JP5924307B2 - Method for determining the conception rate of cattle - Google Patents

Description

  The present invention relates to a method for determining the conception rate of a cow, a determination reagent, and a determination kit.

  It is important for dairymen to improve fertility as well as improving the milking ability of dairy cows. In recent years, Holstein's lactation ability has improved dramatically due to improved feeding management and genetic improvements, while the conception rate has declined significantly. The decrease in conception rate is thought to be due to the fact that genetic improvements were not made to improve fertility, as well as the influence of environmental factors such as stress due to increased milk yield.

  The inventors of the present application have previously confirmed that the bovine Cortactin binding protein 2 N-terminal-like (CTTNBP2NL) gene is involved in the conception rate, and that the conception rate can be evaluated using the base substitution in the 3′UTR of the gene as an index. (Non-Patent Documents 1 and 2). However, genes other than the CTTNBP2NL gene that serve as an index for evaluating the conception rate are not known.

Mayumi Sugimoto et al., "Cortactin binding protein 2 N-terminal-like is involved in bovine conception", 34th Annual Meeting of the Molecular Biology Society of Japan (Web), 1T11PI-4, 2011 Mayumi Sugimoto et al., "Cortactin binding protein 2 N-terminal-like inhibits implantation through regulating permeability of gap junction", 35th Annual Meeting of the Molecular Biology Society of Japan (Web), 1P- Issued 0338, 2012

  Establishing a genetic diagnostic method that detects gene mutations that cause a difference in conception rate enables rapid detection of individuals with good conception rate. Therefore, an object of the present invention is to provide a new index other than the CTTNBP2NL gene, which can contribute to the rapid detection of cattle with good conception rate.

  As a result of intensive studies, the inventors of the present invention succeeded in newly identifying PKP2, CACNB2, and SETD6 genes as genes closely related to conception rate, and completed the present invention.

That is, the present invention relates to a method for determining the conception rate of a bovine, comprising detecting the presence or absence of a mutation in at least one gene region of a PKP2 gene, a CACNB2 gene and a SETD6 gene for a nucleic acid sample isolated from a bovine. a is said in each gene, the presence of a mutant allele is Ri Do an indicator of low conception rates, mutations in PKP2 gene region, of the 3476 th nucleotide and the 3477 th base of the base sequence represented in SEQ ID NO: 1 TA is inserted between them, and the mutation in the CACNB2 gene region is a mutation in which AT is inserted between the 2457th base and the 2458th base in the base sequence shown in SEQ ID NO: 2, and the SETD6 gene The mutation in the region provides a method in which the 3002nd base in the nucleotide sequence shown in SEQ ID NO: 3 is a mutation that replaces cytosine with adenine . The present invention also includes a primer set for amplifying a region including a mutation site in the PKP2 gene region, a primer set for amplifying a region including a mutation site in the CACNB2 gene region, and a region including a mutation site in the SETD6 gene region. A reagent for determining the conception rate of a bovine, comprising at least one of the primer sets to be amplified, the primer set for amplifying a region containing a mutation site in the PKP2 gene region is the 3477th position in SEQ ID NO: 1. A primer set that amplifies a region on the bovine genome containing a base, and a primer set that amplifies a region containing a mutation site in the CACNB2 gene region is a primer that amplifies a region on the bovine genome containing the 2458th base in SEQ ID NO: 2 A primer set that amplifies a region including a mutation site in the SETD6 gene region is shown in SEQ ID NO: 3. It is a primer set for amplifying a region of the bovine genomic containing 3002 th base that provides a determination reagent. Furthermore, this invention provides the determination kit of the conception rate of a cow containing the determination reagent of the said invention.

  The present invention provides a new method capable of rapidly detecting a cow with a high conception rate. Conception rate is a quantitative trait involving multiple genes. By investigating a plurality of gene mutations that serve as an index for determining the conception rate, the accuracy of screening for cows with good conception rate can be further improved, so that genetic improvement for improving fertility can be promoted more rapidly.

