JP7690175B2 - DNA marker for use in determining risk of developing mastitis and method for determining risk of mastitis using the same - Google Patents

Description

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

Mastitis occurs when pathogenic microorganisms invade the udder, grow, and stimulate the milk ducts and mammary glands, causing inflammation. Mastitis is also known as an "occupational disease of dairy cows," and is one of the most difficult diseases of dairy cows to treat worldwide. Although various countermeasures have been proposed, progress in eradicating the disease has been limited (Non-Patent Document 1). When inflammation occurs due to mastitis, blood flow increases and vascular permeability increases,
White blood cells migrate from the blood and migrate into the milk. Also, epithelial cells damaged by inflammation are shed and migrate into the milk. For this reason, an increase in the number of somatic cells, such as white blood cells and shed epithelial cells, contained in milk is used worldwide as an indicator of mastitis infection (Non-Patent Document 2).

Patent Document 1 discloses the identification of a mastitis resistance gene (FEZL gene) closely related to bovine mastitis as a means for easily, reliably and quickly detecting/diagnosing bovine mastitis resistance. According to Patent Document 1, bovine mastitis resistance is diagnosed by identifying a genetic mutation in the FEZL gene. Furthermore, 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 α chain as a method for determining resistance or susceptibility to bovine mastitis. Furthermore, Patent Document 3 discloses a mutation in which 1 to 4 bp of cytosine (c) is inserted at position 1110 in the base sequence of the IGF1R gene of mastitis-resistant bovines, which is found in mastitis-susceptible bovines. Patent Document 3 discloses a method for diagnosing mastitis resistance using the mutation.

Furthermore, Patent Document 4 discloses the following genetic markers that indicate mastitis resistance in cattle:
Polymorphic microsatellite markers C6orf93 and inra084 on bovine chromosome BTA9, bovine chromosome BT
Polymorphic microsatellite markers HELMTT43 and BM3501 on A11 are disclosed. Furthermore, Patent Document 5 discloses a number of SNPs as genetic markers associated with a trait indicating mastitis resistance in cattle.

JP 2006-67928 A JP 2007-295844 A JP 2008-72946 A Special Publication No. 2009-525733 Special Publication No. 2015-526099

Journal of Dairy Science Vol. 100 No. 12, 2017 LIAJ News No.122, pp1-7

As mentioned above, although genetic markers for diagnosing bovine mastitis resistance/susceptibility have been known in the past, the diagnostic accuracy is not sufficient, and effective factors for identifying mastitis risk have not been identified. At present, diagnosis using specific markers is not being performed, and it has not yet been put to practical use.

In view of the above-mentioned circumstances, the present invention aims to provide a DNA marker that can identify mastitis risk with high accuracy and a method for determining mastitis risk using the DNA marker.

In order to achieve the above-mentioned objective, the inventor conducted intensive research and conducted a multifaceted evaluation through genomics analysis of the differences between populations that repeatedly develop mastitis and healthy populations, identifying DNA mutations that enable the identification of animals at high risk of developing mastitis, thereby completing the present invention.
The present invention encompasses the following:

(1) collecting a biological sample from a test animal;
A step of identifying the genotype of a DNA marker for determining a risk of mastitis in genomic DNA contained in the biological sample, wherein the DNA marker for determining the risk of mastitis is at least one of the following base mutations: a base mutation at position 101 in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to said base mutation; a base mutation at position 101 in a base sequence selected from the group consisting of SEQ ID NO: 2 to 8 located in a region sandwiched between the base sequences of SEQ ID NO: 2 and SEQ ID NO: 8 or a base mutation linked to said base mutation; and a base mutation at position 101 in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation;
and determining the mastitis risk of a test animal based on the genotype of a mastitis risk determining DNA marker.

(2) 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 mastitis risk.
1) A method for determining mastitis risk as described above.

(3) The 101st base mutation in the base sequence of SEQ ID NO: 1 is a major allele.
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 A and the minor allele is G, and the 101st base mutation in the base sequence of SEQ ID NO: 3 is G and the minor allele is T,
The 101st base mutation in the base sequence of SEQ ID NO: 4 is a major allele A and a minor allele G, the 101st base mutation in the base sequence of SEQ ID NO: 5 is a major allele A and a minor allele G, the 101st base mutation in the base sequence of SEQ ID NO: 6 is a major allele A and a minor allele T, the 101st base mutation in the base sequence of SEQ ID NO: 7 is a major allele C and a minor allele A, the 101st base mutation in the base sequence of SEQ ID NO: 8 is a major allele G and a minor allele A, and the 101st base mutation in the base sequence of SEQ ID NO: 9 is a
A method for determining mastitis risk described in (1), characterized in that the first base mutation has a major allele of A and a minor allele of G.

(4) The mastitis risk assessment method described in (1) is characterized in that in the step of identifying the genotype of the mastitis risk assessment DNA marker, the genotype is identified for the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to said base mutation, the 101st base mutation in the base sequence shown in SEQ ID NO: 2 or a base mutation linked to said base mutation, the 101st base mutation in the base sequence shown in SEQ ID NO: 7 or a base mutation linked to said base mutation, and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation.

