DNA markers used for determining the risk of mastitis onset and a method for determining the risk of mastitis using the same

Abstract

To determine the risk of mammitis with high accuracy.SOLUTION: The 101st base mutation in a specific base sequence and a base mutation linked to the base sequence is used as a DNA marker for determining the risk of mammitis.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a DNA marker that can be used to determine the onset risk of mastitis, which frequently occurs in dairy cows, and a method for determining the mastitis risk using the same.

Background Art

[0002] Mastitis is considered to be caused by pathogenic microorganisms invading and multiplying in the mammary gland, stimulating the milk ducts and mammary glands, and causing inflammation. Mastitis is also called "the occupational disease of dairy cows" and is one of the most intractable diseases common to dairy cows worldwide. However, despite various countermeasures being proposed, its control has not progressed (Non-Patent Document 1). When inflammation occurs due to mastitis, blood flow increases, vascular permeability increases, white blood cells in the blood migrate, and white blood cells migrate into the milk. In addition, epithelial cells damaged by inflammation detach and also migrate into the milk. For this reason, an increase in the number of somatic cells such as white blood cells and exfoliated epithelial cells contained in the milk is globally used as an indicator of mastitis infection (Non-Patent Document 2). detach and also migrate into the milk. For this reason, an increase in the number of somatic cells such as white blood cells and exfoliated epithelial cells contained in the milk is globally used as an indicator of mastitis infection (Non-Patent Document 2). detach and also migrate into the milk. For this reason, an increase in the number of somatic cells such as white blood cells and exfoliated epithelial cells contained in the milk is globally used as an indicator of mastitis infection (Non-Patent Document 2). Non-Patent Document 1 discloses that a mastitis resistance gene (FEZL gene) closely related to bovine mastitis was identified as a means for simply, reliably, and rapidly detecting / diagnosing bovine mastitis resistance. According to Non-Patent Document 1, the mastitis resistance of cows is diagnosed by identifying gene mutations in the FEZL gene. Further, Non-Patent Document 2 discloses a method for detecting the DQA1*1201 allele and / or the DQA1*0101 allele in the DQA1 gene encoding the bovine major histocompatibility antigen DQ molecule alpha chain as a method for determining the onset resistance or susceptibility to bovine mastitis. Furthermore, Non-Patent Document 3 shows that

[0003] Non-Patent Document 1 discloses that a mastitis resistance gene (FEZL gene) closely related to bovine mastitis was identified as a means for simply, reliably, and rapidly detecting / diagnosing bovine mastitis resistance. Non-Patent Document 1 discloses that a mastitis resistance gene (FEZL gene) closely related to bovine mastitis was identified as a means for simply, reliably, and rapidly detecting / diagnosing bovine mastitis resistance. According to Non-Patent Document 1, the mastitis resistance of cows is diagnosed by identifying gene mutations in the FEZL gene. According to Non-Patent Document 1, the mastitis resistance of cows is diagnosed by identifying gene mutations in the FEZL gene. , a cytosine (c) of 1 to 4 bp is inserted at position 1110 of the nucleotide sequence of the IGF1R gene of mastitis-resistant cows The mutation is disclosed. Patent Document 3 discloses a method for diagnosing mastitis resistance using the mutation.

[0004] Furthermore, Patent Document 4 discloses polymorphic microsatellite markers C6orf93 and inra084 on bovine chromosome BTA9, and polymorphic microsatellite markers HELMTT43 and BM3501 on bovine chromosome BTA11 as genetic markers indicating mastitis tolerance in cows. on bovine chromosome BTA9, and polymorphic microsatellite markers HELMTT43 and BM3501 on bovine chromosome BTA11 are disclosed. Furthermore, Patent Document 5 discloses a number of SNPs as genetic markers related to the trait indicating mastitis resistance in cows.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] As described above, conventionally, although gene markers for diagnosing bovine mastitis resistance / susceptibility have been known, their diagnostic accuracy has not been sufficient, and effective factors for identifying mastitis risk have not been identified. Currently, diagnosis using specific markers has not been carried out and has not reached practical application. Therefore, in view of the above-described circumstances, an object of the present invention is to provide a DNA marker capable of accurately identifying mastitis risk and a method for determining mastitis risk using the DNA marker.

[0008] Therefore, in view of the above-described circumstances, the present invention provides a DNA marker capable of accurately identifying mastitis risk and a method for determining mastitis risk using the DNA marker.

Means for Solving the Problems

[0009] As a result of intensive studies by the present inventors to achieve the above object, differences between a population of individuals with repeated mastitis onset and a population of healthy individuals were comprehensively evaluated through genomic analysis, and DNA mutations enabling discrimination of animals with a high risk of mastitis onset were identified, leading to the completion of the present invention. The present invention includes the following.

[0010] (1) A step of collecting a biological sample from a test animal, and a step of identifying the genotype of a mastitis risk determination DNA marker in genomic DNA contained in the biological sample, wherein the mastitis risk determination DNA marker is a base mutation at the 101st position in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation; a locus in a region sandwiched by the base sequences of SEQ ID NO: 2 and SEQ ID NO: 8, and SEQ ID NOs: 2 to 8 ​​​​​​​​​​a 101st base mutation in one base sequence selected from the following group or a base mutation linked to the base mutation; a step of being at least one base mutation among the base mutations linked to the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or the base mutation; a step of determining the mastitis risk of a test animal based on the genotype of the mastitis risk determination DNA marker; and a method for determining mastitis risk, comprising:

[0011] (2) When the genotype of the mastitis risk determination DNA marker is heterozygous or homozygous for the minor allele, it is determined that the mastitis risk of the test animal is high, characterized in that it is the method for determining mastitis risk according to (1).

[0012] (3) The 101st base mutation in the base sequence of SEQ ID NO: 1 is such that the major allele is A and the minor allele is C, the 101st base mutation in the base sequence of SEQ ID NO: 2 is such that the major allele is A and the minor allele is G, the 101st base mutation in the base sequence of SEQ ID NO: 3 is such that the major allele is G and the minor allele is T, the 101st base mutation in the base sequence of SEQ ID NO: 4 is such that the major allele is A and the minor allele is G, the 101st base mutation in the base sequence of SEQ ID NO: 5 is such that the major allele is A and the minor allele is G, the 101st base mutation in the base sequence of SEQ ID NO: 6 is such that the major allele is A and the minor allele is T, the 101st base mutation in the base sequence of SEQ ID NO: 7 is such that the major allele is C and the minor allele is A, the 101st base mutation in the base sequence of SEQ ID NO: 8 is such that the major allele is G and the minor allele is A, and the 101st base mutation in the base sequence of SEQ ID NO: 9 is such that the major allele is A and the minor allele is G, the 101st base mutation in the base sequence of SEQ ID NO: 10 is such that the major allele is A and the minor allele is T, the 101st base mutation in the base sequence of SEQ ID NO: 11 is such that the major allele is C and the minor allele is A, the 101st base mutation in the base sequence of SEQ ID NO: 12 is such that the major allele is G and the minor allele is A, and the 101st base mutation in the base sequence of SEQ ID NO: 13 is such that the major allele is A and the minor allele is T, the 101st base mutation in the base sequence of SEQ ID NO: 14 is such that the major allele is C and the minor allele is A, the 101st base mutation in the base sequence of SEQ ID NO: 15 is such that the major allele is G and the minor allele is A, and the 101st base mutation in the base sequence of SEQ ID NO: 16 is such that the major allele is A and the minor allele is T, the 101st base mutation in the base sequence of SEQ ID NO: 17 is such that the major allele is C and the minor allele is A, the 101st base mutation in the base sequence of SEQ ID NO: 18 is such that the major allele is G and the minor allele is A, and the 101st base mutation in the base sequence of SEQ ID NO: 19 is​ The first base mutation is characterized in that the major allele is A and the minor allele is G for the method for determining the risk of mastitis described in (1).

[0013] (4) In the step of identifying the genotype of the mastitis risk determination DNA marker, the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation, and the 101st base mutation in the base sequence shown in SEQ ID NO: 2 or a base mutation linked to the base mutation, and the 101st base mutation in the base sequence shown in SEQ ID NO: 7 or a base mutation linked to the base mutation, and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation are identified for their genotypes, which is characterized in that it is the method for determining the risk of mastitis described in (1).