It is a figure which shows the variation | mutation part of the PKP2 gene identified in the Example. Squares indicate the corresponding positions of the PCR primers PKP2-F and PKP2-R for detecting the insertion part, and → indicates the direction from 5 ′ to 3 ′ of the primer. It is the waveform data of the base sequence of the mutation site vicinity of the PKP2 gene identified in the Example. When the nucleotide sequence of the PCR-amplified fragment in the region shown in FIG. 1 is determined, the 519th base is guanine (G) in the amplified fragment using the high conception-type genomic DNA as a template, and the low conception-type genomic DNA. In the amplified fragment using as a template, thymine (T) is seen at the 519th base. It is a figure which shows the variation | mutation part of CACNB2 gene. Squares indicate the corresponding positions of PCR primers CACNB2-F and CACNB2-R for detecting the insertion part, and → indicates the direction from 5 ′ to 3 ′ of the primer. It is the waveform data of the base sequence of the mutation site | part vicinity of the CACNB2 gene identified in the Example. When the nucleotide sequence of the PCR amplified fragment in the region shown in FIG. 3 is determined, the 189th base is cytosine (C) in the highly fragmented genomic DNA template, and the low fertility genomic DNA. In the amplified fragment using as a template, adenine (A) is seen at the 189th base. It is a figure which shows the variation | mutation part of the SETD6 gene identified in the Example. The squares indicate the corresponding positions of the PCR primers SETD6-F and SETD6-R for detecting the inserted portion, and the → indicates the 5 ′ to 3 ′ direction of the primer. It is the waveform data of the base sequence of the mutation site vicinity of the SETD6 gene identified in the Example. When the nucleotide sequence of the PCR-amplified fragment in the region shown in FIG. 5 is determined, the 126th base is cytosine (C) in the amplified fragment using the high conception-type genomic DNA as a template, and the low conception-type genomic DNA. In the amplified fragment using as a template, adenine (A) is seen at the 126th base.

  In the present invention, the `` gene region '' includes a region encoding a protein, an intron region, a promoter region, a region that can be involved in the control of expression of the gene, such as 3′- and 5′-UTR, A continuous region on the genome. Specifically, the “PKP2 gene region” is a region on the genome containing the sequence shown in SEQ ID NO: 1, and the “CACNB2 gene region” is a region on the genome containing the sequence shown in SEQ ID NO: 2. The “SETD6 gene region” is a region on the genome containing the sequence shown in SEQ ID NO: 3.

  In the present invention, gene mutation refers to a portion that differs from a base sequence serving as a reference. The reference base sequence in the present invention is the base sequence shown in SEQ ID NO: 1 in the sequence table in the PKP2 gene region, the base sequence shown in SEQ ID NO: 2 in the CACNB2 gene region, and shown in SEQ ID NO: 3 in the SETD6 gene region. It is a base sequence. Hereinafter, in this specification, when referring to a site in each gene region, it is represented by a position in the reference sequence shown in SEQ ID NOs: 1 to 3. The amino acid sequence serving as a reference for the SETD6 protein is the amino acid sequence shown in SEQ ID NO: 14, and when referring to the amino acid site in the SETD6 protein, it is represented by the position in the reference sequence shown in SEQ ID NO: 14.

  The PKP2 (plakophilin 2) gene is a gene encoding an intercellular adhesion protein. The mutation in the PKP2 gene region in the present invention is a mutation that decreases the function of the PKP2 gene in bovine. The decrease in the function of the PKP2 gene in the bovine body includes a decrease in the accumulated amount of wild-type PKP2 protein in the bovine body due to a decrease in the expression level of the PKP2 gene, and a mutant PKP2 protein with decreased physiological activity. It is included that it is produced in the bovine body. The mutation of the PKP2 gene region in the present invention can typically be a mutation that suppresses the expression of the PKP2 gene. A specific example of such a mutation is an insertion mutation between the 3476th base and the 3477th base in the base sequence shown in SEQ ID NO: 1, and a specific example of the insertion mutation is 2 bases (typical Includes insertion of TA).

  CACNB2 (calcium channel, voltage-dependent, beta 2 subunit) gene is a gene encoding a calcium channel. The mutation of the CACNB2 gene region in the present invention is a mutation that reduces the function of the CACNB2 gene in bovine. CACNB2 gene function in the bovine body is reduced due to a decrease in the amount of wild-type CACNB2 protein accumulated in the bovine body due to a decrease in the expression level of the CACNB2 gene, and a mutant CACNB2 protein with reduced physiological activity. It is included that it is produced in the bovine body. The mutation in the CACNB2 gene region in the present invention may typically be a mutation that suppresses the expression of the CACNB2 gene. A specific example of such a mutation is an insertion mutation between the 2457th base and the 2458th base in the base sequence shown in SEQ ID NO: 2, and a specific example of the insertion mutation is 2 bases (typical Includes AT).