(5) In the step of identifying the genotype of the mastitis risk determining DNA marker, a combination of the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to said base mutation and the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation, a combination of the 101st base mutation in the base sequence shown in SEQ ID NO: 7 ...2 or a base mutation linked to said base mutation,
A method for assessing mastitis risk described in (1), characterized in that the genotype is identified for a combination of a base mutation at position 01 or a base mutation linked to said base mutation and a base mutation at position 101 in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation.

(6) A method for assessing mastitis risk described in (1), characterized in that in the step of identifying the genotype of the mastitis risk-assessing DNA marker, a nucleic acid fragment containing the mastitis risk-assessing DNA marker is amplified by a nucleic acid amplification method using genomic DNA derived from the test animal as a template.

(7) A method for determining mastitis risk described in (6), 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.

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

(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,
The primer set according to (8) is characterized in that it is at least one combination of the following: 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: 18 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 19; a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 20 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 21; a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 22 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 23; a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 24 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 25; and a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 26 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 27.

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

FIG. 1 is a characteristic diagram showing the results of plotting p-values for each region of the X chromosome.

The present invention will be described in detail below.
The DNA marker for determining a risk of mastitis according to the present invention is a base mutation at a specific position on the genome that can be used to determine the risk of developing mastitis. Here, the base mutation serving as the DNA marker for determining a risk of mastitis means a single nucleotide variant (SNV).
A single nucleotide variant (SNV) is a single nucleotide difference (substitution mutation) in a genome sequence. A single nucleotide polymorphism (SNP) is a mutation occurring at a frequency of 1% or more in a population.
This includes alleles (e.g., rphisms, SNPs).

1. DNA markers for determining mastitis risk DNA markers for determining mastitis risk can be identified based on the bovine reference genome. The bovine reference genome is ARS-UCD1.2 (Hereford, 2018)
April 11, 2011, https://www.ncbi.nlm.nih.gov/assembly/GCF_002263795.1/) can be used. In the following explanation, the mastitis risk determination DNA markers will be described based on the base sequence of the reference genome in cattle.
The NA marker can be identified not only in cattle but also in any animal including humans. That is, when the genomic base sequence (e.g., the base sequence of the reference genome) of the animal to be tested is known, the mastitis risk determining DNA marker in the animal can be identified based on the genomic base sequence of the animal to be tested.

The DNA marker for determining a mastitis risk according to the present invention is a nucleotide sequence represented by SEQ ID NO:1.
At least one of the following base mutations: a base mutation at position 01 or a base mutation linked to said base mutation; a base mutation at position 101 or a base mutation linked to said base mutation in a base sequence selected from the group consisting of SEQ ID NOs: 2 to 8, located in a region sandwiched between the base sequences of SEQ ID NOs: 2 and 8; and a base mutation at position 101 or a base mutation linked to said base mutation in the base sequence shown in SEQ ID NO: 9.

Here, the 101st base mutation in the base sequence shown in SEQ ID NO: 1 is a mutation site located at 25087716 in chromosome 15 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 C. This base mutation is also listed in the database (
For example, they are registered in the Ensembl.

Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 2 is a mutation site located at 88209479 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. This base mutation is also registered in a database (e.g., Ensembl) as a SNP identified by the rs number: rs208854668.

Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 3 is a mutation site located at 89239121 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 G and the minor allele is T.
In addition, this base mutation is registered in a database (eg, Ensembl) as a SNP identified by rs number: rs381839020.

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. This base mutation is also listed in the database (
For example, they are registered in the Ensembl.

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. This base mutation is also listed in the database (
For example, they are registered in the Ensembl.

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. This base mutation is also listed in the database (
For example, they are registered in the Ensembl.

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 in which the major allele is C and the minor allele is A. This base mutation is also listed in the database (
For example, they are registered in the Ensembl.

Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 8 is a mutation site located at 89289439 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 G and the minor allele is A. This base mutation is also registered in a database (e.g., Ensembl) as a SNP identified by rs number: rs135732164.

Furthermore, the 101st base mutation in the base sequence shown in SEQ ID NO: 9 is a mutation site located at 12219701 in chromosome 24 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. This base mutation is also registered in a database (e.g., Ensembl) as a SNP identified by rs number: rs110369890.

As described above, the DNA markers for determining mastitis risk according to the present invention are a base substitution present in a predetermined region of chromosome 15, a base substitution present in a predetermined region of chromosome X, and a base substitution present in a predetermined region of chromosome 24.

Here, the predetermined region of chromosome 15 means a region containing a nucleotide mutation linked to the above-mentioned mutation site located at 25087716 (the 101st position in SEQ ID NO: 1, rs110036757). That is, the mastitis risk determination DNA marker according to the present invention contains a mutation site (single nucleotide variation (SNV)) that is in linkage disequilibrium with the mutation site located at 25087716 (the 101st position in SEQ ID NO: 1, rs110036757).

