the base, as a primer set for amplifying a DNA fragment containing the 271st base of the base sequence shown in SEQ ID NO: 24 Can be mentioned. Specifically, a combination of the polynucleotide represented by the base sequence represented by SEQ ID NO: 99 and the polynucleotide represented by the base sequence represented by SEQ ID NO: 100 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 25 as a primer set for amplifying a DNA fragment containing the 141st base of the base sequence shown in SEQ ID NO: 25, giving a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 101 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 102 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 137th base of the base sequence shown in SEQ ID NO: 26, a primer set designed based on the base sequence shown in SEQ ID NO: 26 that gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 103 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 104 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 27, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 344th base of the base sequence shown in SEQ ID NO: 27 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 105 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 106 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 28, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 246th base of the base sequence shown in SEQ ID NO: 28 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 107 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 108 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 29, which gives a gene amplification product capable of detecting the base, as a primer set for amplifying a DNA fragment containing the 108th base of the base sequence shown in SEQ ID NO: 29 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 109 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 110 can be preferably exemplified.

  A primer set for amplifying a DNA fragment containing the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30 is designed based on the base sequence shown in SEQ ID NO: 30 to give a gene amplification product capable of detecting the base. And primer sets. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 111 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 112 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 31 as a primer set for amplifying a DNA fragment containing the 62nd base of the base sequence shown in SEQ ID NO: 31 to give a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 113 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 114 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 32, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 156th base of the base sequence shown in SEQ ID NO: 32 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 115 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 116 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 669th base of the base sequence shown in SEQ ID NO: 33, a primer set designed based on the base sequence shown in SEQ ID NO: 33, which gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 117 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 118 can be preferably exemplified.

  As a primer set for amplifying a DNA fragment containing the 183rd base of the base sequence shown in SEQ ID NO: 34, a primer set designed based on the base sequence shown in SEQ ID NO: 34 that gives a gene amplification product capable of detecting the base Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 119 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 120 can be preferably exemplified.

  A primer set for amplifying a DNA fragment containing the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35 is designed based on the base sequence shown in SEQ ID NO: 35, which gives a gene amplification product capable of detecting the base. And primer sets. Specifically, a combination of the polynucleotide represented by the base sequence represented by SEQ ID NO: 121 and the polynucleotide represented by the base sequence represented by SEQ ID NO: 122 can be preferably exemplified.

  A primer set for amplifying a DNA fragment containing the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36 is designed based on the base sequence shown in SEQ ID NO: 36, which gives a gene amplification product capable of detecting the base. And primer sets. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 123 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 124 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 37, which gives a gene amplification product capable of detecting the base, as a primer set for amplifying a DNA fragment containing the 361st base of the base sequence shown in SEQ ID NO: 37 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 125 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 126 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 38, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 139th base of the base sequence shown in SEQ ID NO: 38 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 127 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 128 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 39, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 320th base of the base sequence shown in SEQ ID NO: 39 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 129 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 130 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 40, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 265th base of the base sequence shown in SEQ ID NO: 40 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 131 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 132 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 41, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 337th base of the base sequence shown in SEQ ID NO: 41 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 133 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 134 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 42, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 312th base of the base sequence shown in SEQ ID NO: 42 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 135 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 136 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 43, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 160th base of the base sequence shown in SEQ ID NO: 43 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 137 and a polynucleotide represented by the base sequence shown in SEQ ID NO: 138 can be preferably exemplified.

  Primer set designed based on the base sequence shown in SEQ ID NO: 44, which gives a gene amplification product capable of detecting the base as a primer set for amplifying a DNA fragment containing the 314th base of the base sequence shown in SEQ ID NO: 44 Can be mentioned. Specifically, a combination of a polynucleotide represented by the base sequence represented by SEQ ID NO: 139 and a polynucleotide represented by the base sequence represented by SEQ ID NO: 140 can be preferably exemplified.

  Any of the above primer sets can be used as a reagent. A reagent kit comprising one or more containers filled with one or more of these primer sets is also included in the scope of the present invention. This reagent kit can further contain substances required for carrying out the measurement, such as a labeling substance for detecting a gene polymorphism, a buffer solution, and a salt. Furthermore, substances such as stabilizers and / or preservatives may be included. In formulating, a known formulation means may be introduced according to each substance to be used, for example, a polynucleotide. Such reagents and reagent kits are preferably used in the bovine individual identification method and parent-child discrimination method according to the present invention.

  We searched for useful DNA markers for individual identification and parent-child differentiation in Japanese black cattle using the AFLP method. In addition, the effectiveness of the developed marker was evaluated by calculating the discrimination ability based on the estimated gene frequency.

[Materials and methods]
The Test animals Japanese Black 111 animals were subjected to the experiment. Liver samples for 20 cattle selected so as not to straddle the same prefecture and the same breed of cattle from the 6th National Wagyu Co-promotion Society held in 1992, and the 8th nationwide held in 2002 Liver samples of 91 heads selected so that there was as little bias as possible in the prefectures of the cattle presented by the Wagyu Ability Promotion Society were used.

Purification of genomic DNA from liver tissue Genomic DNA from tissue was extracted as follows. About 1.0 g of tissue was homogenized with a homogenizer, filtered with gauze while adding 20 ml of TNE solution (10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 1 mM EDTA), and centrifuged at 3,000 rpm for 10 minutes. Thereafter, the supernatant was removed, 15 ml of TNE solution was added and mixed well, and centrifuged again at 3,000 rpm for 10 minutes to remove the supernatant. Mix well with 1 ml of physiological saline / EDTA (0.16M NaCl, 1 mM EDTA), and add 15 ml of sarkosyl solution (0.5% N-lauroylsarcosine sodium salt, 10 mM Tris-HCl pH8.0, 10 mM EDTA) to avoid cleaving the DNA. Shake slowly to elute the DNA. Then, 200 μl of Proteinase K solution (10 mg / ml in water) was added and incubated at 37 ° C. overnight. Add an equivalent amount of TE-phenol (dissolve phenol at 60 ° C, add 8-hydroxyquinoline to 0.1%, and water-saturate with TE buffer (10 mM Tris-HCl; pH 8.0, 1 mM EDTA)) for 10 minutes. Mix slowly. Centrifugation was performed at 3,000 rpm for 10 minutes, and the aqueous layer was separated so as not to remove proteins formed between the aqueous layer and the phenol layer. Phenol / chloroform / isoamyl alcohol (25: 24: 1, containing 0.05% 8-hydroxyquinoiine) was added to the aqueous layer, and the mixture was centrifuged for 10 minutes to separate the aqueous layer. To this aqueous layer, an equivalent amount of chloroform / isoamyl alcohol (24: 1) was added and mixed for 10 minutes, followed by centrifugation at 3,000 rpm for 5 minutes. Next, the aqueous layer was separated, an equivalent amount of diethyl ether was added, and the mixture was shaken slightly until the white aqueous layer became transparent, then centrifuged at 3,000 rpm for several seconds, and ether was removed by ventilation. Then, 1/10 volume of 3M sodium acetate solution (adjusted to pH 5.2 with acetic acid) was added, and 2 volumes of 100% ethanol cooled at −20 ° C. were mixed to precipitate DNA. The DNA was washed with 70% ethanol, dried at room temperature, dissolved in 2 ml TE buffer, and stored at 4 ° C.

DNA quantification
DNA was quantified by measuring absorbance at 260 nm. OD ₂₆₀ = 1.00 corresponds to a concentration of 50 μg / ml of double-stranded DNA. At the same time, the absorbance at 280 nm was also measured. If the OD ₂₆₀ / OD ₂₈₀ ratio was 1.8 ± 0.1, it was considered that high-purity DNA free from impurities such as proteins, surfactants, and phenols could be recovered.

AFLP method was used to detect polymorphic bands of AFLP method. The operation in the AFLP method was basically performed according to the method of Vos et al. (“Nucleic Acids Research”, 1995, Vol. 23, No. 21, p. 4407-4414). The outline is shown in FIG. Details of the method are shown below.