  The SETD6 (SET domain containing 6) gene is a region encoding methyltransferase. The SETD6 gene region mutation in the present invention is a mutation that increases the function of the SETD6 gene in bovine. The increase in the function of the SETD6 gene in the bovine body includes an increase in the accumulated amount of wild-type SETD6 protein in the bovine body due to an increase in the expression level of the SETD6 gene, and a mutant SETD6 protein with increased physiological activity. It is included that it is produced in the bovine body. The mutation of the SETD6 gene region in the present invention can typically be the latter, that is, a mutation that increases the methyl group transfer activity of the SETD6 protein. A specific example of such a mutation is an amino acid substitution mutation in which the 360th amino acid of SETD6 protein is changed from alanine to glutamic acid. A specific example of the nucleotide sequence mutation that causes such amino acid substitution is a single nucleotide substitution mutation in which the 3002nd base in the nucleotide sequence shown in SEQ ID NO: 3 is substituted from cytosine to adenine.

  Whether a mutation in a gene region suppresses (or increases) the expression of the gene can be examined, for example, by a reporter assay used also in the following Examples. The reporter assay is a well-known conventional method, and various kits are commercially available. For example, in the case of a luciferase reporter assay, a construct is constructed in which a certain region including a mutation site in a gene region is linked downstream of a luciferase gene, the construct is expressed in vitro, and the luciferase is expressed based on the enzyme activity of the expressed luciferase. What is necessary is just to evaluate whether gene expression is suppressed (or increased). If the enzyme activity of luciferase is lower in the mutant construct than in the wild construct, it can be determined that the mutation is a mutation that suppresses gene expression.

  The physiological activity of a protein having a mutant sequence can be examined by a person skilled in the art by selecting an appropriate assay method based on the properties of the original protein. For example, the SETD6 protein is a methyltransferase, but the methyltransferase activity of a SETD6 protein having an arbitrary mutated sequence can be examined by a method as described in the following examples. That is, a cDNA encoding the mutant amino acid sequence is expressed in an appropriate cultured cell to produce a SETD6 protein mutant, which is recovered (added with a tag sequence such as a His tag and secreted outside the cultured cell. If it is expressed as such, it can be easily recovered by immunoprecipitation using a His tag), and the enzyme activity may be evaluated using a commercially available methyltransferase assay kit.

  The nucleic acid sample is not particularly limited, and a genomic DNA sample, an RNA sample (total RNA or mRNA), or a cDNA sample can be used. A cDNA sample can be prepared by reverse transcription using RNA isolated from bovine as a template. As the nucleic acid sample, a genomic DNA sample can be preferably used because of the simplicity of the work process.

  As a specific method for examining a mutation in a gene region using a nucleic acid sample, for example, a method in which a nucleic acid sample is subjected to a nucleic acid amplification reaction such as PCR and the presence or absence of a mutation is examined based on the base sequence of the amplified fragment. It is done. If there is a difference in the restriction enzyme recognition site between the wild-type sequence and the mutant sequence due to mutation, analyze the restriction fragment length polymorphism of the amplified fragment (ie, using the well-known PCR-RFLP method). Although the presence or absence of mutation can be examined, in general, the method of examining the presence or absence of mutation by determining the base sequence of the amplified fragment is more preferred.

  As a mutation in the PKP2 gene region, when detecting the presence or absence of a TA insertion mutation between the 3476th base and the 3477th base in SEQ ID NO: 1, a part of the PKP2 gene region (50 bp to It is sufficient to amplify a region of about 1000 bp) from a genomic DNA sample by a nucleic acid amplification method, determine the base sequence of the amplified fragment, and confirm whether there is TA insertion. When PCR is performed using the primer sets shown in SEQ ID NOs: 4 and 5, the region shown in FIG. 1 (SEQ ID NO: 10) in the PKP2 gene region is amplified. When the 519th base (3477th in SEQ ID NO: 1) in this amplified fragment is compared, it is guanine when there is no TA insertion, and thymine when there is a TA insertion. Therefore, the presence or absence of the TA insertion mutation can be easily detected by confirming what is the 3477th base in SEQ ID NO: 1 (519th base in SEQ ID NO: 10).

  As a mutation in the CACNB2 gene region, when detecting the presence or absence of an AT insertion mutation between the 2457th base and the 2458th base in SEQ ID NO: 2, a part of the CACNB2 gene region containing the site (50 bp to It is sufficient to amplify a region of about 1000 bp) from a genomic DNA sample by a nucleic acid amplification method, determine the base sequence of the amplified fragment, and confirm whether there is AT insertion. When PCR is performed using the primer sets shown in SEQ ID NOs: 6 and 7, the region shown in FIG. 3 (SEQ ID NO: 11) in the CACNB2 gene region is amplified. When the 189th base (2458th in SEQ ID NO: 2) in this amplified fragment is compared, it is cytosine when there is no AT insertion, and adenine when there is an AT insertion. Therefore, the presence or absence of the AT insertion mutation can be easily detected by confirming what is the 2458th base in SEQ ID NO: 2 (the 189th base in SEQ ID NO: 11).