The predetermined region of the X chromosome refers to the region sandwiched between the base sequence of SEQ ID NO: 2 and the base sequence of SEQ ID NO: 8. This region includes the above-mentioned mutation site located at 88209479 (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, rs385943123), and the mutation site located at 88319202 (the 101st position in SEQ ID NO: 6, rs385943123).
rS109551898), the mutation site located at 88700655 (the 101st position in SEQ ID NO: 6, r
s137152785), the mutation site located at 89067208 (the 101st position in SEQ ID NO: 7, rs110688
314) and the mutation site located at 89289439 (position 101 in SEQ ID NO: 8, rs135732164)
Therefore, the predetermined region of the X chromosome is a 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 variation (SNV)) that is in linkage disequilibrium with any one of these mutation sites.

More specifically, the predetermined region of the X chromosome includes a mutation site (major allele T, minor allele A) located at 88209483 on the X chromosome as a mutation site (single nucleotide variation (SNV)) linked to 88209479 on the X chromosome. Also, the predetermined region of the X chromosome includes a mutation site (single nucleotide variation (SNV)) linked to 89239121 on the X chromosome as a mutation site (single nucleotide variation (SNV)) linked to 89239121 on the X chromosome.
The chromosomes include 235533 (major allele T, minor allele C), 89237555 (major allele G, minor allele A), and 89237780 (major allele G, minor allele A). In addition, the X chromosome includes a mutation site (single base mutation (S
89259615 (major allele C, minor allele T) on the X chromosome, 8926967
7 (major allele T, minor allele C) and 89280192 (major allele C, minor allele G). Furthermore, the predetermined region of the X chromosome includes 88313029 (major allele C, minor allele G), 88338299 (major allele T, minor allele C) and 883390 (major allele T, minor allele C) on the X chromosome as mutation sites (single nucleotide variation (SNV)) linked to 88319202 on the X chromosome.
19 (major allele T, minor allele G). Furthermore, a specific region of the X chromosome contains X chromosome 88700655 as a mutation site (single nucleotide variation (SNV)) linked to 88700655 on the X chromosome.
The predetermined region of the X chromosome includes 88664079 (major allele T, minor allele C) in the X chromosome. Furthermore, the predetermined region of the X chromosome includes 89073474 (major allele G, minor allele C) in the X chromosome as a mutation site (single nucleotide variation (SNV)) linked to 89067208 in the X chromosome. Furthermore, the predetermined region of the X chromosome includes 89278639 (major allele G, minor allele A) in the X chromosome as a mutation site (single nucleotide variation (SNV)) linked to 89289439 in the X chromosome.

Furthermore, the predetermined region of chromosome 24 refers to a region in which a base mutation linked to the above-mentioned mutation site located at 12219701 (the 101st position in SEQ ID NO: 9, rs110369890) exists. In other words, the mastitis risk assessment DNA marker of the present invention contains a mutation site (single nucleotide variation (SNV)) that is in linkage disequilibrium with the mutation site located at 12219701 (the 101st position in SEQ ID NO: 9, rs110369890).

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.
It does not mean that the technical scope of the DNA marker is limited to cattle (i.e., mastitis risk determination DNA marker in cattle), but it can be identified in any mammal including human, and the mastitis risk determination DNA marker can be applied to any mammal. For example, mastitis risk determination DNA markers can be identified in buffalo, goat, sheep, pig, horse, yak, mouse, rat, guinea pig, hamster, rabbit, dog, cat, monkey, and human. To identify a mastitis risk determination DNA marker in a mammal other than cattle, it can be identified as a region having high homology with the base sequence shown in SEQ ID NO: 1 to 9 by homology search based on the base sequence shown in SEQ ID NO: 1 to 9 described above. In this case, as long as it can be identified as a highly homologous sequence such as a homolog, paralog, or ortholog, the position on the chromosome of the genome does not matter.

The degree of homology is not particularly limited, and can be, for example, 70% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more with respect to the base sequences of SEQ ID NOs: 1 to 9. A region having such high homology can be identified in the genome of a mammal other than cattle, and a base mutation contained in the identified region can be used as a DNA marker for determining the risk of mastitis in the mammal other than cattle. When identifying a DNA marker for determining the risk of mastitis in a mammal other than cattle, the entire length of SEQ ID NOs: 1 to 9 does not need to be used for the homology search, and only the upstream or downstream of the base mutation site may be used, or for example, 200 bases, 50 bases, 20 bases, or 10 bases upstream and/or downstream may be used. In addition, the reference genome is updated as data is accumulated, and the position for identifying the genomic region may change, but the region can be identified by using the sequence shown in the SEQ ID NO.

For example, buffalo (Bubalus bubalis breed Mediterranean chromosome X, ASM312139v1, water buffalo)
hole genome shotgun sequence, NC_037569.1), goat (Capra hircus breed San Clemen
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.
For 1), a homology search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) can be performed to identify DNA markers for determining mastitis risk in buffaloes, goats, and sheep.

Specifically, for 25087716 (the 101st position in SEQ ID NO: 1) on chromosome 15, 59845877 on chromosome 16 in buffalo was identified, and 57 on chromosome 15 in goats was identified.
243975 on chromosome 15 in sheep and 24820077 on chromosome 15 in sheep have been identified and can be used as DNA markers for determining mastitis risk in buffaloes, goats and sheep, respectively.