1) Cleavage of DNA with restriction enzymes
500 ng of DNA was cut with a combination of TaqI, a frequent cutter, and EcoRI, a rare cutter. Each restriction enzyme recognizes and cleaves a unique base sequence. The base sequence of the restriction enzyme recognition site is shown in Table 1. Both EcoRI and TaqI form overhanging ends when cleaving double-stranded DNA.

  Specifically, mix 1.0 μl of ultrapure water, 2.5 μl of 10 × restriction enzyme buffer, 1.0 μl of 10 × BSA (Bovine Serum Albumin), 0.5 μl of TaqI (10 unit / μl), and 20 μl of DNA solution, and mix at 65 ° C. Incubated for 1 hour. Thereafter, 12.5 μl of ultrapure water, 1.5 μl of 10 × restriction enzyme buffer and 1.0 μl of EcoRI (10 unit / μl) were added and mixed well, and incubated at 37 ° C. for 1 hour.

2) Addition of adapters TaqI adapter and EcoRI adapter were added to the fragments treated with restriction enzymes. The adapter sequence is shown in Table 2. Each adapter was prepared by annealing single-stranded DNA to make it double-stranded. 100 pmol / μl equivalent amount of single-stranded solution was mixed well, heated in a water bath at 65 ° C., turned off, and left overnight to be double-stranded.

  In Table 2, N at the 3 ′ end of each primer represents a selective base, and any base of A, C, G, T is entered.

  Add 10 μl solution containing 10 μl ligase buffer 1.0 μl, 1 mM ATP 1.0 μl, TaqI adapter 50 pmol, EcoRI adapter 5.0 pmol, T4 DNA ligase (7.5 unit / μl) 0.2 μl to 40 μl of DNA solution to make a total volume of 50 μl, Incubated at 37 ° C for 3 hours. Thereafter, it was diluted 10-fold with 10 mM Tris-HCl / 0.1 mM EDTA (pH 8.0), and this was used as template DNA for PCR. The obtained product was stored at -20 ° C.

3) 1st PCR (Pre-amplification PCR)
5.0 μl template DNA with adapter added, 5.0 μl 10 × PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl; pH 8.6), 4.0 μl dNTP Mixture (2.5 mM each), TaKaRa Ex Taq ^™ Hot Start Version (5 unit / μl) 0.2 μl, 75 ng each of TaqI primer and EcoRI primer that specifically bind to the adapter to which one base was added were added, and the reaction was carried out with ultrapure water to a total volume of 50 μl. The reaction was performed using a thermal cycler under the following conditions. First, 20 cycles of 94 ° C. for 30 seconds, 56 ° C. for 1 minute, and 72 ° C. for 1 minute were performed, followed by extension reaction at 72 ° C. for 10 minutes. After the PCR reaction, it was diluted 10 times with ultrapure water. The PCR product was then stored at -20 ° C.

4) 2nd PCR (Selective-amplification PCR)
The product obtained by pre-amplification PCR was amplified by adding TaqI primer and EcoRI primer to which 3 bases were added. PCR products 5.0μl, 10 × PCR buffer 2.0μl, dNTP Mixture 1.6μl, TaKaRa Ex Taq TM Hot Start Version 0.08μl, 3 added base added was the TaqI primer 30ng and EcoRI primer 5ng respectively, the total amount with ultrapure water The reaction was performed at 20 μl. Primers added depend on the type of adapter added. A thermal cycler was used for the reaction. The conditions were 13 cycles of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 1 minute. Further, a cycle of 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 23 cycles. At that time, touchdown was performed to lower the temperature by 0.7 ° C. every cycle. After the reaction, 8 μl of Stop / Loading buffer (95% Formamid 95 ml, 1M EDTA 2 ml, ultrapure water 3 ml, Xylen Cyanol FF 0.1 g, Bromophenol Blue 0.1 g in 100 ml) was added and mixed. The sequences of the 1st amplification primer and the 2nd amplification primer are shown in Table 2.

5) Band detection
A silver staining method was used to detect the band amplified by the AFLP method. SILVER SEQUENCE ^™ DNA Staining Reagents (Promega, Tokyo, Japan) was used for silver staining. The method followed the protocol.

Creating gel solution, (Tris 108 g in _{1L, Boric Acid 55g, Na 3} EDTA · 2H 2 O 9.3g) urea 35g, 10 × TBE 10ml, Long Ranger registered TM Gel Solution 10 ml, this ultrapure water In addition, the mixture was stirred well and after the urea was completely dissolved, ultrapure water was added to make the total volume 70 ml. The solution was filtered through a 0.45 μm filter and degassed for 10 minutes. To this, 350 μl of 10% APS and 35 μl of TEMED were added, and the mixture was slowly poured into the gel plate. For electrophoresis, a slab type electrophoresis apparatus was used. The buffer used was 0.6 × TBE, and it was performed at 1,800 V for 2 hours.

After electrophoresis, the gel plate was peeled off and stained. For staining, 500 μl of 10% acetic acid solution (stop solution), 1 g AgNO ₃ · 37% Formaldehyde 1 L (stain solution), and 30 g NaCO ₃ 1 L (developer) were used. Further, the developer was stored on ice, and immediately before use, 1.5 ml of 37% formaldehyde and 200 μl of sodium thiosulfate were added.

  The gel plate was placed in a tray, stop solution was added, and shaken for 20 minutes. Thereafter, the plate was washed three times with ultrapure water, the gel plate was immersed in the staining solution, and shaken for 30 minutes. The gel plate after dyeing was dipped lightly in ultrapure water to such an extent that silver did not fall off too much, quickly dipped in the developer, and shaken until a band appeared. Thereafter, a stop solution was added at an appropriate point to stop the reaction, and then the plate was washed again with ultrapure water, and a DNA band was observed.

Extraction of AFLP polymorphic fragments AFLP polymorphic fragments detected by silver staining were excised from polyacrylamide gel using a scalpel well wiped with ultrapure water. Then, it was immersed in 20.0 μl of TE buffer, and AFLP polymorphic fragments were leached from the gel slices. Selective amplification using the DNA solution as a template, 40% glycerol solution (40% glycerol, 0.25% Bromophenol Blue, 0.25% Xylene Cyanol FF) was added and 1.5% agarose gel (Agarose S 60 mg, 1 × TBE buffer 40 ml) , Ethidium bromide solution (10 mg / ml in water 2.0 μl) at 100 V for 20 minutes. Thereafter, the gel was cut out under UV irradiation, transferred to a 1.5 ml tube, and the gel was collected. The DNA band was recovered from the agarose gel using an Ultra Clean ^™ 15 DNA Purification Kit (Mo Bio Laboratories, Inc. Carlsbad, CA, USA). All procedures followed the protocol. The extracted DNA was dissolved in 15 μl of ultrapure water.

Base sequence determination of AFLP polymorphic fragment The base sequence of the obtained polymorphic fragment was determined by a capillary sequencer.

  First, the concentration of DNA obtained by extraction of AFLP polymorphic fragments was measured using Nano Drop ND-1000 (NanoDrop Technologies, Wilmington DE, USA). The DNA whose concentration was measured was adjusted to 10 to 20 ng / μl, and used as a template DNA for the sequencing reaction. The reaction was performed by mixing 1.0 μl of template DNA, 2.0 μl of Ready Reaction Premix, 3.0 μl of BigDye Sequencing Buffer, 3.2 μl of primer (1.0 pmol), and 20 μl of sterilized ultrapure water so that the total amount was 20 μl. This operation was performed in the dark. The reaction was performed using a thermal cycler under the following conditions. First, after heat denaturation at 94 ° C. for 1 minute, 25 cycles of 96 ° C. for 10 seconds, annealing temperature for 5 seconds and 60 ° C. for 4 minutes were performed and stored at 4 ° C.