  When detecting the presence or absence of a substitution mutation (substitution of cytosine to adenine) at the 3002nd base in SEQ ID NO: 3 as a mutation in the SETD6 gene region, a part of the SETD6 gene region (50 bp to 1000 bp) containing the site It is sufficient to amplify a region of a certain degree) from a genomic DNA sample by a nucleic acid amplification method, determine the base sequence of the amplified fragment, and confirm whether or not there is the above substitution mutation. When PCR is performed using the primer sets shown in SEQ ID NOs: 8 and 9, the region shown in FIG. 5 (SEQ ID NO: 12) in the SETD6 gene region is amplified. Since the base substitution site corresponds to the 126th base in this amplified fragment, the presence or absence of the above-described single base substitution mutation in the SETD6 gene region can be detected by confirming whether the site is cytosine or adenine. .

  In the method for determining the conception rate of cattle of the present invention, the presence or absence of a mutation in at least one of the PKP2 gene, CACNB2 gene and SETD6 gene is detected. Any one gene may be examined, or a plurality of genes may be examined. You may investigate combining further CTTNBP2NL gene (nonpatent literature 1, 2) known to become an index of a fertility rate. In any gene, the mutant allele is an indicator of low conception rate. When examining the presence or absence of mutations in one gene for a cow, if one allele is a mutant allele, the cow has a slightly lower conception rate, and if both alleles are mutant alleles, the cow Can be determined to have a low conception rate. If multiple genes are studied, the higher the number of mutant alleles, the lower the conception rate, and conversely, the higher the number of wild-type alleles without mutation, the higher the conception rate. it can. The conception rate can be determined for bulls in the same way as cows. If a bull has a large number of mutant alleles, it is determined that the calf fetus donated by the bull has a low conception rate in cows. be able to. The effect of determining the conception rate when combining a plurality of genes is additive. Therefore, when it is desired to select cattle with high conception rate by the method of the present invention, both bulls and cows are selected and mated (individual fertilization is also performed) by selecting individuals having a large amount of wild-type alleles (individuals having fewer mutant alleles). Include)

  Mutations as defined above in the PKP2 gene, CACNB2 gene and SETD6 gene are indicators of the conception rate of cattle, so a primer set that amplifies a region on the bovine genome including each mutation site, that is, a mutation in the PKP2 gene region The primer set that amplifies the region containing the site, the primer set that amplifies the region that contains the mutation site in the CACNB2 gene region, and the primer set that amplifies the region that contains the mutation site in the SETD6 gene region are It can be provided as a determination reagent. One of the primer sets may be provided as a determination reagent, or a plurality of primer sets may be combined and provided as a determination reagent. When these primer sets are used for PCR using a nucleic acid sample derived from a bovine wild-type sequence that does not have mutations as a template, it is natural to obtain amplified fragments of the mutation-free sequence including the site corresponding to the mutation site. It is done.

  The primer set for amplifying a region containing a mutation site in the PKP2 gene region can be, for example, a primer set for amplifying a region on the bovine genome containing the 3477th base in SEQ ID NO: 1. Such a primer set includes a forward primer that specifically hybridizes to a partial region within the region of the 1st to 3476th bases in the base sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 1. And a reverse primer that specifically hybridizes to a partial region within the region of the 3479th to 3985th bases. The “forward primer” is a primer that hybridizes to the complementary strand of the base sequence actually shown in the sequence listing, and the “reverse primer” is the base actually shown in the sequence listing. A primer that hybridizes to a sequence.

  “Specifically hybridize” means that under normal hybridization conditions, it hybridizes only to the region of interest and does not substantially hybridize to other regions. “Normal hybridization conditions” refer to the conditions used for normal PCR annealing and probe detection. For example, in the case of PCR using Taq polymerase, 50 mM KCl, 10 mM Tris- The reaction is performed using a general buffer solution such as HCl (pH 8.3 to 9.0) and 1.5 mM MgCl2 at an appropriate annealing temperature of about 54 ° C to 60 ° C. For example, in the case of Northern hybridization , 5 x SSPE, 50% formamide, 5 x Denhardt's solution, 0.1 to 0.5% SDS, and the like, and the reaction is performed at an appropriate hybridization temperature of about 42 ° C to 65 ° C. However, the appropriate annealing temperature and hybridization temperature are not limited to the above examples, and are determined based on the Tm value of the primer or probe and the empirical rule of the experimenter, and can be easily determined by those skilled in the art. “Substantially does not hybridize” means that it does not hybridize at all, or even if it is significantly less than the amount hybridized to the target region, and hybridizes only in a relatively negligible amount. is there.