[0014] (5) In the step of identifying the genotype of the mastitis risk determination DNA marker, the combination of the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation, the combination of the 101st base mutation in the base sequence shown in SEQ ID NO: 7 or a base mutation linked to the base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation, and the combination of the 101st base mutation in the base sequence shown in SEQ ID NO: 2 or a base mutation linked to the base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation are identified for their genotypes, which is characterized in that it is the method for determining the risk of mastitis described in (1).

[0015] (6) In the step of identifying the genotype of the mastitis risk determination DNA marker, the genomic DNA derived from the test animal is used as a template, and a nucleic acid fragment containing the mastitis risk determination DNA marker is amplified by a nucleic acid amplification method. The mastitis risk determination method according to (1) is characterized in that.

[0016] (7) In the step of identifying the genotype of the mastitis risk determination DNA marker, the nucleotide sequence of the nucleic acid fragment is determined to identify the genotype. The mastitis risk determination method according to (6) is characterized in that.

[0017] (8) A primer set for amplifying a nucleic acid fragment containing a mastitis risk determination DNA marker consisting of at least one base mutation among the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation; the 101st base mutation in one base sequence selected from the group consisting of SEQ ID NOs: 2 to 8 located in the region sandwiched by the base sequences of SEQ ID NO: 2 and SEQ ID NO: 8 or a base mutation linked to the base mutation; the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation.

[0018] (9) A combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 10 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 11, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 12 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 13, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 14 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 15, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 16 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 17, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO:. ​ An oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 18 and an oligo nucleotide combination, an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 20 and an oligonucleotide combination containing the nucleotide sequence shown in SEQ ID NO: 21, an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 22 and an oligonucleotide combination containing the nucleotide sequence shown in SEQ ID NO: 23, an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 24 and an oligonucleotide combination containing the nucleotide sequence shown in SEQ ID NO: 25, an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 26 and an oligonucleotide combination containing the nucleotide sequence shown in SEQ ID NO: 27, at least one combination selected from the group consisting of combinations of oligonucleotides containing the nucleotide sequence shown in SEQ ID NO: 27, characterized in that it is a primer set according to (8). imer set.

Advantages of the Invention

[0019] According to the method for determining the risk of mastitis according to the present invention, by using a novel DNA marker for determining the risk of mastitis, the risk of mastitis in an animal to be examined can be determined with extremely high accuracy.

Brief Description of the Drawings

[0020]

Figure 1

Embodiments for Carrying Out the Invention

[0021] Hereinafter, the present invention will be described in detail. The DNA marker for determining the risk of mastitis according to the present invention is a base mutation at a predetermined position on the genome that can be used to determine the risk of developing mastitis. Here, the DNA marker for determining the risk of mastitis ​​​​The base mutation that becomes a causative factor means a single nucleotide variant (SNV). A single nucleotide variant (SNV) means a difference of one base (substitution mutation) in the genomic base sequence, and in a certain population, it means including a single nucleotide polymorphism (SNP) where the frequency of the substitution mutation is 1% or more.

[0022] 1. Mastitis risk determination DNA marker The mastitis risk determination DNA marker can be identified based on the reference genome in cattle. The reference genome of cattle can use ARS-UCD1.2 (Hereford breed, April 11, 2018 version, https: / / www.ncbi.nlm.nih.gov / assembly / GCF_002263795.1 / ). In the following description, the mastitis risk determination DNA marker will be described based on the base sequence of the reference genome in cattle, but the mastitis risk determination D NA marker according to the present invention is not limited to cattle and can be identified in any animal including humans. That is, when the genomic base sequence (for example, the base sequence of the reference genome) in the animal to be examined is known, the mastitis risk determination DNA marker in the animal can be identified based on the genomic base sequence of the animal to be examined.

[0023] The mastitis risk determination DNA marker according to the present invention is the 1 01st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation; located in the region sandwiched by the base sequences of SEQ ID NO: 2 and SEQ ID NO: 8, selected from the group consisting of SEQ ID NOs: 2 to 8 ​The 101st base mutation in a disclosed base sequence or a base mutation linked to the base mutation ; among the 101st base mutations in the base sequence shown in SEQ ID NO: 9 or base mutations linked to the base mutation is at least one base mutation.

[0024] Here, the 101st base mutation in the base sequence shown in SEQ ID NO: 1 is a mutation site located at 25087716 on chromosome 15 in the base sequence of the bovine reference genome . This base mutation is a substitution mutation where the major allele is A and the minor allele is C . Further, this base mutation is registered in a database (for example , Ensembl) as an SNP identified by the rs number: rs110036757 .

[0025] Also, the 101st base mutation in the base sequence shown in SEQ ID NO: 2 is a mutation site located at 88209479 on chromosome X in the base sequence of the bovine reference genome. This base mutation is a substitution mutation where the major allele is A and the minor allele is G. Moreover , this base mutation is registered in a database (for example , Ensembl) as an SNP identified by the rs number: rs208854668 .

[0026] Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 3 is a mutation site located at 89239121 on chromosome X in the base sequence of the bovine reference genome. This base mutation is a substitution mutation where the major allele is G and the minor allele is T . Also, this base mutation is registered in a database (for example , Ensembl) as an SNP identified by the rs number: rs381839020 .

[0027] Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 4 is a mutation site located at 89242713 on the X chromosome in the base sequence of the bovine reference genome. This base mutation is a substitution mutation in which the major allele is A and the minor allele is G. Also, this base mutation is registered in a database ( e.g., Ensembl) as an SNP identified by rs number: rs385943123.

[0028] Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 5 is a mutation site located at 88319202 on the X chromosome in the base sequence of the bovine reference genome. This base mutation is a substitution mutation in which the major allele is A and the minor allele is G. Also, this base mutation is registered in a database ( e.g., Ensembl) as an SNP identified by rs number: rs109551898.

[0029] Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 6 is a mutation site located at 88700655 on the X chromosome in the base sequence of the bovine reference genome. This base mutation is a substitution mutation in which the major allele is A and the minor allele is T. Also, this base mutation is registered in a database ( e.g., Ensembl) as an SNP identified by rs number: rs137152785.

[0030] Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 7 is a mutation site located at 89067208 on the X chromosome in the base sequence of the bovine reference genome. This base mutation is a substitution mutation where the major allele is C and the minor allele is A. In addition, this base mutation is registered in a database ( e.g., Ensembl) as an SNP identified by the rs number: rs110688314.

[0031] Furthermore, the 101st base mutation in the nucleotide sequence shown in SEQ ID NO: 8 is a mutation site located at 89289439 on the X chromosome in the nucleotide sequence of the bovine reference genome. This base mutation is a substitution mutation where the major allele is G and the minor allele is A. In addition, this base mutation is registered in a database (e.g., Ensembl) as an SNP identified by the rs number: rs135732164.

[0032] Furthermore, the 101st base mutation in the nucleotide sequence shown in SEQ ID NO: 9 is a mutation site located at 12219701 on chromosome 24 in the nucleotide sequence of the bovine reference genome. This base mutation is a substitution mutation where the major allele is A and the minor allele is G. In addition, this base mutation is registered in a database (e.g., Ensembl) as an SNP identified by the rs number: rs110369890.

[0033] As described above, the mastitis risk determination DNA marker according to the present invention is a base substitution existing in a predetermined region of chromosome 15, a base substitution existing in a predetermined region of the X chromosome, and a base substitution existing in a predetermined region of chromosome 24. Here, the predetermined region of chromosome 15 refers to the mutation site located at 25087716 described above (sequence

[0034] It means the region where there is a base mutation linked to the 101st position (rs110036757) in No. 1. That is, the mastitis risk determination DNA marker according to the present invention is in linkage disequilibrium with the mutation site located at 25087716 (the 101st position in SEQ ID NO: 1, rs110036757). It will include a mutation site (single nucleotide polymorphism (SNV)) that is in a relationship of linkage disequilibrium with the mutation site located at 25087716 (the 101st position in SEQ ID NO: 1, rs110036757).