Next, in order to obtain more accurate data from the reacted product, the sequencing product was purified by carrying out Ethanol / EDTA precipitation method to remove unreacted fluorescent terminator. According to the protocol of BigDye ^{registered TM} Terminator v3.1 Cycle Sequencing Kit, first, 5 μl of 125 mM EDTA and 60 μl of 100% ethanol were added to the sequence product and mixed by inverting 4 times, and incubated at room temperature for 15 minutes. Next, it was centrifuged at 14,000 rpm for 25 minutes at room temperature, and the supernatant was removed (at this time, no precipitate was visible). Further, 60 μl of 70% ethanol was added and mixed by inversion several times. After centrifugation again at 14,000 rpm for 5 minutes at room temperature, the supernatant was removed. This was allowed to air dry for 15 minutes at room temperature. Care was taken to shield from light in the dry state. When the experiment was interrupted, it was stored in a light-shielded state at 4 ° C. Next, 20 μl of injection buffer (Hi-Di formamide) was added to the precipitate and vortexed for 4 minutes to dissolve the precipitate. The solution was boiled at 95 ° C. for 2 minutes and immediately cooled on ice for 5 minutes. The whole amount was transferred to a 96-well plate for a capillary sequencer and set in the capillary sequencer ABI PRISM ^{registered TM} 3100 Genetic Analyzer (Applied Biosystems, Tokyo, Japan), and the base sequence was determined.

The specific polymorphic site of the polymorphic site may be EcoRI, TaqI restriction enzyme recognition site, 3-base selection site, and insertion deletion (indel) within the AFLP polymorphic fragment. Mutations in restriction enzyme recognition sites and 3-base selection sites cannot be identified by sequencing in AFLP fragments. In order to identify these mutations, the base sequence around the outside of the AFLP fragment is required. The polymorphic site and the base sequence around the outside were identified by BLAST homology search (http://www.ncbi.nlm.nih.gov/blast/index.shtml) against the bovine genome sequence. When the polymorphic site could not be identified from the AFLP fragment, the polymorphic site was identified by determining the base sequence of the individual sample from which the AFLP fragment was not obtained.

Using the mutation identified by the comparison of the genotyping base sequences, 113 Japanese black breeds were genotyped. The method was PCR or PCR-RFLP depending on the type of mutation. The PCR conditions were basically set as follows. That is, after 2 minutes at 94 ° C., 35 cycles were repeated with 94 ° C. for 30 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 1 minute, followed by extension reaction at 72 ° C. for 7 minutes. The mutations identified at the restriction enzyme recognition sites were basically treated with restriction enzymes under the following conditions. In other words, 5.0 μl of PCR product, 1.5 μl of 10 × restriction enzyme buffer, 0.25 μl of the relevant restriction enzyme, and if necessary, BSA, SAM (S-Adenosyl-L-methionine) etc. are added to make a total volume of 15.0 μl. Ultra sterilized water was added as follows. They were incubated for 8 hours or more using a water bath at an appropriate temperature for each restriction enzyme. Moreover, it detected by agarose gel or polyacrylamide gel electrophoresis according to the size of the PCR product and the restriction enzyme fragment.

Calculation of individual identification probability and paternity denial probability using SNP markers
Based on the genotype frequency and gene frequency obtained as a result of the above genotype determination, the individual identification probability and the paternity denial probability of Japanese black hair were calculated using the SNPs marker obtained by AFLP method. The individual identification probability (PI) is basically calculated by Holt et al. (“Forensic Science International”), 2000, Vol. 112, No. 2-3, p.91. -109). In Equation (a), pp, pq, and qq indicate the genotype frequency values obtained with each SNP marker.

  In addition, the calculation of the fatherhood negation probability (PE) basically followed the formulas shown in formulas (b) and (c). In the formulas (b) and (c), pi and pj represent the allele frequency values obtained with the respective SNPs markers.

  That is, if the genotype of the mother cow is known, Jamieson et al. (“Heredity”, 1965, Volume 20, p. 419-441); “Proceedings of the 16th International Conference on Animal Blood” Group and Biochemical Polymorphism, Leningrad 1978 (Proceedings of the XVIth International Conference on Animal Blood Groups and Biochemical Polymorphism, Leningrad 1978), 1979, Vol. 4, p. 27) Negative probability. Alternatively, if the genotype of the mother cow is not known, Grabar & Morrice et al. (“Inclusion Probabilities in Parentage Testing”, 1983, p. 277-280) (c ) Is the paternity denial probability.

[result]
Detection of polymorphic fragments by AFLP method Genomic DNA was cleaved with rare cutter EcoRI and frequent cutter TaqI. The polymorphisms in Japanese black hairs were investigated in a total of 220 primer sets by combining 16 types of EcoRI primers and 16 types of TaqI primers. First, we tested 20 Japanese black cattle selected from the 6th nationwide Japanese beef performance co-promotion cattle so as not to be biased to the same prefecture and the same bull, and detected AFLP polymorphic fragments. Details of the primer sets used are shown in Table 3. The solid areas in Table 3 indicate the combinations of selective bases used.

  When the SNP marker has a biallelic gene, a marker whose gene frequency is close to 0.5 is considered to be more useful in individual identification and parent-child determination. The purpose of this study is to develop SNPs markers with a minor allele frequency of 0.3 or more in the population. The selection criteria for AFLP polymorphic fragments are 65% to 85% of AFLP polymorphic fragments (total 20 animals). 13 to 17 of them). As a result of screening 20 Japanese black hairs, 115 polymorphic fragments satisfying the criteria were obtained. AFLP polymorphic fragments meeting the criteria were detected at a rate of approximately 1 per 0.52 primer set. They were named TSA1-TSA115 (Traseability SNPs markers from AFLP) and were subsequently converted to these SNPs markers.

In order to identify mutation sites in 115 polymorphic fragments that are candidates for conversion of AFLP polymorphic fragments into SNPs markers, the base sequences of the fragments were determined. A capillary sequencer was used to determine the base sequence. After determining the base sequence, we attempted to identify the sequence around the fragment by BLAST homology search. As a result, 28 polymorphic bands were point mutations at the TaqI restriction enzyme recognition site, 4 polymorphic bands were point mutations at the EcoRI restriction enzyme recognition site, and 8 polymorphic bands were 3 bases. Point mutation at the selected site, 4 polymorphic bands were insertion / deletion (indel) of 4 bp or less. Conversion to SNP markers was made in a total of 44 polymorphic bands that allowed identification of mutations that caused polymorphisms. In addition, primers prepared for detecting SNPs, annealing temperatures, and the like are collectively shown in Table 4-1, Table 4-2, Table 4-3, Table 4-4, and Table 4-5.

  For the other 71 bands that were not converted to markers, the DNA sequence of 16 was extracted from the polymorphic fragment was very thin and difficult to clone, so the nucleotide sequence could not be determined. In addition, 27 of the re-amplified bands were difficult to analyze due to DNA congestion and deviation from the appropriate concentration. For bands whose base sequence has been determined and the cause of the polymorphism has been identified, indel (6) of 5 bp or more, repetitive sequence (17), mutation on chromosome X (3), one allele for genotyping There were no individuals with homozygous (1), but the analysis was abandoned due to reasons such as low reproducibility (1), which was thought to be a mutation at the 3 base selection site (the number of candidate bands in parentheses) Show).

  The details of the mutation site of each SNP marker are described below. In addition, the chromosome in which each marker exists and the position on the chromosome are shown in Table 5-1 and Table 5-2.

  TSA 1: A sequence of AFLP fragments detected in 17 Japanese black hairs, present on chromosome 29 in Bos taurus. There was an overlapping sequence of TaqI restriction enzyme recognition site and EcoRI restriction enzyme recognition site inside, and it was point-mutated from (TCGAATTC) to (TCAAATTC) at that site. PCR-RFLP was performed using TaqI. The nucleotide sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 1.