In addition, 88209479 (SEQ ID NO: 2) was used as a DNA marker for determining mastitis risk located on the X chromosome.
For the 101st position in SEQ ID NO:3, 54070123 in buffalo, 15916117 in goat, and 54912361 in sheep were identified, and these can be used as mastitis risk assessment DNA markers for buffalo, goat, and sheep, respectively.
These can be used as DNA markers for determining the risk of mastitis in buffalo, goat and sheep, respectively. Furthermore, 89242713 (1 in SEQ ID NO: 4) is a DNA marker for determining the risk of mastitis located on the X chromosome.
For the 88319202 (101st position in SEQ ID NO: 5), 53045801 in buffalo and 14829743 in goats were identified, and these can be used as mastitis risk assessment DNA markers for buffalo and goats, respectively. Furthermore, for the 88319202 (101st position in SEQ ID NO: 5) located on the X chromosome, 53958808 in buffalo and 15774295 in goats were identified.
and 54778777 in sheep have been identified, which can be used as DNA markers for determining the risk of mastitis in buffalo, goat, and sheep, respectively. Furthermore, as a DNA marker for determining the risk of mastitis located on the X chromosome, 88700655 (the 101st position in SEQ ID NO: 6) is
53590316 in buffalo has been identified, and this can be used as a DNA marker for determining the risk of mastitis in buffalo. Furthermore, as a DNA marker for determining the risk of mastitis located on the X chromosome for 89067208 (the 101st position in SEQ ID NO: 7), 53220380 in buffalo, 14849521 in goats, and 54011154 in sheep have been identified, and these can be used as DNA markers for determining the risk of mastitis in buffalo, goats, and sheep, respectively. Furthermore, as a DNA marker for determining the risk of mastitis located on the X chromosome for 89289439 (the 101st position in SEQ ID NO: 8), 53002464 and 53052853 in buffalo, and 14787290 and 150674 in goats have been identified.
37, 53754307 and 53805622 in sheep were identified, which can be used as DNA markers for determining mastitis risk in buffalo, goat and sheep, respectively.

Furthermore, for 12219701 (the 101st position in SEQ ID NO: 9) located on chromosome 24, 49964421 on chromosome 22 in buffalo was identified, and 1237 on chromosome 24 in goats was identified.
6430 have been identified and can be used as DNA markers for determining mastitis risk in buffaloes and goats, respectively.

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

The biological sample collected from the subject animal is not particularly limited, and examples thereof include hair roots, skin, milk, saliva, nasal secretions, blood, meat, organs, sperm, eggs, fertilized eggs, etc.
In consideration of animal welfare, it is preferable to use biological samples that can be collected without causing pain to individuals.

When identifying the genotype of the mastitis risk determining DNA marker in the genomic DNA contained in a biological sample, the genomic DNA may be extracted from the biological sample first. The extraction of the genomic DNA can be performed by applying a conventional method for recovering polynucleotides. For example, a method using phenol or chloroform, or a method using sodium iodide can be used.
The genotype of the mastitis risk determining DNA marker in the genomic DNA contained in the biological sample may be identified without extracting the genomic DNA contained in the biological sample. For example, according to the direct PCR method, the genotype of the mastitis risk determining DNA marker in the genomic DNA contained in the cell can be identified without extracting the genomic DNA.
PCR can also be performed using A as a template to identify the genotype of the mastitis risk determining DNA marker.

One method for determining the genotype of a mastitis risk determining DNA marker is to determine the base sequence of a region containing the mastitis risk determining DNA marker. Any conventionally known method can be selected for determining the base sequence. Examples include Sanger sequencing, microarray, next-generation sequencing, real-time PCR, PCR-RFLP, mass spectrometry, LAMP, and nanopore sequencing. When using a method involving a nucleic acid amplification reaction among these various methods, a pair of primers is designed to amplify a nucleic acid fragment containing a mastitis risk determining DNA marker.

A pair of primers for amplifying a nucleic acid fragment containing a mastitis risk assessment DNA marker is
When the base sequence of the genomic DNA of the test animal is known, the pair of primers can be designed based on the base sequence. When the base sequence of the genomic DNA of the test animal is not known, the pair of primers may be designed after determining the base sequence of the genomic DNA, or after determining the base sequence near the mastitis risk determining DNA marker.

When designing a primer, the primer may be, but is not limited to, 20 to 1000 bases, 20 to 800 bases, 20 to 600 bases, 20 to 400 bases, 20 to 200 bases, 20 to 300 bases, 20 to 400 bases, 20 to 500 bases, 20 to 600 bases, 20 to 800 bases, 20 to 900 bases, 20 to 1000 bases, 20 to 120 bases, 20 to 200 bases, 20 to 300 bases, 20 to 400 bases, 20 to 500 bases, 20 to 600 bases, 20 to 700 bases, 20 to 800 bases, 20 to 900 bases, 20 to
A pair of primers can be designed to generate a nucleic acid fragment of 100 bases or 20 to 50 bases. When designing a pair of primers, the length of the primers can be, for example, 17 to 25 mer, and the GC content can be 40 to 60%, preferably 45 to 55%. In addition, it is preferable that the high Tm value of the pair of primers does not differ greatly, and the difference in Tm value is preferably within 2°C, for example, within 1°C.