[0035] Also, the predetermined region of the X chromosome means the region sandwiched between the base sequence of SEQ ID NO: 2 and the base sequence of SEQ ID NO: 8. In this region, there are the mutation sites located at 88209479 described above (the 101st position in SEQ ID NO: 2, rs208854668), the mutation site located at 89239121 (the 101st position in SEQ ID NO: 3, rs381839020), the mutation site located at 89242713 (the 101st position in SEQ ID NO: 4, rs385943123), the mutation site located at 88319202 (the 101st position in SEQ ID NO: 5, rs109551898), the mutation site located at 88700655 (the 101st position in SEQ ID NO: 6, rs137152785), the mutation site located at 89067208 (the 101st position in SEQ ID NO: 7, rs110688314), and the mutation site located at 89289439 (the 101st position in SEQ ID NO: 8, rs135732164). Therefore, the predetermined region of the X chromosome is the region sandwiched between the base sequence of SEQ ID NO: 2 and the base sequence of SEQ ID NO: 8, and includes these mutation sites and a mutation site (single nucleotide polymorphism (SNV)) that is in a relationship of linkage disequilibrium with any of these mutation sites. More specifically, the predetermined region of the X chromosome has a mutation site linked to 88209479 on the X chromosome.

[0036] ​​​​​​​​​​​A mutation site (measure - allele T, minor allele A) located at 88209483 on the X chromosome is included as a locus (single - nucleotide variant (SNV)). Also, in a predetermined region of the X chromosome, mutation sites (single - nucleotide variants (SNVs)) linked to 89239121 on the X chromosome include 89235533 (major allele T, minor allele C), 89237555 (major allele G, minor allele A), and 89237780 (major allele G, minor allele A) on the X chromosome. Further, in a predetermined region of the X chromosome, mutation sites (single - nucleotide variants (SNVs)) linked to 89242713 on the X chromosome include 89259615 (major allele C, minor allele T), 89269677 (major allele T, minor allele C), and 89280192 (major allele C, minor allele G) on the X chromosome. Additionally, in a predetermined region of the X chromosome, mutation sites (single - nucleotide variants (SNVs)) linked to 88319202 on the X chromosome include 88313029 (major allele C, minor allele G), 88338299 (major allele T, minor allele C), and 88339019 (major allele T, minor allele G) on the X chromosome. Moreover, in a predetermined region of the X chromosome, a mutation site (single - nucleotide variant (SNV)) linked to 88700655 on the X chromosome includes 88664079 (major allele T, minor allele C) on the X chromosome. Furthermore, in a predetermined region of the X chromosome, a mutation site (single - nucleotide variant (SNV)) linked to 89067208 on the X chromosome includes 89073474 (major allele G, minor allele C) on the X chromosome. Additionally, in a predetermined region of the X chromosome, a mutation site (single - nucleotide variant (SNV)) linked to 89289439 on the X chromosome contains the minor allele A). Also, in a predetermined region of the X chromosome, there are mutation sites (single - nucleotide variants (SNVs)) linked to 89239121 on the X chromosome. As a mutation site (single - nucleotide variant (SNV)) linked to 89239121 on the X chromosome, 89 235533 (major allele T, minor allele C), 89237555 (major allele G, minor allele A), and 89237780 (major allele G, minor allele A) are included. Further, in a predetermined region of the X chromosome, there are mutation sites (single - nucleotide variants (SNVs)) linked to 89242713 on the X chromosome. As a mutation site (single - nucleotide variant (SNV)) linked to 89242713 on the X chromosome, 89259615 (major allele C, minor allele T), 8926967 7 (major allele T, minor allele C), and 89280192 (major allele C, minor allele G) are included. Additionally, in a predetermined region of the X chromosome, there are mutation sites (single - nucleotide variants (SNVs)) linked to 88319202 on the X chromosome. As a mutation site (single - nucleotide variant (SNV)) linked to 88319202 on the X chromosome, 88313029 (major allele C, minor allele G), 88338299 (major allele T, minor allele C), and 883390 19 (major allele T, minor allele G) are included. Moreover, in a predetermined region of the X chromosome, there is a mutation site (single - nucleotide variant (SNV)) linked to 88700655 on the X chromosome. As a mutation site (single - nucleotide variant (SNV)) linked to 88700655 on the X chromosome, 88664079 (major allele T, minor allele C) on the X chromosome is included. Additionally, in a predetermined region of the X chromosome, a mutation site (single - nucleotide variant (SNV)) linked to 89067208 on the X chromosome includes 89073474 (major allele G, minor allele C) on the X chromosome. Furthermore, in a predetermined region of the X chromosome, there is a mutation site (single - nucleotide variant (SNV)) linked to 89289439 on the X chromosome. As a different site (single nucleotide variant (SNV)), 89278639 on the X chromosome (major allele G, minor allele A) is included.

[0037] Furthermore, a predetermined region of chromosome 24 means a region where a base mutation linked to the mutation site (the 101st position in SEQ ID NO: 9, rs110369890) located at 12219701 described above exists. That is, the mastitis risk determination DNA marker according to the present invention includes a mutation site (single nucleotide variant (SNV)) in a linkage disequilibrium relationship with the mutation site (the 101st position in SEQ ID NO: 9, rs110369890) located at 12219701.

[0038] As described above, the mastitis risk determination DNA marker according to the present invention can be specified based on the base sequence of the bovine reference genome. This does not mean that the technical scope of the mastitis risk determination DNA marker according to the present invention is limited to cattle (that is, the mastitis risk determination DNA marker in cattle), but rather that the mastitis risk determination DNA marker can be specified in any mammal including humans, and the mastitis risk determination DNA marker can be applied to any mammal. For example, in buffalo, goats, sheep, pigs, horses, yaks, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, monkeys, and humans, the mastitis risk determination DNA marker can be specified. In order to specify the mastitis risk determination DNA marker in mammals other than cattle, it can be specified as a region having high homology with the base sequences shown in SEQ ID NOs: 1 to 9 described above by homology search based on the base sequences shown in SEQ ID NOs: 1 to 9 described above. At this time, as long as it can be specified as a sequence with high homology such as homolog, paralog, or ortholog, the position on the chromosome of the genome does not matter. ​​​​​​​​​​​​

[0039] The degree of homology is not particularly limited, but for the nucleotide sequences of SEQ ID NOs: 1 to 9, for example , 70% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more can be used. A region having such high homology is identified in the genome of mammals other than cattle , and the nucleotide mutations contained in the identified region can be used as a DNA marker for determining the risk of mastitis in mammals other than cattle . When identifying a DNA marker for determining the risk of mastitis in mammals other than cattle , it is not necessary to use the full lengths of SEQ ID NOs: 1 to 9 for homology search, and it may be only upstream or downstream of the nucleotide mutation site, or it can be, for example, 200 nucleotides, 50 nucleotides, 20 nucleotides, or 10 nucleotides upstream and / or downstream. In addition, the reference genome is updated as data accumulates, and the positions for identifying genomic regions may vary , but the region can be identified by using the sequences indicated by SEQ ID NOs. For example, water buffalo (Bubalus bubalis breed Mediterranean chromosome X, ASM312139v1, w hole genome shotgun sequence, NC_037569.1), goat (Capra hircus breed San Clemen

[0040] te chromosome X unlocalized genomic scaffold, ASM170441v1, whole genome shotgun sequence, NW_017189516.1), and sheep (Ovis aries strain OAR_USU_Benz2616 breed Rambo te chromosome X unlocalized genomic scaffold, ASM170441v1, whole genome shotgun sequence, NW_017189516.1), and sheep (Ovis aries strain OAR_USU_Benz2616 breed Rambo uillet chromosome X, ARS-UI_Ramb_v2.0, whole genome shotgun sequence, NC_056080. Regarding (1), by performing a homology search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), it is possible to identify mastitis risk determination DNA markers in water buffalo, goats, and sheep.

[0041] Specifically, for 25087716 on chromosome 15 (the 101st in SEQ ID NO: 1), 59845877 on chromosome 16 in water buffalo is identified, 57243975 on chromosome 15 in goats is identified, and 24820077 on chromosome 15 in sheep is identified. These can be used respectively as mastitis risk determination DNA markers for water buffalo, goats, and sheep.