  TSA2: A sequence of AFLP fragments detected in 12 Japanese black hairs, present on the X chromosome in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 2.

  TSA 3: A sequence of AFLP fragments detected in 12 Japanese black hairs, present on the X chromosome in Bos taurus. It was a point mutation from (TCGA) to (TGGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 3.

  TSA4: AFLP fragment sequence detected in 15 Japanese black hairs, present on chromosome 21 in Bos taurus. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 4.

  TSA 6: AFLP fragment sequence detected from 15 Japanese black hairs, present on chromosome 15 in Bos taurus. It was a point mutation from (TCGA) to (CCGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 5.

  TSA10: AFLP fragment sequence detected from 16 Japanese black hairs, and the chromosomes present are unknown. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 6.

  TSA12: AFLP fragment sequence detected from 11 Japanese black hairs, present on chromosome 29 in Bos taurus. It was a point mutation from (CCT) to (CCA) at the 3-base selection site. Since there was a mutation thought to be linked to the mutation part of the 3 base selection site within the sequence, and the restriction enzyme BsrI recognition site (CCAGT, AFLP fragment sequence T was C point mutation), PCR- Genotyping was performed by RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 7.

  TSA18: A sequence of AFLP fragments detected in 17 Japanese black breeds and is located on chromosome 3 in Bos taurus. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 8.

  TSA22: A sequence of AFLP fragments detected from 14 Japanese black hairs, in which 4 bp indel was detected. Present on chromosome 15 in Bos taurus. Genotyping was performed by PCR. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 9.

  TSA34: A sequence of AFLP fragments detected from 16 Japanese black hairs, present on chromosome 17 in Bos taurus. It was a point mutation from (TCGA) to (TCCA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 10.

  TSA35: AFLP fragment sequence detected from 14 Japanese black hairs, present on chromosome 29 in Bos taurus. It was a point mutation from (ACC) to (ATC) at the 3 base selection site. Since the mutation site contained a recognition site for restriction enzyme HphI (TCACC), genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 11.

  TSA36: A sequence of AFLP fragments detected from 15 Japanese black hairs, present on chromosome 28 in Bos taurus. It was a point mutation from (TCGA) to (TCCA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 12.

  TSA37: A sequence of AFLP fragments detected from 16 Japanese black hairs, present on chromosome 8 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 13.

  TSA38: A sequence of AFLP fragments detected from 16 Japanese black hairs, and the chromosomes present are unknown. It was a point mutation from (TCGA) to (TCTA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 14.

  TSA39: AFLP fragment sequence detected from 16 Japanese black hairs, present on chromosome 5 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 15.

  TSA40: A sequence of AFLP fragments detected in 15 Japanese black hairs, present on chromosome 7 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 16.

  TSA43: AFLP fragment sequence detected from 16 Japanese black hairs, present on chromosome 21 in Bos taurus. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 17.

  TSA44: A sequence of AFLP fragments detected in 14 Japanese black hairs, present on chromosome 21 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 18.

  TSA45: A sequence of AFLP fragments detected from 16 Japanese black hairs, and the chromosomes present are unknown. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 19.

  TSA48: A sequence of AFLP fragments detected from 15 Japanese black hairs, present on chromosome 10 in Bos taurus. It was a point mutation from (GAATTC) to (GAATCC) at the restriction enzyme recognition site of EcoRI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 20.

  TSA49: AFLP fragment sequence detected from 14 Japanese black hairs, present on chromosome 19 in Bos taurus. It was a point mutation from (TCGA) to (TCGG) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 21.

  TSA55: An AFLP fragment sequence detected from 14 Japanese black hairs, present on chromosome 22 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 22.

  TSA57: A sequence of AFLP fragments detected in 13 Japanese black hairs, present on chromosome 9 in Bos taurus. It was a point mutation from (AGG) to (AGA) at the 3-base selection site. Since the recognition site (TCNGA) of the restriction enzyme Hpy188I was included in the mutated portion, genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 23.

  TSA59: AFLP fragment sequence detected from 16 Japanese black hairs, present on chromosome 16 in Bos taurus. It was a point mutation from (ACG) to (ATG) at the 3-base selection site. Since the mutation site contained a recognition site (CATG) for the restriction enzyme Nla3, genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 24.

  TSA60: AFLP fragment sequence detected from 16 Japanese black hairs, present on chromosome 24 in Bos taurus. It was a point mutation from (ACG) to (ACA) at the 3-base selection site. There was a mutation thought to be linked to this mutation in the vicinity, and the mutation site contained a recognition site (CATG) for the restriction enzyme RsaI, so genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 25.

  TSA63: AFLP fragment sequence detected in 14 Japanese black hairs, present on chromosome 23 in Bos taurus. It was a point mutation from (GAATTC) to (AAATTC) at the restriction enzyme recognition site of EcoRI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 26.

TSA67: A sequence of AFLP fragments detected in 17 Japanese black hairs, present on chromosome 16 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 27.

  TSA68: AFLP fragment sequence detected from 14 Japanese black hairs, present on chromosome 9 in Bos taurus. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 28.

  TSA69: A sequence of AFLP fragments detected in 16 Japanese black hairs, present on chromosome 9 in Bos taurus. It was a point mutation from (GAATTC) to (GAATTT) at the restriction enzyme recognition site of EcoRI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 29.

  TSA70: AFLP fragment sequence detected from 16 Japanese black hairs, with 4 bp indel detected inside. Present on chromosome 2 in Bos taurus. Genotyping was performed by PCR. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 30.

  TSA71: AFLP fragment sequence detected from 17 Japanese black hairs, present on chromosome 25 in Bos taurus. It was a point mutation from (ATG) to (ACG) at the 3-base selection site. Since the mutation site contained a recognition site (CATG) for the restriction enzyme Nla3, genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 31.

  TSA72: AFLP fragment sequence detected in 13 Japanese black hairs, present on chromosome 24 in Bos taurus. It was a point mutation from (TCGA) to (TCTA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 32.

  TSA77: A sequence of AFLP fragments detected in 14 Japanese black hairs, and the chromosomes present are unknown. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 33.

TSA78: AFLP fragment sequence detected from 13 Japanese black hairs, present on chromosome 10 in Bos taurus. It was a point mutation from (AAG) to (AAC) at the 3-base selection site. Since the mutation site contained a recognition site for restriction enzyme BspCNI (N ₈ CTGAG (SEQ ID NO: 141)), genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 34.

  TSA81: AFLP fragment sequence detected from 15 Japanese black hairs, with 2 bp indel detected inside. Present on chromosome 16 in Bos taurus. Genotyping was performed by PCR. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 35.

  TSA82: AFLP fragment sequence detected in 13 Japanese black hairs, with 2 bp indel detected inside. Present on chromosome 3 in Bos taurus. Genotyping was performed by PCR. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 36.

  TSA84: AFLP fragment sequence detected from 16 Japanese black hairs, present on chromosome 17 in Bos taurus. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 37.

  TSA87: A sequence of AFLP fragments detected in 15 Japanese black hairs, present on chromosome 16 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 38.

  TSA90: AFLP fragment sequence detected from 14 Japanese black hairs, present on chromosome 27 in Bos taurus. It was a point mutation from (TCGA) to (TTGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 39.

  TSA91: A sequence of AFLP fragments detected in 14 Japanese black hairs, present on chromosome 4 in Bos taurus. It was a point mutation from (TCGA) to (TCAA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 40.

  TSA92: A sequence of AFLP fragments detected from 16 Japanese black hairs, and the chromosomes present are unknown. It was a point mutation from (ACA) to (ACC) at the 3-base selection site. Since the mutation site contained a recognition site (CCGG) for the restriction enzyme MspI, genotyping was performed by PCR-RFLP. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 41.

  TSA102: AFLP fragment sequence detected from 14 Japanese black hairs, present on chromosome 2 in Bos taurus. It was a point mutation from (TCGA) to (TCGC) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 42.