Specifically, based on the bovine reference genome sequence,
A pair of primers (SEQ ID NOs: 10 and 11) for amplifying a nucleic acid fragment containing 087716 (the 101st position in SEQ ID NO: 1) can be designed. In addition, based on the base sequence of the bovine reference genome, 88209479 (the 101st position in SEQ ID NO: 2) in the X chromosome can be designed.
A pair of primers (SEQ ID NOs: 12 and 13) for amplifying a nucleic acid fragment containing 89239121 (the 101st position in SEQ ID NO: 3) in the X chromosome can be designed based on the base sequence of the bovine reference genome. A pair of primers (SEQ ID NOs: 14 and 15) for amplifying a nucleic acid fragment containing 89242713 (the 101st position in SEQ ID NO: 4) in the X chromosome can be designed based on the base sequence of the bovine reference genome.
) can be designed. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 18 and 19) for amplifying a nucleic acid fragment containing 88319202 (101st in SEQ ID NO: 5) in the X chromosome can be designed. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 20 and 21) for amplifying a nucleic acid fragment containing 88700655 (101st in SEQ ID NO: 6) in the X chromosome can be designed. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 22 and 23) for amplifying a nucleic acid fragment containing 89067208 (101st in SEQ ID NO: 7) in the X chromosome can be designed. Furthermore, based on the base sequence of the bovine reference genome, a pair of primers (SEQ ID NOs: 24 and 25) for amplifying a nucleic acid fragment containing 89289439 (101st in SEQ ID NO: 8) in the X chromosome can be designed. Furthermore, a pair of primers (SEQ ID NOs: 26 and 27) can be designed for amplifying a nucleic acid fragment containing 12219701 (the 101st position in SEQ ID NO: 9) in chromosome 24 based on the base sequence of the bovine reference genome.

In addition, the method of determining the genotype of a DNA marker using a nucleic acid fragment containing a mastitis risk determining DNA marker is not limited to the above-mentioned method of determining a base sequence, and a method using a probe can be applied. In the method of identifying the genotype of a DNA marker using a probe, a major allele probe that specifically hybridizes to a major allele and a minor allele probe that specifically hybridizes to a minor allele are used. In this method, a form using a microarray in which the major allele probe and the minor allele probe are fixed on a substrate can be applied. The major allele probe and the minor allele probe may have a configuration in which only the base mutation of the mastitis risk determining DNA marker is different, or may have different lengths. The length of the major allele probe and the minor allele probe is not particularly limited, but
For example, it may be 10 bases, 20 bases, 30 bases, 40 bases, or 50 bases. Furthermore, the major allele probe and the minor allele probe may be designed so that the bases of the mastitis risk determination DNA marker are located approximately in the center of the entire length, or may be designed to be biased toward the 5' end or the 3' end.

In the step of determining the risk of developing mastitis, the risk of mastitis is determined from the genotype of the mastitis risk determining DNA marker. As described above, the mastitis risk determining DNA marker according to the present invention contains multiple base mutations. When determining the risk of mastitis, all of these multiple mastitis risk determining DNA markers may be used, or one of these mastitis risk determining DNA markers may be used. The number of mastitis risk determining DNA markers used to determine the risk of mastitis is not particularly limited, and may be one type, two types, three types,
There can be 4 types, 5 types, 6 types, 7 types, 8 types, 9 types, 10 types, or 11 types.

More specifically, among the above-mentioned mastitis risk determination DNA markers, the risk of mastitis may be determined using the 101st base mutation in the base sequence shown in SEQ ID NO: 1 to 9 or a base mutation linked to the base mutation (i.e., 9 types of base mutations), or the risk of mastitis may be determined using one, two, three, four, five, six, seven, or eight types of base mutations among these 9 types of base mutations. In particular, among these 9 types of base mutations, it is preferable to determine the risk of mastitis using 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 a 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. By using these base mutations, the detection rate of mastitis can be extremely high and the incidence rate of false positives can be extremely low.

More preferably, the risk of mastitis is determined by combining the 101st base mutation in the base sequence shown in SEQ ID NO: 1 or a base mutation linked to said base mutation with the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation, or by combining the 101st base mutation in the base sequence shown in SEQ ID NO: 7 or a base mutation linked to said base mutation with the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation, or by combining the 101st base mutation in the base sequence shown in SEQ ID NO: 2 or a base mutation linked to said base mutation with the 101st base mutation in the base sequence shown in SEQ ID NO: 9 or a base mutation linked to said base mutation. In this case as well, the detection rate of mastitis can be extremely high and the incidence of false positives can be extremely low.