[0042] Also, as mastitis risk determination DNA markers located on the X chromosome, for 88209479 (the 101st in SEQ ID NO: 2), 54070123 in water buffalo, 15916117 in goats, and 54912361 in sheep are identified. These can be used respectively as mastitis risk determination DNA markers for water buffalo, goats, and sheep. Also, as mastitis risk determination DNA markers located on the X chromosome, for 89239121 (the 101st in SEQ ID NO: 3), 53049056 in water buffalo, 14833575 in goats, and 53801786 in sheep are identified. These can be used respectively as mastitis risk determination DNA markers for water buffalo, goats, and sheep. Furthermore, as mastitis risk determination DNA markers located on the X chromosome, for 89242713 (the 1st in SEQ ID NO: 4), ​​​​​​​​​Regarding the first one (No. 01), 53045801 in buffalo and 14829743 in goat were identified, and these can be used respectively as DNA markers for mastitis risk determination in buffalo and goat. Furthermore, as a DNA marker for mastitis risk determination located on the X chromosome, for 88319202 (No. 101 in SEQ ID NO: 5), 53958808 in buffalo, 15774295 in goat, and 54778777 in sheep were identified, and these can be used respectively as DNA markers for mastitis risk determination in buffalo, goat and sheep. Furthermore, as a DNA marker for mastitis risk determination located on the X chromosome, for 88700655 (No. 101 in SEQ ID NO: 6), 53590316 in buffalo was identified, and this can be used as a DNA marker for mastitis risk determination in buffalo. Furthermore, as a DNA marker for mastitis risk determination located on the X chromosome, for 89067208 (No. 101 in SEQ ID NO: 7), 53220380 in buffalo, 14849521 in goat, and 54011154 in sheep were identified, and these can be used respectively as DNA markers for mastitis risk determination in buffalo, goat and sheep. Furthermore, as a DNA marker for mastitis risk determination located on the X chromosome, for 89289439 (No. 101 in SEQ ID NO: 8), 53002464 and 53052853 in buffalo, 14787290 and 15067437 in goat, and 53754307 and 53805622 in sheep were identified, and these can be used respectively as DNA markers for mastitis risk determination in buffalo, goat and sheep.

[0043] Furthermore, regarding 12219701 (No. 101 in SEQ ID NO: 9) located on chromosome 24 49964421 on chromosome 22 in buffalo is identified, and 1237 6430 on chromosome 24 in goat is identified, and these can be used respectively as DNA markers for mastitis risk determination in buffalo and goat.

[0044] 2. Method for determining mastitis risk The method for determining mastitis risk according to the present invention includes the steps of collecting a biological sample from a test animal, identifying the genotype of the DNA marker for mastitis risk in the genomic DNA contained in the biological sample, and determining the mastitis risk of the test animal based on the genotype of the DNA marker for mastitis risk.

[0045] The biological sample collected from the test animal is not particularly limited, and examples include hair roots, skin, milk, saliva, nasal mucus, blood, meat, organs, sperm, eggs, fertilized eggs, etc. Preferably, in consideration of animal welfare, a biological sample that can be collected without causing pain to the individual is used. This is preferred.

[0046] When identifying the genotype of the DNA marker for mastitis risk in the genomic DNA contained in the biological sample, first, genomic DNA may be extracted from the biological sample. The extraction of genomic DNA can apply methods for recovering conventionally known polynucleotides. For example, methods using phenol, chloroform, and methods using sodium iodide can be mentioned. Also, without extracting the genomic DNA contained in the biological sample, the genotype of the DNA marker for mastitis risk in the genomic DNA contained in the biological sample may be identified. For example, according to the lectin PCR method, without performing the extraction operation of genomic DNA, the genomic DN contained in the cells A can be identified. PCR can also be performed using A as a template to identify the genotype of the mastitis risk determination DNA marker. It is possible.

[0047] As one method for determining the genotype of the mastitis risk determination DNA marker, a method of determining the nucleotide sequence of the region containing the mastitis risk determination DNA marker can be mentioned. The determination of the nucleotide sequence can select any conventionally known method. For example, methods such as Sanger sequencing, microarray, next-generation sequencing, real-time PCR, PCR-RFLP, mass spectrometry, etc., the LAMP method and nanopore sequencing can be mentioned. Among these various methods, when using a method involving a nucleic acid amplification reaction, a pair of primers for amplifying the nucleic acid fragment containing the mastitis risk determination DNA marker is designed. When using a method involving a nucleic acid amplification reaction, a pair of primers for amplifying the nucleic acid fragment containing the mastitis risk determination DNA marker is designed. of primers for amplifying the nucleic acid fragment containing the mastitis risk determination DNA marker is designed.

[0048] A pair of primers for amplifying the nucleic acid fragment containing the mastitis risk determination DNA marker can be designed based on the nucleotide sequence of the genomic DNA of the test animal when the nucleotide sequence of the genomic DNA of the test animal is known. Also, when the nucleotide sequence of the genomic DNA of the test animal is not known it is possible to design a pair of primers after determining the nucleotide sequence of the genomic DNA, and it is also possible to design a pair of primers after determining the nucleotide sequence near the mastitis risk determination DNA marker. When designing the primers, although not particularly limited, a pair of primers can be designed to be a nucleic acid fragment of 20 to 1000 bases, 20 to 800 bases, 20 to 600 bases, 20 to 400 bases, 20 to 200 bases, 20 to 100 bases or 20 to 50 bases containing the site of the mastitis risk determination DNA marker.

[0049] When designing the primers, although not particularly limited, a pair of primers can be designed to be a nucleic acid fragment of 20 to 1000 bases, 20 to 800 bases, 20 to 600 bases, 20 to 400 bases, 20 to 200 bases, 20 to 100 bases or 20 to 50 bases containing the site of the mastitis risk determination DNA marker. 100 bases or 20 to 50 bases containing the site of the mastitis risk determination DNA marker. It is possible. When designing a pair of primers, for example, the length of the primers can be 17 - 25 mers and the GC content can be 40 - 60%, preferably 45 - 55%. Also, the pair of primers preferably have Tm values that do not differ greatly, and the difference in Tm values is, for example, within 2°C , preferably within 1°C.

[0050] Specifically, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 10 and 11) for amplifying a nucleic acid fragment containing 25087716 (the 101st position in SEQ ID NO: 1) on chromosome 15 can be designed. Also, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 12 and 13) for amplifying a nucleic acid fragment containing 88209479 (the 101st position in SEQ ID NO: 2) on the X chromosome can be designed. Furthermore, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 14 and 15) for amplifying a nucleic acid fragment containing 89239121 (the 101st position in SEQ ID NO: 3) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 16 and 17) for amplifying a nucleic acid fragment containing 89242713 (the 101st position in SEQ ID NO: 4) on the X chromosome can be designed. Moreover, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (the 101st position in SEQ ID NO: 5) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. reference genome, a pair of primers (SEQ ID NOs: 10 and 11) for amplifying a nucleic acid fragment containing 25087716 (the 101st position in SEQ ID NO: 1) on chromosome 15 can be designed. Also, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 12 and 13) for amplifying a nucleic acid fragment containing 88209479 (the 101st position in SEQ ID NO: 2) on the X chromosome can be designed. Furthermore, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 14 and 15) for amplifying a nucleic acid fragment containing 89239121 (the 101st position in SEQ ID NO: 3) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 16 and 17) for amplifying a nucleic acid fragment containing 89242713 (the 101st position in SEQ ID NO: 4) on the X chromosome can be designed. Moreover, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (the 101st position in SEQ ID NO: 5) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. reference genome, a pair of primers (SEQ ID NOs: 12 and 13) for amplifying a nucleic acid fragment containing 88209479 (the 101st position in SEQ ID NO: 2) on the X chromosome can be designed. Furthermore, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 14 and 15) for amplifying a nucleic acid fragment containing 89239121 (the 101st position in SEQ ID NO: 3) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 16 and 17) for amplifying a nucleic acid fragment containing 89242713 (the 101st position in SEQ ID NO: 4) on the X chromosome can be designed. Moreover, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (the 101st position in SEQ ID NO: 5) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. reference genome, a pair of primers (SEQ ID NOs: 14 and 15) for amplifying a nucleic acid fragment containing 89239121 (the 101st position in SEQ ID NO: 3) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 16 and 17) for amplifying a nucleic acid fragment containing 89242713 (the 101st position in SEQ ID NO: 4) on the X chromosome can be designed. Moreover, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (the 101st position in SEQ ID NO: 5) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. reference genome, a pair of primers (SEQ ID NOs: 16 and 17) for amplifying a nucleic acid fragment containing 89242713 (the 101st position in SEQ ID NO: 4) on the X chromosome can be designed. Moreover, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (the 101st position in SEQ ID NO: 5) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (the 101st position in SEQ ID NO: 5) on the X chromosome can be designed. Additionally, based on the nucleotide sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88700655 (the nucleotide sequence of SEQ ID NO: 5) on the X chromosome can be designed. A pair of primers (sequences Numbers 20 and 21) can be designed to amplify a nucleic acid fragment containing the 101st at column number 6. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (sequence numbers 22 and 23) can be designed to amplify a nucleic acid fragment containing 89067208 (the 101st in sequence number 7) on the X chromosome. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (sequence numbers 24 and 25) can be designed to amplify a nucleic acid fragment containing 89289439 (the 101st in sequence number 8) on the X chromosome. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (sequence numbers 24 and 25) can be designed to amplify a nucleic acid fragment containing 89289439 (the 101st in sequence number 8) on the X chromosome. A pair of primers (sequence numbers 24 and 25) can be designed to amplify a nucleic acid fragment containing 89289439 (the 101st in sequence number 8) on the X chromosome. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (sequence numbers 26 and 27) can be designed to amplify a nucleic acid fragment containing 12219701 (the 101st in sequence number 9) on chromosome 24. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (sequence numbers 26 and 27) can be designed to amplify a nucleic acid fragment containing 12219701 (the 101st in sequence number 9) on chromosome 24.