  TSA110: AFLP fragment sequence detected from 16 Japanese black hairs, present on chromosome 2 in Bos taurus. It was a point mutation from (GAATTC) to (AAATTC) at the restriction enzyme recognition site of EcoRI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 43.

  TSA113: AFLP fragment sequence detected from 17 Japanese black hairs and present on chromosome 27 in Bos taurus. It was a point mutation from (TCGA) to (CCGA) at the restriction enzyme recognition site of TaqI. The base sequence of the AFLP fragment containing this mutation site is shown in SEQ ID NO: 44.

Genotyping using SNPs marker The genotype frequency and gene frequency in Japanese black cattle were determined using each SNPs marker. The primers shown in Tables 4-1 to 4-5 were used for genotyping, and PCR-RFLP or PCR was performed. 111 Japanese black cattle were used for genotyping by SNP markers. The results are shown in Table 6-1 and Table 6-2. In the display method, allele P indicates a sequence in which a polymorphic fragment is detected when AFLP is performed, and allele Q indicates a sequence in which no polymorphic fragment is detected. P represents the gene frequency of allele P, and q represents the gene frequency of allele Q. When comparing the results of the AFLP method in individuals and the results of RFLP genotyping, there was a discrepancy of about 1 for each marker, but the presence or absence of AFLP fragments and the genotype of SNPs were almost the same. It was consistent.

Calculation of individual identification probability and paternity denial probability using SNP markers
Based on the genotype frequency and gene frequency obtained as a result of the above genotype determination, the individual identification probability and the paternity denial probability of Japanese black hair were calculated using the SNPs marker obtained by AFLP method. Specifically, for 43 markers excluding TSA3 from among the above 44 markers, the individual identification probability and paternity denial probability were calculated from the genotype frequency and gene frequency. The reason for removing TSA3 is that the marker exists on the X chromosome, and all the cattle listed in the National Wagyu Cooperative Society used in this study are male. There is usually only one male X chromosome, all from the mother, and the genes on the male parent's X chromosome are not inherited by male children. Therefore, TSA3 was excluded in the calculation because the same probability calculation method cannot be applied to TSA3. TSA2 is also present on the X chromosome as TSA3, but since heterogeneity is observed in genotyping, it is considered that the polymorphic site has a homologous region on the Y chromosome. Therefore, it was used in the probability calculation as being effective for parent-child determination.

  The results are shown in Table 7. The individual identification probability (PI) is defined as the probability that two unrelated individuals randomly selected have exactly the same genotype. The paternity denial probability (PE) is defined as the probability that a bull who is not a paternal bull will be excluded from bulls considered as paternal candidates.

In Table 7, the paternity denial probability when the genotype of only one parent is used is shown as P _E1 , and the paternity denial probability when the parental genotype is available is shown as P _E2 .

As shown in Table 7, the calculated individual identification probability was 1.31 × 10 ⁻¹⁶ , and it was found that all the numbers of Japanese black breeds (about 1.64 million) could be identified.

Of the above 44 markers, 17 markers are TSA102, TSA4, TSA55, TSA68, TSA113, TSA49, TSA78, TSA70, TSA63, TSA57, TSA72, TSA69, TSA35, TSA84, TSA10, TSA39 and TSA91. Using this, the individual identification probability was similarly calculated. As a result, the individual identification probability is 7.29 × 10 ⁻⁸ , which matches 1 in 13.7 million. Since the number of Japanese Black Black breeds is less than 2 million, individual identification methods using these 17 markers can be considered to have sufficient individual identification ability.

  On the other hand, as shown in Table 7, the paternity denial probability has two probabilities: the probability under which the mother cow's genotype is not available (PE1) and the probability under which it is available (PE2). Probability was calculated. The probability of the former (PE1) was 0.999619 and the probability of the latter (PE2) was 0.999653. It can be considered that the parent-child differentiation method using 43 types of markers other than TSA3 among the above 44 types of markers has such high appraisal ability.

It is a figure which shows the outline | summary of operation in AFLP method.

SEQ ID NO: 45: polynucleotide designed for use as an EcoRI adapter.
SEQ ID NO: 46: polynucleotide designed for use as an EcoRI adapter.
SEQ ID NO: 47: polynucleotide designed for use as a TaqI adapter.
SEQ ID NO: 48: polynucleotide designed for use as TaqI adapter.
SEQ ID NO: 49: polynucleotide designed for use as an EcoRI primer. (17). . (17): “n” may be “a” or “c” or “g” or “t”.
SEQ ID NO: 50: polynucleotide designed for use as TaqI primer. (17). . (17): “n” may be “a” or “c” or “g” or “t”.
SEQ ID NO: 51: polynucleotide designed for use as an EcoRI primer. (17). . (19): “n” may be “a” or “c” or “g” or “t”.
SEQ ID NO: 52: polynucleotide designed for use as TaqI primer. (17). . (19): “n” may be “a” or “c” or “g” or “t”.
SEQ ID NOs: 53-140: polynucleotides designed for use as primers.
SEQ ID NO: 141: (1). . (8): “n” may be “a” or “c” or “g” or “t”.

Claims (9)