Furthermore, mastitis risk can be assessed by determining whether there is a high possibility of developing mastitis as high risk or a low possibility of developing mastitis as low risk, or by using a numerical value or ranking to indicate the level of risk. For example, when assessing mastitis risk using a score, risk can be assessed in stages from 0 to 10, with 10 being the highest risk and 0 being the lowest risk. Different scores can also be defined depending on the genotype of the mastitis risk assessment DNA marker. For example, 0 points are assigned when the genotype of the mastitis risk assessment DNA marker is homozygous for the major allele, 1 point is assigned when it is heterozygous, and 0 points is assigned when it is homozygous for the minor allele.
The risk of mastitis can be defined as 2 points. When the genotypes of multiple mastitis risk determination DNA markers are used to determine the risk of mastitis, the risk of mastitis can be determined based on the total score. In addition, for example, when evaluating the risk of mastitis by ranking, five levels from A to E can be defined, with E being the highest risk and A being the lowest risk, and the risk can be evaluated in stages from A to E.

In addition, in a form in which multiple mastitis risk determination DNA markers are used, weighting may be performed in advance for each DNA marker to determine the risk of mastitis. In other words, weighting may be performed so that a genetic model or a mastitis risk determination DNA marker that contributes highly to mastitis risk is evaluated higher than other markers, thereby making it possible to determine the risk of mastitis with higher accuracy. If weighting is performed using a genetic model, it may be performed using any coefficient in the form of a recessive model, dominant model, additive model, synergistic model, or the like. For example, in the case of a recessive model, it is possible to consider that having an allele that is not a risk allele has the same effect, and only mutant homozygous types can be evaluated. In the case of a dominant model, it is possible to consider that having a risk allele has the same effect,
Heterozygotes and mutant homozygotes can be equally evaluated. In the additive model, the influence of having a risk allele is considered to increase additively, and mutant homozygotes can be evaluated as having twice the influence of heterozygotes. In the multiplicative model, the influence of having a risk allele is considered to increase synergistically, and mutant homozygotes can be evaluated as having a squared influence of heterozygotes. In addition, when determining using multiple DNA markers, weighting can be performed by combining different genetic models, such as recessive models for some and additive models for others. In addition, when weighting the contribution to mastitis risk, a higher coefficient can be set for mastitis risk determination DNA markers with a high contribution than for other DNA markers. For example, weighting can be performed by calculating the genotype frequency or allele frequency of each individual in a case-control study, and multiplying the value by the mutant homozygote or heterozygote according to the genetic model.

3. Animals to be Determined for Mastitis Risk The mastitis risk determination method using the above-mentioned DNA markers for determining mastitis risk can be applied to mammals including humans, or to mammals other than humans (non-human mammals). Examples of non-human mammals include cows, buffaloes, goats, sheep, pigs, horses, yaks, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, and monkeys, and animal individuals obtained as a result of mating using these individuals can also be used as the determination subject.

The above-mentioned mastitis risk determination DNA markers are identified based on the base sequence of genomic DNA, so the functioning of these mastitis risk determination DNA markers means that many cases of mastitis are likely to be hereditary diseases. In that case, if many individuals tend to have frequent mastitis, there is a possibility that individuals with a high risk of developing mastitis are prevalent in the field.
For example, in the case of cattle, the spread of artificial insemination technology and embryo transfer has led to the intensive use of specific individuals for breeding improvement, and it is assumed that hereditary diseases have spread rapidly. Therefore, animals that have been judged to be at risk of developing mastitis and animals obtained as a result of breeding using such individuals are highly valuable in reducing mastitis in the entire population.

By using the above-mentioned mastitis risk determination DNA markers to determine the risk of mastitis, non-human mammals can be used for individual management and breeding improvement, taking into account the risk of mastitis. For example, it is possible to determine that individuals determined to be at high risk will not be used for breeding improvement at that point. In addition, by carrying out more careful feeding management of high-risk individuals, measures to avoid mastitis can be efficiently taken. In addition, individuals determined to be at low risk can be actively produced as successor cows, thereby reducing the incidence of mastitis in the field in the future, and can contribute to efficient breeding improvement. In addition, by performing random testing and calculating the genotype frequency, it is possible to investigate the extent to which high-risk individuals are widespread in a population, which can be used as a reference for breeding improvement.

From the viewpoint of efficient breeding and improvement, the risk of mastitis can be rapidly reduced within a population by artificially breeding the semen and eggs of individuals determined to be at risk of developing mastitis or individuals born from mating using such individuals. For example, semen can be distributed in the form of ejaculated semen, frozen semen, or non-frozen diluted semen. Eggs can be distributed in the form of unfertilized or fertilized eggs in a fresh or frozen state. When distributing, they can be distributed by methods such as the straw method or pellet method.

The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[Example 1] DNA marker 1
In Example 1, a DNA marker for determining mastitis risk was selected as an index for determining mastitis risk. In this example, a case-control study was performed using whole genome sequencing.
A controlled study was conducted. The test cows were 25 each of cows with no history of clinical mastitis (free group) and cows with recurrent clinical mastitis (recurrent group). The free group consisted of cows that had not contracted mastitis within 610 days of lactation. The recurrent group consisted of cows that had contracted clinical mastitis three or more times within 305 days of lactation. The affected quarters differed depending on the cow. Details of the test cows are shown in Table 1. There were no significant differences between the free and recurrent groups in calving, number of milking days prior to calving, or number of dry days prior to calving, but the average 305-day somatic cell count was significantly higher in the recurrent group.