[0051] In addition, as a method for determining the genotype of the DNA marker containing the mastitis risk determination DNA marker, it is not limited to the method for determining the above-described base sequence, and a method using a probe can be applied. In the method of identifying the genotype of the DNA marker using a probe, a major allele probe that specifically hybridizes to the major allele and a minor allele probe that specifically hybridizes to the minor allele are used. In this method, a form using a microarray in which these major allele probes and minor allele probes are fixed on a substrate can be applied. These major allele probes and minor allele probes are the mastitis risk determination DNA markers ​​​​They may have a configuration that differs only in one base mutation, or they may have different lengths from each other. The lengths of the major allele probe and the minor allele probe are not particularly limited, but for example, they can be 10 bases, 20 bases, 30 bases, 40 bases, or 50 bases. Also, the major allele probe and the minor allele probe may be designed so that the base of the mastitis risk determination DNA marker is located approximately at the center of the full length, or may be designed at a position biased towards the 5' end side, or may be designed at a position biased towards the 3' end side.

[0052] In the step of determining the risk of mastitis onset, the risk of mastitis is determined from the genotype of the mastitis risk determination DNA marker. As described above, the mastitis risk determination DNA marker according to the present invention contains a plurality of base mutations. When determining the mastitis risk, all of these plurality of mastitis risk determination DNA markers may be used, or one of these mastitis risk determination DNA markers may be used. The number of mastitis risk determination DNA markers used for determining the mastitis risk is not particularly limited, and can be 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types, 10 types, 11 types.

[0053] More specifically, among the above-described mastitis risk determination DNA markers, the 101st base mutation in the base sequence shown in SEQ ID NOs: 1 to 9 or a base mutation linked to the base mutation (that is, 9 types of base mutations) may be used to determine the risk of mastitis, or 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, or 8 types of these 9 types of base mutations may be used. The risk of mastitis may be determined using base mutations. In particular, among these 9 types of base mutations of these, the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation, the 101st base mutation in the base sequence shown in SEQ ID NO: 2 or the base mutation linked to the base mutation, the 101st base mutation in the base sequence shown in SEQ ID NO: 7 or a base mutation linked to the base mutation, and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation are preferably used to determine the risk of mastitis. By using these base mutations, the detection rate of mastitis can be extremely increased, and the false positive rate can be extremely decreased.

[0054] More preferably, the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to the base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation are combined, or the 101st base mutation in the base sequence shown in SEQ ID NO: 7 or a base mutation linked to the base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation are combined, or the 101st base mutation in the base sequence shown in SEQ ID NO: 2 or a base mutation linked to the base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to the base mutation are combined to determine the mastitis risk. In this case also, the detection rate of mastitis can be extremely increased, and the false positive rate can be extremely decreased.

[0055] In addition, for the determination of mastitis risk, when the likelihood of developing mastitis is high, it can be determined as high risk, and when the likelihood of developing is low, it can be determined as low risk. However, it is also possible to determine it using numerical values or rankings indicating the level of risk. For example, when evaluating mastitis risk in terms of scores, 10 points represents the highest risk, 0 points represents the lowest risk, and the risk can be evaluated stepwise with numerical values from 0 to 10. Also, different scores can be defined based on the genotypes of the mastitis risk determination DNA markers. For example, when the genotype of the mastitis risk determination DNA marker is homozygous for the major allele, it can be defined as 0 points; when it is heterozygous, it can be defined as 1 point; and when it is homozygous for the minor allele, it can be defined as 2 points. When using the genotypes of multiple mastitis risk determination DNA markers to determine the risk of mastitis, the risk of mastitis can be determined based on the sum of the scores. Also, for example, when evaluating mastitis risk in terms of rankings, a five-level ranking from A to E can be defined, where E represents the highest risk, A represents the lowest risk, and the risk can be evaluated stepwise at the levels of A, B, C, D, and E. It is also possible to determine the level of risk using numerical values or rankings indicating the level of risk. For example, when evaluating mastitis risk in terms of scores, 10 points represents the highest risk, 0 points represents the lowest risk, and the risk can be evaluated stepwise with numerical values from 0 to 10. It is also possible to determine the level of risk using numerical values or rankings indicating the level of risk. For example, when evaluating mastitis risk in terms of scores, 10 points represents the highest risk, 0 points represents the lowest risk, and the risk can be evaluated stepwise with numerical values from 0 to 10. Also, different scores can be defined based on the genotypes of the mastitis risk determination DNA markers. For example, when the genotype of the mastitis risk determination DNA marker is homozygous for the major allele, it can be defined as 0 points; when it is heterozygous, it can be defined as 1 point; and when it is homozygous for the minor allele, it can be defined as 2 points. When using the genotypes of multiple mastitis risk determination DNA markers to determine the risk of mastitis, the risk of mastitis can be determined based on the sum of the scores. It is also possible to determine the level of risk using numerical values or rankings indicating the level of risk. For example, when evaluating mastitis risk in terms of rankings, a five-level ranking from A to E can be defined, where E represents the highest risk, A represents the lowest risk, and the risk can be evaluated stepwise at the levels of A, B, C, D, and E. For example, when evaluating mastitis risk in terms of rankings, a five-level ranking from A to E can be defined, where E represents the highest risk, A represents the lowest risk, and the risk can be evaluated stepwise at the levels of A, B, C, D, and E. It is also possible to determine the level of risk using numerical values or rankings indicating the level of risk.

[0056] In addition, in the form of using multiple mastitis risk determination DNA markers, it is also possible to pre-weight each DNA marker and then determine the risk of mastitis. That is, by weighting the mastitis risk determination DNA markers with a higher contribution to the genetic model or mastitis risk more highly than other markers, it is possible to determine the risk of mastitis with higher accuracy. If the weighting is based on the genetic model, it can be performed in the form of a recessive model, a dominant model, an additive model, a multiplicative model, etc., using arbitrary coefficients. For example, in the case of a recessive model, the risk In addition, in the form of using multiple mastitis risk determination DNA markers, it is also possible to pre-weight each DNA marker and then determine the risk of mastitis. That is, by weighting the mastitis risk determination DNA markers with a higher contribution to the genetic model or mastitis risk more highly than other markers, it is possible to determine the risk of mastitis with higher accuracy. If the weighting is based on the genetic model, it can be performed in the form of a recessive model, a dominant model, an additive model, a multiplicative model, etc., using arbitrary coefficients. For example, in the case of a recessive model, the risk In addition, in the form of using multiple mastitis risk determination DNA markers, it is also possible to pre-weight each DNA marker and then determine the risk of mastitis. It is considered that the effect is the same by having an allyl other than quaryl, and only the mutant homozygous type is targeted for evaluation. In the case of a dominant model, it is considered that the effect is the same by having a risk allele, and both the heterozygous type and the mutant homozygous type can be equally targeted for evaluation. In the case of an additive model it is considered that the effect increases additively by having a risk allele, and the mutant homozygous type can be targeted for evaluation as having twice the effect of the heterozygous type. In the case of a multiplicative model, it is considered that the effect increases multiplicatively by having a risk allele, and the mutant homozygous type can be targeted for evaluation as having a squared effect of the heterozygous type. In addition, when making a determination using multiple DNA markers, it is possible to weight them in combinations of different genetic models, such as in part a recessive model and in part an additive model. Also, when weighting the contribution to the mastitis risk, a higher coefficient can be set for DNA markers for mastitis risk determination with a higher contribution than for other DNA markers. For example, in a case-control study, the genotype frequency and allele frequency of each individual are calculated and weighted by multiplying the values by the mutant homozygous type or heterozygous type according to the genetic model.