Analyzing the DNA of a test cow using at least 10 polymorphisms of the following 44 gene polymorphisms as an index, and determining the polymorphism as an index, comprising the step of identifying a bovine individual or parent and child Identification method:
1. A single nucleotide polymorphism at the 291st base of the base sequence shown in SEQ ID NO: 1,
2. A single nucleotide polymorphism at the 505th base of the base sequence shown in SEQ ID NO: 2,
3. A single nucleotide polymorphism at the 64th base of the base sequence shown in SEQ ID NO: 3,
4). A single nucleotide polymorphism at the 93rd base of the base sequence shown in SEQ ID NO: 4,
5. A single nucleotide polymorphism at the 27th base of the base sequence shown in SEQ ID NO: 5,
6). A single nucleotide polymorphism at the 147th base of the base sequence shown in SEQ ID NO: 6,
7). A single nucleotide polymorphism at the 113th base of the base sequence shown in SEQ ID NO: 7,
8). A single nucleotide polymorphism at the 594th base of the base sequence shown in SEQ ID NO: 8,
9. An insertion deletion (indel) polymorphism at the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9;
10. A single nucleotide polymorphism at the 446th base of the base sequence shown in SEQ ID NO: 10,
11. A single nucleotide polymorphism at the 185th base of the base sequence shown in SEQ ID NO: 11,
12 A single nucleotide polymorphism at the 194th base of the base sequence shown in SEQ ID NO: 12,
13. A single nucleotide polymorphism at the 143rd base of the base sequence shown in SEQ ID NO: 13,
14 A single nucleotide polymorphism at the 206th base of the base sequence shown in SEQ ID NO: 14,
15. A single nucleotide polymorphism at the 217th base of the base sequence shown in SEQ ID NO: 15,
16. A single nucleotide polymorphism at the 130th base of the base sequence shown in SEQ ID NO: 16,
17. A single nucleotide polymorphism at the 221nd base of the base sequence shown in SEQ ID NO: 17,
18. A single nucleotide polymorphism at the 382nd base of the base sequence shown in SEQ ID NO: 18,
19. A single nucleotide polymorphism at the 148th base of the base sequence shown in SEQ ID NO: 19,
20. A single nucleotide polymorphism at the 179th base of the base sequence shown in SEQ ID NO: 20,
21. A single nucleotide polymorphism at the 129th base of the base sequence shown in SEQ ID NO: 21,
22. A single nucleotide polymorphism at position 151 of the base sequence shown in SEQ ID NO: 22,
23. A single nucleotide polymorphism at the 252nd base of the base sequence shown in SEQ ID NO: 23,
24. A single nucleotide polymorphism at the 271st base of the base sequence shown in SEQ ID NO: 24,
25. A single nucleotide polymorphism at the 141st base of the base sequence shown in SEQ ID NO: 25,
26. A single nucleotide polymorphism at the 137th base of the base sequence shown in SEQ ID NO: 26,
27. A single nucleotide polymorphism at the 344th base of the base sequence shown in SEQ ID NO: 27,
28. A single nucleotide polymorphism at the 246th base of the base sequence shown in SEQ ID NO: 28,
29. A single nucleotide polymorphism at the 108th base of the base sequence shown in SEQ ID NO: 29,
30. An insertion deletion (indel) polymorphism at the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30;
31. A single nucleotide polymorphism at the 62nd base of the base sequence shown in SEQ ID NO: 31,
32. A single nucleotide polymorphism at the 156th base of the base sequence shown in SEQ ID NO: 32,
33. A single nucleotide polymorphism at the 669th base of the base sequence shown in SEQ ID NO: 33,
34. A single nucleotide polymorphism at the 183rd base of the base sequence shown in SEQ ID NO: 34,
35. An insertion deletion (indel) polymorphism at the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35,
36. An insertion deletion (indel) polymorphism in the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36;
37. A single nucleotide polymorphism at the 361st base of the base sequence shown in SEQ ID NO: 37,
38. A single nucleotide polymorphism at the 139th base of the base sequence shown in SEQ ID NO: 38,
39. A single nucleotide polymorphism at the 320th base of the base sequence shown in SEQ ID NO: 39,
40. A single nucleotide polymorphism at the 265th base of the base sequence shown in SEQ ID NO: 40,
41. A single nucleotide polymorphism at the 337th base of the base sequence shown in SEQ ID NO: 41,
42. A single nucleotide polymorphism at the 312th base of the base sequence shown in SEQ ID NO: 42,
43. A single nucleotide polymorphism at the 160th base of the base sequence shown in SEQ ID NO: 43,
44. A single nucleotide polymorphism at the 314th base of the base sequence shown in SEQ ID NO: 44. The bovine individual identification method or parent-child discrimination method according to claim 1, wherein the 44 gene polymorphisms are the following gene polymorphisms:
1. A single nucleotide polymorphism represented by whether the 291st base of the base sequence shown in SEQ ID NO: 1 is "G" or "A";
2. A single nucleotide polymorphism represented by whether the 505th base of the base sequence shown in SEQ ID NO: 2 is “G” or “A”;
3. A single nucleotide polymorphism represented by whether the 64th base of the base sequence shown in SEQ ID NO: 3 is “C” or “G”;
4). A single nucleotide polymorphism represented by whether the 93rd base of the base sequence shown in SEQ ID NO: 4 is “C” or “T”;
5. A single nucleotide polymorphism represented by whether the 27th base of the base sequence shown in SEQ ID NO: 5 is "T" or "C";
6). A single nucleotide polymorphism represented by whether the 147th base of the base sequence shown in SEQ ID NO: 6 is “G” or “A”;
7). A single nucleotide polymorphism represented by whether the 113th base of the base sequence shown in SEQ ID NO: 7 is "A" or "T";
8). A single nucleotide polymorphism represented by whether the 594th base of the base sequence shown in SEQ ID NO: 8 is “C” or “T”;
9. An insertion deletion (indel) polymorphism represented by whether the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9 are “GGAC” or “deletion”;
10. A single nucleotide polymorphism represented by whether the 446th base of the base sequence shown in SEQ ID NO: 10 is “G” or “C”;
11. A single nucleotide polymorphism represented by whether the 185th base of the base sequence shown in SEQ ID NO: 11 is “C” or “T”;
12 A single nucleotide polymorphism represented by whether the 194th base of the base sequence shown in SEQ ID NO: 12 is “G” or “C”;
13. A single nucleotide polymorphism represented by whether the 143rd base of the base sequence shown in SEQ ID NO: 13 is "G" or "A";
14 A single nucleotide polymorphism represented by whether the 206th base of the base sequence shown in SEQ ID NO: 14 is “G” or “T”;
15. A single nucleotide polymorphism represented by whether the 217th base of the base sequence shown in SEQ ID NO: 15 is "G" or "A";
16. A single nucleotide polymorphism represented by whether the 130th base of the base sequence shown in SEQ ID NO: 16 is "G" or "A";
17. A single nucleotide polymorphism represented by whether the 221nd base of the base sequence shown in SEQ ID NO: 17 is “C” or “T”;
18. A single nucleotide polymorphism represented by whether the 382nd base of the base sequence shown in SEQ ID NO: 18 is "G" or "A";
19. A single nucleotide polymorphism represented by whether the 148th base of the base sequence shown in SEQ ID NO: 19 is “C” or “T”;
20. A single nucleotide polymorphism represented by whether the 179th base of the base sequence shown in SEQ ID NO: 20 is "T" or "C";
21. A single nucleotide polymorphism represented by whether the 129th base of the base sequence shown in SEQ ID NO: 21 is "A" or "G";
22. A single nucleotide polymorphism represented by whether the 151st base of the base sequence shown in SEQ ID NO: 22 is "G" or "A";
23. A single nucleotide polymorphism represented by whether the 252nd base of the base sequence shown in SEQ ID NO: 23 is “G” or “A”;
24. A single nucleotide polymorphism represented by whether the 271st base of the base sequence shown in SEQ ID NO: 24 is "C" or "T";
25. A single nucleotide polymorphism represented by whether the 141st base of the base sequence shown in SEQ ID NO: 25 is "G" or "A";
26. A single nucleotide polymorphism represented by whether the 137th base of the base sequence shown in SEQ ID NO: 26 is “G” or “A”;
27. A single nucleotide polymorphism represented by whether the 344th base of the base sequence shown in SEQ ID NO: 27 is “G” or “A”;
28. A single nucleotide polymorphism represented by whether the 246th base of the base sequence shown in SEQ ID NO: 28 is “C” or “T”;
29. A single nucleotide polymorphism represented by whether the 108th base of the nucleotide sequence shown in SEQ ID NO: 29 is “C” or “T”;
30. An insertion deletion (indel) polymorphism represented by whether the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30 are “TAAT” or “deletion”;
31. A single nucleotide polymorphism represented by whether the 62nd base of the base sequence shown in SEQ ID NO: 31 is "T" or "C";
32. A single nucleotide polymorphism represented by whether the 156th base of the base sequence shown in SEQ ID NO: 32 is “G” or “T”;
33. A single nucleotide polymorphism represented by whether the 669th base of the base sequence shown in SEQ ID NO: 33 is “G” or “A”;
34. A single nucleotide polymorphism represented by whether the 183rd base of the base sequence shown in SEQ ID NO: 34 is "G" or "C";
35. An insertion deletion (indel) polymorphism represented by whether the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35 are “GA” or “deletion”;
36. An insertion deletion (indel) polymorphism represented by whether the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36 are “AG” or “deletion”;
37. A single nucleotide polymorphism represented by whether the 361st base of the base sequence shown in SEQ ID NO: 37 is “C” or “T”;
38. A single nucleotide polymorphism represented by whether the 139th base of the base sequence shown in SEQ ID NO: 38 is "G" or "A";
39. A single nucleotide polymorphism represented by whether the 320th base of the base sequence shown in SEQ ID NO: 39 is “C” or “T”;
40. A single nucleotide polymorphism represented by whether the 265th base of the base sequence shown in SEQ ID NO: 40 is "G" or "A";
41. A single nucleotide polymorphism represented by whether the 337th base of the base sequence shown in SEQ ID NO: 41 is "A" or "C";
42. A single nucleotide polymorphism represented by whether the 312th base of the base sequence shown in SEQ ID NO: 42 is "A" or "C";
43. A single nucleotide polymorphism represented by whether the 160th base of the base sequence shown in SEQ ID NO: 43 is “G” or “A”;
44. A single nucleotide polymorphism represented by whether the 314th base of the nucleotide sequence shown in SEQ ID NO: 44 is "T" or "C". Analyzing the DNA of the test cow using the gene polymorphism as an index, and determining the polymorphism as the index, the step of amplifying a DNA fragment containing each polymorphism by a gene amplification method and determining the polymorphism as the index The individual identification method or the parent-child differentiation method of the cow according to claim 1 or 2. The said process of amplifying the DNA fragment containing each polymorphism by a gene amplification method is a process of amplifying the DNA fragment containing each polymorphism using the primer set selected from the following primer set. Cattle individual identification method or parent-child differentiation method:
1. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 1, which gives a gene amplification product capable of detecting the 291st base of the base sequence shown in SEQ ID NO: 1.
2. A primer set designed based on the base sequence shown in SEQ ID NO: 2 to give a gene amplification product capable of detecting the 505th base of the base sequence shown in SEQ ID NO: 2;