For 25 cattle from each of the unaffected and recurrent areas, whole blood was centrifuged to collect the buffy coat layer, and DNA was extracted by standard methods and whole genome sequencing was performed using NovaSeq6000 (Illumina, Inc.). Data of more than 100 billion base pairs was obtained, with an average depth of more than ×30 against a reference genome of approximately 2.7 billion base pairs (ARS-UCD1.2, Hereford breed, April 11, 2018 version). Data analysis was performed using CLC Genomics Workbench, and DNA was analyzed by comparing the unaffected and recurrent areas.
A mutation analysis was performed. The mutation analysis consisted of: (1) importing Illumina Fastq data, (2) quality checking and trimming of low-reliability data, (3) mapping to the reference genome, (4) removing PCR duplicates, (5) local realignment, and (6)
The steps were mutation detection, (7) annotation, and (8) filtering.

There were 43 single nucleotide variants (SNVs) compared to the reference genome.
0895 sites, 11871 sites of multi-nucleotide variants (MNV), and 11871 sites of deletions.
A total of 38,916 mutations (n), 41,664 insertions, and 2,843 replacements were identified. Of these, only homozygous mutations were detected in 1,568 SNVs, 62 MNVs, 208 deletions, 210 insertions, and 8 replacements. When the DNA mutations that could be candidates for mastitis risk were narrowed down by p-value, 20 SNVs (p<1x10E-3) and 10 MNVs (p<1x10E-3) were identified.
6 sites (p<1x10E-2), deletions at 20 sites (p<5x10E-3), insertions at 14 sites (p<5x10E-3),
There were 8 replacements (p<5x10E-2). These may not be identified as mutations that cause nonsynonymous substitutions, but in any case, they can be used as indicators for determining mastitis risk. In addition, since linkage disequilibrium occurs in the regions close to these mutations, there are also indicators for determining mastitis risk in the surrounding regions of the identified mutations, and they can be used as indicators for determining mastitis risk.

We further narrowed down the p-values of these mutations, including those with unknown zygosity, homozygosity, and heterozygosity, and identified the mutations with the lowest p-values in Table 2, with the p<1x10E-7 region frequently seen on the X chromosome. These mutation sites and the mutation sites in their surrounding regions were particularly useful indicators for determining mastitis risk.

[Example 2] DNA marker 2
In Example 2, SNV DNA markers for determining mastitis risk were selected from the candidates for mastitis risk determination DNA markers serving as indicators for determining mastitis risk described in Example 1. As in Example 1, 25 milking cows with no history of clinical mastitis (free group) and 25 milking cows with recurrent clinical mastitis (recurrent group) were used as test cows.

For SNVs, DNA markers that were candidates for DNA mutations that were risk factors for mastitis were narrowed down using the p-value calculated from the number of cattle with homozygous or heterozygous mutations for 25 cattle in each group.
62 DNA markers with p<4.8x10E-6 were identified (Table 3). The DNA markers were arranged in ascending order of p value, with 48 in the range 88209379-89369844 on the X chromosome and 23 in the range 89369844 on the X chromosome.
Four were identified in the range of 51376086-51627177 on chromosome 1, three in the range of 18570840-18580407 on chromosome 27, two in the range of 12219601-14118517 on chromosome 24, two in the range of 12406826-12407723 on chromosome 25, one in the range of 25087616-25087816 on chromosome 15, one in the range of 2036632-2036832 on chromosome 26, and one 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.

Furthermore, when p-values were plotted for each region of the X chromosome using SNVs, as shown by the arrow, there was a region in which SNVs with p<1x10E-6 were concentrated in the region 89067208-89369744 (
(Figure 1) Therefore, mutations in this region were particularly useful as indicators for determining mastitis risk.

Furthermore, when we investigated genes that affect mastitis risk using the Ensembl Genome Browser (https://www.ensembl.org), we found that the region of 89067208-89369744 on the X chromosome contains G
Three proteins were encoded: SPT2, ENSBTAG00000052977, and ENSBTAG00000053722. The GSPT2 gene has a transcript ID of ENSBTAT00000017703.5 and is located on the X chromosome at position 89,276,444.
It was present in the reverse strand in the region of ~89,278,960. G1 to S phase transition 2 (2410bp,
It encodes a Tr-type G domain-containing protein, GTPase activity, and G
Its functions, such as TP binding and translation release factor activity, are presumed, but it is an unreviewed protein. The significant SNV g.89279441 was located 500 bp upstream of the start codon, and g.89278639C>T was a synonymous substitution of the 41st amino acid (alanine).

The ENSBTAG00000052977 gene has a transcript ID of ENSBTAT00000083094.1 and is located in the forward strand of the X chromosome in the region 89,245,181-89,245,990.
It encodes a protein (810 bp, 269 aa) that encodes polyadenylate-binding protein, RNA binding
ng is a predicted function, but it is an unreviewed protein. ENSBTAG00000052977
There is one significant mutation, an SNV and a deletion, located 2-3 kbp upstream of g.89242713A>G.
had a low p-value of 4.01x10E-7.