[0057] 3. Animals to be targeted for mastitis risk determination The method for determining the mastitis risk using the above-described DNA markers for mastitis risk determination can target mammals including humans or mammals excluding humans (non-human mammals). Examples of non-human mammals include cows, water buffalo, goats, sheep, pigs, horses, yaks, mice , rats, guinea pigs, hamsters, rabbits, dogs, cats, and monkeys, but it is also possible to target animal individuals obtained as a result of mating using these individuals. ​​​​

[0058] The above-mentioned mastitis risk assessment DNA markers were identified based on the base sequence of genomic DNA. Therefore, the function of this mastitis risk DNA marker means that many mastitis cases are inherited. It is highly likely that this is a hereditary disease. If this is the case, mastitis will tend to occur frequently in many animals. If there is a trend, there may be an influx of individuals at high risk of developing mastitis in the field. For example, with regard to cattle, the spread of artificial insemination technology and embryo transfer has led to the intensive use of specific individuals. It is also assumed that the rapid spread of hereditary diseases due to the breeding of cattle. Animals for which risk of developing the disease has been assessed, and animals obtained as a result of breeding using such animals is highly useful in reducing mastitis in the entire herd.

[0059] Non-human mammals whose mastitis risk was determined using the above-mentioned mastitis risk determination DNA markers By using animals, it is possible to use them for individual management and breeding improvement, taking into account the risk of mastitis. For example, individuals that are judged to be at high risk may not be used for breeding at that time. It is also possible to carry out more careful husbandry and management of high-risk individuals. This allows for efficient measures to be taken to avoid mastitis. The individuals who were selected will be actively breeding replacement cows, which will reduce the incidence of mastitis in the field in the future. This can reduce the rate of random mutations, which contributes to efficient breeding and improvement. By performing tests and calculating genotype frequencies, it is possible to determine the prevalence of high-risk individuals in a population. This can be used to check whether the plant is in good condition and can be used as a reference for breeding improvement.

[0060] In addition, from the perspective of efficient breeding improvement, individuals who have been judged to be at risk of developing mastitis and their individual For individuals born by mating using the body, using their semen or eggs for artificial breeding can rapidly reduce the risk of mastitis within the population. For example, semen can be distributed in the form of ejaculated semen, frozen semen, or diluted semen in a non-frozen state. Eggs can be distributed in the form of unfertilized eggs or fertilized eggs in a fresh state or a frozen state. When distributing, it can be done using techniques such as the straw method or the pellet method.

Example

[0061] Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0062] 〔Example 1〕DNA Marker 1 In Example 1, a mastitis risk determination DNA marker, which is an index for determining the risk of mastitis, was selected. In this example, it was carried out by a case-control study using whole-genome sequencing. The test cows used were 25 milking cows without a history of clinical mastitis (unrelated group) and 25 milking cows with a recurrence of clinical mastitis (recurrence group) respectively. The unrelated group consisted of cows that did not contract mastitis within 610 days of the lactation period. The recurrence group consisted of cows that had suffered from clinical mastitis three or more times within 305 days of the lactation period. Note that the affected quarters varied among cows. The details of the test cows are shown in Table 1. There was no significant difference in parity, prepartum milking days, and prepartum dry days between the unrelated group and the recurrence group, but the average somatic cell count at 305 days was significantly higher in the recurrence group.

[0063]

Table 1

[0064] For each of the 25 animals in the non-recurrence group and the recurrence group, whole blood was centrifuged to collect the buffy coat layer. Subsequently, DNA was extracted by a conventional method, and whole genome sequencing was performed using NovaSeq6000 (Illumina, Inc.). Sequencing was carried out. Data of over 100 billion base pairs were obtained, and for a reference genome of approximately 2.7 billion base pairs (ARS-UCD1.2, Holstein breed, version of April 11, 2018), the average depth was made 30-fold or more. Data analysis was performed using CLC Genomics Workbench, and DNA mutation analysis was carried out by comparing the non-recurrence group and the recurrence group. The mutation analysis was performed in the order of: (1) importing Illumina's Fastq data, (2) performing quality checks and trimming low-reliability ones, (3) mapping against the reference genome, (4) removing PCR duplicates, (5) local realignment, (6) mutation detection, (7) annotation, and (8) filtering. For the reference genome, 4,308,955 single nucleotide variants (SNVs), 11,871 multi-nucleotide variants (MNVs), 38,916 deletions, 41,664 insertions, and 2,843 replacements were identified. Among them, for those where only mutant homozygotes were detected, there were 1,568 SNVs, 62 MNVs, 208 deletions, 210 insertions, and 8 replacements. When DNA mutations that are candidates for the risk of mastitis onset were narrowed down by p-value, there were 20 SNVs (p < 1x10E-3) and

[0065] 6 sites (p < 1x10E-2), 20 sites of Deletion (p < 5x10E-3), 14 sites of Insertion (p < 5x10E-3), 8 sites of Replacement (p < 5x10E-2). Although these may not be identified as mutations causing non-synonymous substitutions, they can be used as indicators for determining the mastitis risk in any case. Also, since linkage disequilibrium occurs in the regions proximal to these mutation sites, there are also sites that can serve as indicators for determining the mastitis risk in the peripheral regions of the identified mutation sites, and they can be used as indicators for determining the mastitis risk.

[0066] Regarding these mutations, when further narrowing down by p-value including those with unknown zygosity, homo, and hetero, as shown in Table 2, the mutations were identified in ascending order of p-value, and regions with p < 1x10E-7 were more frequently observed on the X chromosome. These mutation sites and the mutation sites in their peripheral regions are particularly powerful as indicators for determining the mastitis risk.

[0067]

Table 2

[0068] 〔Example 2〕DNA Marker 2 In Example 2, from among the mastitis risk determination DNA marker candidates serving as indicators for determining the mastitis risk described in Example 1, SNV mastitis risk determination DNA markers were selected. The test cows were the same as in Example 1, with 25 milking cows without a history of clinical mastitis (unrelated group) and 25 milking cows with recurrent clinical mastitis (recurrent group) each.

[0069] ​​Regarding SNV, DNA markers were narrowed down using p-values calculated with the number of animals with homozygous or heterozygous mutations for each section of 25 animals. As DNA markers, 62 were identified with p < 4.8x10E-6 (Table 3). Each DNA marker was arranged in ascending order of p-value. There were 48 in the range of 88209379 - 89369844 on the X chromosome, 4 in the range of 51376086 - 51627177 on chromosome 23, 3 in the range of 18570840 - 18580407 on chromosome 27, 2 in the range of 12219601 - 14118517 on chromosome 24, 2 in the range of 12406826 - 12407723 on chromosome 25, 1 in the range of 25087616 - 25087816 on chromosome 15, 1 in the range of 2036632 - 2036832 on chromosome 26, and 1 in the range of 30165014 - 30165214 on chromosome 28. The mutation sites identified in Table 3 and the mutation sites in the surrounding regions could be used as indicators for determining mastitis risk.

[0070]

Table 3

[0071] Furthermore, when p-values were plotted for each region of the X chromosome using SNV, as shown by the arrow, there was a region where SNV with p < 1x10E-6 concentrated between 89067208 and 89369744 ( Figure 1). Therefore, the mutation sites in this region were particularly powerful as indicators for determining mastitis risk.