3. A primer set designed based on the base sequence shown in SEQ ID NO: 3 to give a gene amplification product capable of detecting the 64th base of the base sequence shown in SEQ ID NO: 3;
4). A primer set designed based on the base sequence shown in SEQ ID NO: 4 to give a gene amplification product capable of detecting the 93rd base of the base sequence shown in SEQ ID NO: 4;
5. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 5, which gives a gene amplification product capable of detecting the 27th base of the base sequence shown in SEQ ID NO: 5;
6). A primer set designed based on the base sequence shown in SEQ ID NO: 6 to give a gene amplification product capable of detecting the 147th base of the base sequence shown in SEQ ID NO: 6;
7). A primer set designed based on the base sequence shown in SEQ ID NO: 7 to give a gene amplification product capable of detecting the 113th base of the base sequence shown in SEQ ID NO: 7;
8). A primer set designed based on the base sequence shown in SEQ ID NO: 8, which gives a gene amplification product capable of detecting the 594th base of the base sequence shown in SEQ ID NO: 8;
9. A primer set designed based on the base sequence shown in SEQ ID NO: 9, which gives a gene amplification product capable of detecting the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9;
10. A primer set designed based on the base sequence shown in SEQ ID NO: 10, which gives a gene amplification product capable of detecting the 446th base of the base sequence shown in SEQ ID NO: 10;
11. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 11, which gives a gene amplification product capable of detecting the 185th base of the base sequence shown in SEQ ID NO: 11;
12 A primer set designed based on the base sequence shown in SEQ ID NO: 12, which gives a gene amplification product capable of detecting the 194th base of the base sequence shown in SEQ ID NO: 12;
13. A primer set designed based on the base sequence shown in SEQ ID NO: 13, which gives a gene amplification product capable of detecting the 143rd base of the base sequence shown in SEQ ID NO: 13;
14 A primer set designed based on the base sequence shown in SEQ ID NO: 14, which gives a gene amplification product capable of detecting the 206th base of the base sequence shown in SEQ ID NO: 14;
15. A primer set designed based on the base sequence shown in SEQ ID NO: 15 to give a gene amplification product capable of detecting the 217th base of the base sequence shown in SEQ ID NO: 15;
16. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 16, which gives a gene amplification product capable of detecting the 130th base of the base sequence shown in SEQ ID NO: 16;
17. A primer set designed based on the base sequence shown in SEQ ID NO: 17, which gives a gene amplification product capable of detecting the 221st base of the base sequence shown in SEQ ID NO: 17;
18. A primer set designed based on the base sequence shown in SEQ ID NO: 18, which gives a gene amplification product capable of detecting the 382nd base of the base sequence shown in SEQ ID NO: 18;
19. A primer set designed based on the base sequence shown in SEQ ID NO: 19, which gives a gene amplification product capable of detecting the 148th base of the base sequence shown in SEQ ID NO: 19;
20. A primer set designed based on the base sequence shown in SEQ ID NO: 20 to give a gene amplification product capable of detecting the 179th base of the base sequence shown in SEQ ID NO: 20;
21. A primer set designed based on the base sequence shown in SEQ ID NO: 21, which gives a gene amplification product capable of detecting the 129th base of the base sequence shown in SEQ ID NO: 21;
22. A primer set designed based on the base sequence shown in SEQ ID NO: 22, which gives a gene amplification product capable of detecting the 151st base of the base sequence shown in SEQ ID NO: 22;
23. A primer set designed based on the base sequence shown in SEQ ID NO: 23, which gives a gene amplification product capable of detecting the 252nd base of the base sequence shown in SEQ ID NO: 23;
24. A primer set designed based on the base sequence shown in SEQ ID NO: 24, which gives a gene amplification product capable of detecting the 271st base of the base sequence shown in SEQ ID NO: 24;
25. A primer set designed based on the base sequence shown in SEQ ID NO: 25, which gives a gene amplification product capable of detecting the 141st base of the base sequence shown in SEQ ID NO: 25;
26. A primer set designed based on the base sequence shown in SEQ ID NO: 26, which gives a gene amplification product capable of detecting the 137th base of the base sequence shown in SEQ ID NO: 26;
27. A primer set designed based on the base sequence shown in SEQ ID NO: 27, which gives a gene amplification product capable of detecting the 344th base of the base sequence shown in SEQ ID NO: 27;
28. A primer set designed based on the base sequence shown in SEQ ID NO: 28, which gives a gene amplification product capable of detecting the 246th base of the base sequence shown in SEQ ID NO: 28;
29. A primer set designed based on the base sequence shown in SEQ ID NO: 29, which gives a gene amplification product capable of detecting the 108th base of the base sequence shown in SEQ ID NO: 29;
30. A primer set designed based on the base sequence shown in SEQ ID NO: 30 to give a gene amplification product capable of detecting the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30;
31. A primer set designed on the basis of the base sequence shown in SEQ ID NO: 31, which gives a gene amplification product capable of detecting the 62nd base of the base sequence shown in SEQ ID NO: 31;
32. A primer set designed based on the base sequence shown in SEQ ID NO: 32, which gives a gene amplification product capable of detecting the 156th base of the base sequence shown in SEQ ID NO: 32;
33. A primer set designed based on the base sequence shown in SEQ ID NO: 33, which gives a gene amplification product capable of detecting the 669th base of the base sequence shown in SEQ ID NO: 33;
34. A primer set designed based on the base sequence shown in SEQ ID NO: 34, which gives a gene amplification product capable of detecting the 183rd base of the base sequence shown in SEQ ID NO: 34;
35. A primer set designed based on the base sequence shown in SEQ ID NO: 35, which gives a gene amplification product capable of detecting the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35;
36. A primer set designed based on the base sequence shown in SEQ ID NO: 36, which gives a gene amplification product capable of detecting the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36;
37. A primer set designed based on the base sequence shown in SEQ ID NO: 37, which gives a gene amplification product capable of detecting the 361st base of the base sequence shown in SEQ ID NO: 37;
38. A primer set designed based on the base sequence shown in SEQ ID NO: 38, which gives a gene amplification product capable of detecting the 139th base of the base sequence shown in SEQ ID NO: 38;
39. A primer set designed based on the base sequence shown in SEQ ID NO: 39, which gives a gene amplification product capable of detecting the 320th base of the base sequence shown in SEQ ID NO: 39;
40. A primer set designed based on the base sequence shown in SEQ ID NO: 40, which gives a gene amplification product capable of detecting the 265th base of the base sequence shown in SEQ ID NO: 40;
41. A primer set designed based on the base sequence shown in SEQ ID NO: 41, which gives a gene amplification product capable of detecting the 337th base of the base sequence shown in SEQ ID NO: 41;
42. A primer set designed based on the base sequence shown in SEQ ID NO: 42, which gives a gene amplification product capable of detecting the 312th base of the base sequence shown in SEQ ID NO: 42;
43. A primer set designed based on the base sequence shown in SEQ ID NO: 43, which gives a gene amplification product capable of detecting the 160th base of the base sequence shown in SEQ ID NO: 43;
44. A primer set designed based on the base sequence shown in SEQ ID NO: 44, which gives a gene amplification product capable of detecting the 314th base of the base sequence shown in SEQ ID NO: 44. The step of amplifying a DNA fragment containing each polymorphism by a gene amplification method is a step of amplifying a DNA fragment containing each polymorphism using a primer set selected from a combination of the following polynucleotides: Cattle individual identification method or parent-child discrimination method:
1. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 53 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 54;
2. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 55 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 56;
3. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 57 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 58;
4). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 59 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 60;
5. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 61 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 62;
6). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 63 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 64;
7). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 65 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 66;
8). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 67 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 68;
9. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 69 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 70;
10. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 71 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 72;
11. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 73 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 74;
12 A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 75 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 76;
13. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 77 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 78;
14 A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 79 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 80;
15. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 81 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 82;
16. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 83 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 84;
17. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 85 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 86;
18. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 87 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 88;
19. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 89 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 90;
20. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 91 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 92;
21. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 93 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 94;
22. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 95 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 96;
23. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 97 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 98;
24. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 99 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 100;
25. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 101 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 102;
26. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 103 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 104;
27. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 105 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 106;
28. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 107 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 108;
29. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 109 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 110;
30. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 111 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 112;
31. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 113 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 114;
32. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 115 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 116;
33. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 117 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 118;
34. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 119 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 120;
35. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 121 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 122;
36. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 123 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 124;
37. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 125 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 126;
38. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 127 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 128;
39. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 129 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 130;
40. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 131 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 132;
41. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 133 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 134;
42. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 135 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 136;
43. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 137 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 138;
44. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 139 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 140. The cow individual identification method or parent-child discrimination method according to any one of claims 1 to 5, wherein the cow is a Japanese black cattle. Use of any one of the following 44 gene polymorphisms as a bovine individual identification or parentage discrimination marker:
1. A single nucleotide polymorphism at the 291st base of the base sequence shown in SEQ ID NO: 1,
2. A single nucleotide polymorphism at the 505th base of the base sequence shown in SEQ ID NO: 2,
3. A single nucleotide polymorphism at the 64th base of the base sequence shown in SEQ ID NO: 3,
4). A single nucleotide polymorphism at the 93rd base of the base sequence shown in SEQ ID NO: 4,
5. A single nucleotide polymorphism at the 27th base of the base sequence shown in SEQ ID NO: 5,
6). A single nucleotide polymorphism at the 147th base of the base sequence shown in SEQ ID NO: 6,
7). A single nucleotide polymorphism at the 113th base of the base sequence shown in SEQ ID NO: 7,
8). A single nucleotide polymorphism at the 594th base of the base sequence shown in SEQ ID NO: 8,
9. An insertion deletion (indel) polymorphism at the 47th to 50th bases of the base sequence shown in SEQ ID NO: 9;
10. A single nucleotide polymorphism at the 446th base of the base sequence shown in SEQ ID NO: 10,
11. A single nucleotide polymorphism at the 185th base of the base sequence shown in SEQ ID NO: 11,
12 A single nucleotide polymorphism at the 194th base of the base sequence shown in SEQ ID NO: 12,
13. A single nucleotide polymorphism at the 143rd base of the base sequence shown in SEQ ID NO: 13,
14 A single nucleotide polymorphism at the 206th base of the base sequence shown in SEQ ID NO: 14,
15. A single nucleotide polymorphism at the 217th base of the base sequence shown in SEQ ID NO: 15,
16. A single nucleotide polymorphism at the 130th base of the base sequence shown in SEQ ID NO: 16,
17. A single nucleotide polymorphism at the 221nd base of the base sequence shown in SEQ ID NO: 17,
18. A single nucleotide polymorphism at the 382nd base of the base sequence shown in SEQ ID NO: 18,
19. A single nucleotide polymorphism at the 148th base of the base sequence shown in SEQ ID NO: 19,
20. A single nucleotide polymorphism at the 179th base of the base sequence shown in SEQ ID NO: 20,
21. A single nucleotide polymorphism at the 129th base of the base sequence shown in SEQ ID NO: 21,
22. A single nucleotide polymorphism at position 151 of the base sequence shown in SEQ ID NO: 22,
23. A single nucleotide polymorphism at the 252nd base of the base sequence shown in SEQ ID NO: 23,
24. A single nucleotide polymorphism at the 271st base of the base sequence shown in SEQ ID NO: 24,
25. A single nucleotide polymorphism at the 141st base of the base sequence shown in SEQ ID NO: 25,
26. A single nucleotide polymorphism at the 137th base of the base sequence shown in SEQ ID NO: 26,
27. A single nucleotide polymorphism at the 344th base of the base sequence shown in SEQ ID NO: 27,
28. A single nucleotide polymorphism at the 246th base of the base sequence shown in SEQ ID NO: 28,
29. A single nucleotide polymorphism at the 108th base of the base sequence shown in SEQ ID NO: 29,
30. An insertion deletion (indel) polymorphism at the 69th to 72nd bases of the base sequence shown in SEQ ID NO: 30;
31. A single nucleotide polymorphism at the 62nd base of the base sequence shown in SEQ ID NO: 31,
32. A single nucleotide polymorphism at the 156th base of the base sequence shown in SEQ ID NO: 32,
33. A single nucleotide polymorphism at the 669th base of the base sequence shown in SEQ ID NO: 33,
34. A single nucleotide polymorphism at the 183rd base of the base sequence shown in SEQ ID NO: 34,
35. An insertion deletion (indel) polymorphism at the 89th to 90th bases of the base sequence shown in SEQ ID NO: 35,
36. An insertion deletion (indel) polymorphism in the 27th to 28th bases of the base sequence shown in SEQ ID NO: 36;
37. A single nucleotide polymorphism at the 361st base of the base sequence shown in SEQ ID NO: 37,
38. A single nucleotide polymorphism at the 139th base of the base sequence shown in SEQ ID NO: 38,
39. A single nucleotide polymorphism at the 320th base of the base sequence shown in SEQ ID NO: 39,
40. A single nucleotide polymorphism at the 265th base of the base sequence shown in SEQ ID NO: 40,
41. A single nucleotide polymorphism at the 337th base of the base sequence shown in SEQ ID NO: 41,
42. A single nucleotide polymorphism at the 312th base of the base sequence shown in SEQ ID NO: 42,
43. A single nucleotide polymorphism at the 160th base of the base sequence shown in SEQ ID NO: 43,
44. A single nucleotide polymorphism at the 314th base of the base sequence shown in SEQ ID NO: 44. Any one combination selected from the following polynucleotide combinations:
1. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 53 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 54;
2. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 55 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 56;
3. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 57 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 58;
4). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 59 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 60;
5. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 61 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 62;
6). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 63 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 64;
7). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 65 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 66;
8). A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 67 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 68;
9. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 69 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 70;
10. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 71 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 72;
11. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 73 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 74;
12 A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 75 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 76;
13. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 77 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 78;
14 A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 79 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 80;
15. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 81 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 82;
16. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 83 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 84;
17. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 85 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 86;
18. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 87 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 88;
19. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 89 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 90;
20. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 91 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 92;
21. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 93 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 94;
22. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 95 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 96;
23. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 97 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 98;
24. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 99 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 100;
25. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 101 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 102;
26. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 103 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 104;
27. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 105 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 106;
28. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 107 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 108;
29. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 109 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 110;
30. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 111 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 112;
31. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 113 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 114;
32. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 115 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 116;
33. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 117 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 118;
34. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 119 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 120;
35. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 121 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 122;
36. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 123 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 124;
37. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 125 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 126;
38. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 127 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 128;
39. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 129 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 130;
40. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 131 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 132;
41. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 133 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 134;
42. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 135 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 136;
43. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 137 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 138;
44. A combination of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 139 and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 140. A reagent kit comprising at least the combination of polynucleotides according to claim 7.

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