The ENSBTAG00000053722 gene has a transcript ID of ENSBTAT00000068858.1 and is located in the forward strand of the X chromosome in the region 89,289,437 to 89,293,543.
8bp, 105aa, DZF domain-containing protein, double-stranded RNA binding, si
The function of this gene is assumed to be single-stranded RNA binding, but the details are unknown. The significant SNV g.89289439G>A has a p-value of 1.52x10E-6, and the start codon ATG becomes ATA.
A nonsynonymous substitution occurred. It was assumed that the expected protein would not be expressed due to the nonsynonymous substitution of the start codon. Therefore, the g.89289439G>A SNV on the X chromosome was a very useful indicator for determining mastitis risk.

[Example 3] Testing using DNA markers In Example 3, a test method was carried out to determine the risk of mastitis by using some of the mastitis risk determining DNA markers identified in Examples 1 and 2. As an example, a form in which the genotype of the mastitis risk determining DNA marker is determined by Sanger sequencing of the PCR amplification product will be described. As in Example 1, 25 milking cows with no history of clinical mastitis (free group) and 25 milking cows with recurrent clinical mastitis (recurrent group) were used as test cows.
In this example, the following nine types of DNA markers for determining mastitis risk were examined.

In this example, PCR was performed using KOD FX Neo (Toyobo Co., Ltd.) to obtain a PCR amplified product. Primers and dNTPs were removed from the PCR amplified product using ExoSAP-IT Express (Thermo Fisher Scientific). The base sequence was determined using SupreDye v3.1 Cycle Sequencing Kit (MS Techno Systems Co., Ltd.) and a 3500xL Genetic Analyzer (Applied Biosystems) to identify the genotype. The results are shown in Table 5.

As shown in Table 5, for the SNV number 2, g.88209479A>G on the X chromosome, among the mastitis risk assessment DNA markers, the genotype frequency was 21 reference homozygotes (WT homo) in the unaffected area, 10 heterozygotes (Hetero) and 10 mutant homozygotes (Mutant
On the other hand, in the recurrence group, the reference homozygous type (WT homo) was
In other words, when only SNV number 2 was used as a DNA marker for determining mastitis risk, WT
When determining that homos have a low risk of mastitis and that heteros and mutant homos have a high risk of mastitis, 21 out of 25 cows (84%) in the control group and 20 out of 25 cows (80%) in the recurrent group were correctly predicted, for a combined accuracy rate of 82%, demonstrating that mastitis risk can be determined with a high degree of accuracy.

In this example, nine types of mastitis risk assessment DNA markers were used, and the reference homozygous type was given a score of 0, the heterozygous type a score of 1, and the mutant homozygous type a score of 2.
The mean ± standard deviation (minimum to maximum) of the total score of the markers was 3.2 ± 3.7 (
In the non-recurrent group, the risk of mastitis was 10.4±2.5 (5-15), and in the recurrent group, the risk of mastitis was 10.4±2.5 (5-15).
In the recurrence group, 2 cows (88%) were correctly predicted, and 22 out of 25 cows (88%) were correctly predicted. This resulted in a combined accuracy rate of 88%, and enabled us to determine the risk of mastitis with a high degree of accuracy.

In addition, for the nine mastitis risk determination DNA markers used in this example, SNV number 1
When SNV number 1 and SNV number 2 were used alone, the detection rates were 0.88 and 0.8, respectively, and the false positive rate was 0.
The results shown in Table 5 were 0.2 and 0.16. In addition, the results showed that by testing SNV number 9 of the nine mastitis risk determination DNA markers, individuals that were determined to be heterozygous for SNV number 7 were tested for SNV number 9.
It was revealed that the detection rate was 88% and the false positive rate was kept close to 0. Similarly, from the results shown in Table 5, it was revealed that by testing SNV number 9 of the nine mastitis risk determination DNA markers and testing individuals determined to be heterozygous for SNV number 1, the detection rate was 80% and the false positive rate was kept close to 0. Furthermore, from the results shown in Table 5, it was revealed that the detection rate was 80% and the false positive rate was kept close to 0.
It was found that by testing DNA markers for SNV number 2 and then testing individuals determined to be heterozygous for SNV number 9, the detection rate was 80% and the false positive rate was reduced to 4%.

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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

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Claims (10)

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. The method for determining mastitis risk according to 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. 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. 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 method for assessing mastitis risk according to 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. The method for determining mastitis risk according to claim 1, characterized in that in the step of identifying the genotype of the mastitis risk determining DNA marker, a nucleic acid fragment containing the mastitis risk determining DNA marker is amplified by a nucleic acid amplification method using genomic DNA derived from the test animal, bovine, as a template. The method for determining mastitis risk 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. 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 in the base sequence shown in SEQ ID NO:2 or rs208854668. 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. 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. The primer set according to claim 9 further includes at least one 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: 18 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 19, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 20 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 21, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 22 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 23, a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 24 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 25, and a combination of an oligonucleotide containing the base sequence shown in SEQ ID NO: 26 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 27.

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