[0072] Furthermore, when examining the genes affecting mastitis using the Ensembl Genome Browser (https: / / www.ensembl.org), in the region of 89067208 - 89369744 on the X chromosome, three proteins, G SPT2, ENSBTAG00000052977, and ENSBTAG00000053722, were encoded. The GSPT2 gene had a Transcript ID of ENSBTAT00000017703.5 and was present on the Reverse strand in the region of 89,276,444 - 89,278,960 on the X chromosome. It encoded G1 to S phase transition 2 (2410bp, 631aa), and functions such as Tr-type G domain-containing protein, GTPase activity, G TP binding, and translation release factor activity were inferred, but it was an un-reviewed protein. The significant SNV g.89279441 was located 500bp upstream of the start codon, and g.89278639C>T was a synonymous substitution of the 41st amino acid (alanine).

[0073] The ENSBTAG00000052977 gene had a Transcript ID of ENSBTAT00000083094.1 and was present on the Forward strand in the region of 89,245,181 - 89,245,990 on the X chromosome. It encoded a PABC domain-containing protein (810bp, 269aa), and functions such as polyadenylate-binding protein and RNA bindi ng were inferred, but it was an un-reviewed protein. ENSBTAG00000052977 There is one SNV and one deletion, which are significant mutations, in the vicinity of 2 - 3 kbp upstream. g.89242713A>G had a low p - value of 4.01x10E - 7.

[0074] The ENSBTAG00000053722 gene has a Transcript ID of ENSBTAT00000068858.1 and is located on the X chromosome in the region of 89,289,437 - 89,293,543 on the Forward strand. ENSBTAG00000053722 is 31 8bp, 105aa, and is speculated to have functions such as DZF domain - containing protein, double - stranded RNA binding, and single - stranded RNA binding, but the details are unknown. The significant SNV g.89289439G>A has a p - value of 1.52x10E - 6, causing the start codon ATG to become ATA, resulting in a non - synonymous substitution. Due to the non - synonymous substitution in the start codon, it was assumed that the expected protein would not be expressed. Therefore, the SNV of g.89289439G>A on the X chromosome was extremely powerful as an indicator for determining mastitis risk.

[0075] 〔Example 3〕Inspection using a DNA marker In Example 3, a test method for determining mastitis risk was implemented using some of the mastitis risk - determining DNA markers identified in Examples 1 and 2. As an example, the method of determining the genotype of the mastitis risk - determining DNA marker using Sanger sequencing of PCR amplification products is described. The test cows were, as in Example 1, 25 cows without a history of clinical mastitis (unrelated group) and 25 cows with recurrent clinical mastitis (recurrent group) each. ​​​In this example, the following nine types of DNA markers for determining mastitis risk were examined.

[0076] [Table 4]

[0077] In this example, PCR was performed using KOD FX Neo (Toyobo Co., Ltd.) to obtain a PCR amplification product. The PCR amplified products were amplified using ExoSAP-IT Express (Thermo Fisher Scientific). SupreDye v3.1 Cycle Sequencing Kit (MS Techno Co., Ltd.) was used. The DNA was analyzed using a 3500xL Genetic Analyzer (Applied Biosystems). The sequences were sequenced and the genotypes were identified, and the results are shown in Table 5.

[0078] [Table 5] TIFF0007690175000008.tif244155TIFF0007690175000009.tif223103

[0079] As shown in Table 5, among the mastitis risk determination DNA markers, g.88209479A>G on the X chromosome For a given SNV number 2, the genotype frequency is 21 were WT homozygous, 21 were heterozygous, and 21 were mutant homozygous. On the other hand, in the recurrence group, the reference homozygous type (WT homo) was There were 5 cases of genotype, 20 cases of heterozygotes and 20 cases of mutant homozygotes. That is, when only this SNV number 2 is used as the mastitis risk determination DNA marker, WT homo has a low mastitis risk, and when Hetero and Mutant homo are determined to have a high mastitis risk, in the non-related group, 21 out of 25 (84%) were correctly predicted, and in the recurrence group, 20 out of 25 (80%) were correctly predicted. Combined, the correct answer rate was 82%, indicating that the mastitis risk can be determined with a high probability. .

[0080] In this example, when using 9 types of mastitis risk determination DNA markers and setting the reference homozygous type to 0 points, the heterozygous type to 1 point, and the mutant homozygous type to 2 points, the average ± standard deviation (minimum value to maximum value) of the total score of these 9 types of DNA markers was 3.2 ± 3.7 ( 0 to 11) in the non-related group and 10.4 ± 2.5 (5 to 15) in the recurrence group, showing a significant difference. Here, when determining that the total score is less than 9 points indicates a low mastitis risk and 9 points or more indicates a high mastitis risk, in the non-related group, 22 out of 25 (88%) were correctly predicted, and in the recurrence group, 22 out of 25 (88%) were correctly predicted. Combined, the correct answer rate was 88%, indicating that the mastitis risk can be determined with a high probability. Also, for the 9 types of mastitis risk determination DNA markers used in this example, when using SNV number 1 and SNV number 2 alone, the detection rates were 0.88 and 0.8 respectively, and the false positive rates were 0

[0081] .2 and 0.16 respectively. Also, from the results shown in Table 5, by testing the individuals determined to be heterozygous by testing SNV number 7 among the 9 types of mastitis risk determination DNA markers with SNV number 9, it was revealed that the detection rate was 88% and the false positive rate could be suppressed to almost 0. Similarly, from the results shown in Table 5, among the 9 types of mastitis risk determination DNA markers, by testing SNV number 1 and determining it to be hetero . Among the 9 types of mastitis risk determination DNA markers, by testing the individuals determined to be heterozygous by testing SNV number 7 and performing the test of SNV number 9, it was revealed that the detection rate was 88% and the false positive rate could be suppressed to almost 0. Similarly, from the results shown in Table 5, among the 9 types of mastitis risk determination DNA markers, by testing SNV number 1 and determining it to be hetero and performing the test of SNV number 9 on the individuals determined to be heterozygous by testing SNV number 1 among the 9 types of mastitis risk determination DNA markers shown in Table 5, By performing the test of SNV No. 9 on the individuals determined as Ro, it was revealed that the detection rate was 80% and the false positive rate could be suppressed to 0. Furthermore, from the results shown in Table 5, by performing the test of SNV No. 9 on the individuals determined as heterozygous by testing SNV No. 2 among the nine kinds of mastitis risk determination DNA markers, it was revealed that the detection rate was 80% and the false positive rate could be suppressed to 4%.

[0082] <Sequence Listing> SEQUENCE LISTING <110> Tokyo University of Agriculture and Technology Hamamatsu University School of Medicine National Federation of Agricultural Cooperative Associations <120> DNA markers used to determine the risk of developing mastitis and the method for determining the risk of mastitis using the same <130> P24-0122 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 201 <212> DNA <213> Bos taurus <400> 1 taaagcatca ctttatccat gagtatttaa taagagccaa ctttcttttt taggtatcct 60 tttaatagag tgtgaaggtg gaggaaaaac cttattgaag matcattctc ttaatggaat 120 agttttcaga aaaaaatttt ctctgataag aattttataa gtgatactca ttaatgttca 180 ttatagataa catgttttct c 201 <210> 2 <211> 201 <212> DNA <213> Bos taurus <400> 2 acagcccttt tagccgcgat ggggaaggag aagatggagc agaaatatta gacaatgtcc 60 tttccctcaa gaatttttag aacttggtga ttgaaacagg rcaattacag gtgaaaagaa 120 atccaagcac agatgtcagt ttatgattat gacttcacac tttgtgcaca tggggtgtag 180 cagtggaaca aagtgtgaca g 201 <210> 3 <211> 201 <212> DNA <213> Bos taurus <400> 3 ttccaaagat attttagttt acatttctgt tactaagagt agatttaatc atctttttat 60 atatttaagg gtcatttgta tgtctttttc taaaatattt kttcagagaa tgtactgcct 120 atgttttcct ctaagagctt tatagttttg ggcattacat ttaagtctgt aagccatttt 180 gagtttattt ttgtgcatgg t 201 <210> 4 <211> 201 <212> DNA <213> Bos taurus <400> 4 taatattaag agtttccttt attacataga tctttaattt ttagatgagt cttttaatga 60 tatgaattaa attaattatg atattaattt attatggtat rtatgatatt aaattatgaa 120 ttaattatga tatgaattaa agattttatc tttaatcaaa tggctgtcca gttgtccaaa 180 aagcatttat taaatgtttt t 201 <210> 5 <211> 201 <212> DNA <213> Bos taurus <400> 5 agaatcagtt tgctttataa caccatacat aaaaatgaac acaaaatgga ctaaagactt 60 aaatgtaaaa catgtaagac gtgaaactat aaaaatccta raagaaaaaa tagacagtat 120 gctctttggc atctgtctta gcaatatttt tttttgccta tgtctcctca gataagggaa 180 acaagagcaa atatttaaaa a 201 <210> 6 <211> 201 <212> DNA <213> Bos taurus <400> 6 tttcaatttt atacaatatg tgctatagaa gaaaggagag acattactac actctcagga 60 tggtagaaca taaatataga aagagccagg gacttttttt waaaattatt tatttatttt 120 aattggagtc taattacttt acaatattta gtggtttttg ccatacattg acatgaatca 180 gccaagggtg tacatgtgtt c 201 <210> 7 <211> 201 <212> DNA <213> Bos taurus <400> 7 attagtgaaa atcctttggg ttgcaaggag cagaaaatta ttttctaggt tggctcaatg 60 gaaaaggaag ttcatgatct cagagcattt gtaatatctg matcactttc cctccctctg 120 gtagattact cctgtcaaaa taaaaattga tttattatta ttatttaaaa aacaaacaga 180 aaaaaaacct tctttttgcc t 201 <210> 8 <211> 201 <212> DNA <213> Bos taurus <400> 8 tagattcatt atttggaaat tgggtaagca gaggcaaata atgaaaccgt tatcctgtgt 60 gtggcacatg aactataaca ttggttacca aacagtggat ragggaagtg tctcatcaga 120 gaagaatttc atctaataaa tgaagaagcg tagatagaat gaaaatacct ccattttcaa 180 cctcaaatga cacaagcact g 201 <210> 9 <211> 201 <212> DNA <213> Bos taurus <400> 9 tactgatgct ctaaactagc aaaataaaaa gtaagttttg tttttatagc aatgatgcat 60 ccatcaatat aaaatttgtt acactctagc ccttacacag ratggaggga tggtcatgga 120 aacacagatt gtcattggag taggaaaaaa aaaaacaaaa cctaaacttc ctgaataggg 180 caaattaata tatgcagggg c 201 <210> 10 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 10 attgcctttt cactggaact gt 22 <210> 11 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 11 tacccagaag gcctttttct ct 22 <210> 12 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 12 ttttcttgag tgaagcattc ca 22 <210> 13 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 13 tttcctttcc acactgtcac ac 22 <210> 14 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 14 gcatggatca caacagatag ga 22 <210> 15 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 15 acacttccta acaccatgca ca 22 <210> 16 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 16 ccccttgatc atgtttgaat tt 22 <210> 17 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 17 tggcacttga atgaacaaac ta 22 <210> 18 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 18 acattgcagg tggattcctt ac 22 <210> 19 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 19 aggttgcatt ttcattttgt tg 22 <210> 20 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 20 aaaaggatat tgaggacacc cc 22 <210> 21 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 21 atagatcacc agtccaggtt cg 22 <210> 22 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 22 tcagtgggca taaaacagaa ga 22 <210> 23 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 23 tgtccccata gggagtaaga aa 22 <210> 24 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 24 agacctcatg gtcgctatga tt 22 <210> 25 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 25 cttctaattt ggcaagaatg gc 22 <210> 26 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 26 cgtccaggtt gtcagaaata ca 22 <210> 27 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 27 ctcaactgtt gtgtggttga ca 22

Claims

1. A step of collecting a biological sample from a subject animal, a bovine; A step of identifying the genotype of a mastitis risk determining DNA marker in genomic DNA contained in the biological sample, the mastitis risk determining DNA marker being a 101st base mutation or rs208854668 in the base sequence shown in SEQ ID NO: 2; and determining the risk of mastitis in a test animal based on the genotype of the mastitis risk determining DNA marker; When the genotype of the mastitis risk determination DNA marker is a heterozygote or a homozygote of a minor allele, the test animal is determined to have a high risk of mastitis. Methods for determining mastitis risk.

2. The method for determining mastitis risk described in claim 1, wherein the 101st base mutation in the base sequence of SEQ ID NO: 2 or rs208854668 has a major allele of A and a minor allele of G.

3. The mastitis marker is The nucleic acid sequence further includes at least one of the following base mutations located in the region sandwiched between the base sequence of SEQ ID NO: 2 and the base sequence of SEQ ID NO: 8: a base mutation at the 101st position in each of the base sequences of SEQ ID NO: 3 to 8, or rs381839020, rs385943123, rs109551898, rs137152785, rs110688314, and rs135732164; and a base mutation at the 101st position in the base sequence shown in SEQ ID NO: 9, or rs110369890. The method for determining mastitis risk according to claim 1.

4. The 101st base mutation or rs381839020 in the base sequence of SEQ ID NO: 3 has a major allele of G and a minor allele of T; the 101st base mutation or rs385943123 in the base sequence of SEQ ID NO: 4 has a major allele of A and a minor allele of G; the 101st base mutation or rs109551898 in the base sequence of SEQ ID NO: 5 has a major allele of A and a minor allele of G; the 101st base mutation or rs137152785 in the base sequence of SEQ ID NO: 6 has a major allele of A and a minor allele of G; The mastitis risk assessment method described in claim 3, characterized in that the major allele is A and the minor allele is T, the 101st base mutation or rs110688314 in the base sequence of SEQ ID NO: 7 has a major allele of C and a minor allele of A, the 101st base mutation or rs135732164 in the base sequence of SEQ ID NO: 8 has a major allele of G and a minor allele of A, and the 101st base mutation or rs110369890 in the base sequence of SEQ ID NO: 9 has a major allele of A and a minor allele of G.

5. The mastitis risk assessment method described in claim 1, characterized in that in the step of identifying the genotype of the mastitis risk assessment DNA marker, a nucleic acid fragment containing the mastitis risk assessment DNA marker is amplified by a nucleic acid amplification method using genomic DNA derived from the test animal, cow, as a template.

6. The method for determining a risk of mastitis according to claim 5, characterized in that in the step of identifying the genotype of the mastitis risk determining DNA marker, the base sequence of the nucleic acid fragment is determined and the genotype is identified.

7. A primer set that can be used to amplify a nucleic acid fragment containing a mastitis risk assessment DNA marker that is the 101st base mutation or rs208854668 in the base sequence shown in SEQ ID NO:

2.

8. The primer set according to claim 7, comprising a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO:12 and an oligonucleotide containing the base sequence shown in SEQ ID NO:

13.

9. The primer set according to claim 7 can be used to amplify a nucleic acid fragment containing a DNA marker for determining mastitis risk, which is located in a region sandwiched between the base sequence of SEQ ID NO: 2 and the base sequence of SEQ ID NO: 8, and which is composed of at least one base mutation selected from the 101st base mutation or rs381839020, rs385943123, rs109551898, rs137152785, rs110688314 and rs135732164 in the base sequences of SEQ ID NO: 3 to 8; and the 101st base mutation or rs110369890 in the base sequence shown in SEQ ID NO:

9.

10. The primer set according to claim 9 further includes at least one combination of the following: a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 14 and an oligonucleotide having a base sequence shown in SEQ ID NO: 15; a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 16 and an oligonucleotide having a base sequence shown in SEQ ID NO: 17; a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 18 and an oligonucleotide having a base sequence shown in SEQ ID NO: 19; a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 20 and an oligonucleotide having a base sequence shown in SEQ ID NO: 21; a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 22 and an oligonucleotide having a base sequence shown in SEQ ID NO: 23; a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 24 and an oligonucleotide having a base sequence shown in SEQ ID NO: 25; and a combination of an oligonucleotide having a base sequence shown in SEQ ID NO: 26 and an oligonucleotide having a base sequence shown in SEQ ID NO: 27.

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