  As a primer that specifically hybridizes under such conditions, a primer having a base sequence that is completely complementary to the hybridizing partial region is preferable, but usually a primer having a sequence in which 10% or less of the base is substituted. In many cases, amplification is caused by hybridizing to the target partial region even when using (“base sequence substituted with 10% or less base” is the base at the 3 ′ end of the original base sequence) It is preferable that a base other than is substituted). As is well known in this field, even if an arbitrary sequence such as a restriction enzyme recognition sequence is added to the 5 ′ end of the primer, it hybridizes specifically to the target partial region and amplifies the target region. can do.

  Therefore, the forward primer that specifically hybridizes to the partial region in the region of the 1st to 3476th bases in the base sequence shown in SEQ ID NO: 1 is used as the 1st in the base sequence shown in SEQ ID NO: 1. To a primer consisting of a base sequence of 15 bases or more, preferably 18 bases or more in the region of the 3476th base, a primer consisting of a base sequence in which 10% or less of the base sequence is substituted, and these A primer having an additional sequence on the 5 ′ end side of the primer is included. That is, in the forward primer, a base sequence of 15 or more bases in the region of the 1st to 3476th bases in the base sequence shown in SEQ ID NO: 1 or 10% or less of the base sequences is substituted. It may be a primer containing the base sequence on the 3 ′ end side.

  Similarly, the reverse primer that specifically hybridizes to a partial region in the region of the 3479th to 3985th bases in the base sequence shown in SEQ ID NO: 1 contains the 3479 in the base sequence shown in SEQ ID NO: 1. Primer consisting of a sequence complementary to a base sequence of 15 bases or more, preferably 18 bases or more in the region of No. 3 to No. 3985, and a base substituted by 10% or less of the complementary sequence Primers comprising sequences as well as primers having additional sequences on the 5 ′ end side of these primers are included. That is, the reverse primer is a sequence complementary to the base sequence consisting of 15 or more bases in the region of the 3479th to 3985th bases in the base sequence shown in SEQ ID NO: 1 or the complementary sequence. Among them, the primer may contain a base sequence substituted with 10% or less of the base on the 3 ′ end side.

  The set of the primer consisting of the base sequence shown in SEQ ID NO: 4 and the primer consisting of the base sequence shown in SEQ ID NO: 5 used in the following example detects a TA insertion mutation in the PKP2 gene region identified in the example However, the primer set for amplifying the PKP2 gene region that can be used as a determination reagent for bovine conception is not limited to this.

  The primer set for amplifying a region containing a mutation site in the CACNB2 gene region can be, for example, a primer set for amplifying a region on the bovine genome containing the 2458th base in SEQ ID NO: 2. Such a primer set includes a forward primer that specifically hybridizes to a partial region within the region of the 1st to 2457th bases in the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO: 2. And a reverse primer that specifically hybridizes to a partial region in the region of the 2460th to 2974th bases.

  The forward primer that specifically hybridizes to the partial region in the region of the 1st to 2457th bases in the base sequence shown in SEQ ID NO: 2 includes the 1st to 2nd in the base sequence shown in SEQ ID NO: 2. A primer consisting of a base sequence of 15 bases or more, preferably 18 bases or more in the region of the 2457th base, a primer consisting of a base sequence substituted with 10% or less of the base sequence, and a primer of these primers A primer having an additional sequence on the 5 ′ end side is included. That is, in the forward primer, 15 or more consecutive base sequences in the region of the 1st to 2457th bases in the base sequence shown in SEQ ID NO: 2 or 10% or less of the base sequences are substituted It may be a primer containing the base sequence on the 3 ′ end side.

  For the reverse primer that specifically hybridizes to the partial region in the region of the 2460th to 2974th bases in the base sequence shown in SEQ ID NO: 2, the 2460th to 2nd in the base sequence shown in SEQ ID NO: 2 are used. A primer consisting of a sequence complementary to a base sequence of 15 bases or more, preferably 18 bases or more in the region of the 2974th base, and a base sequence in which 10% or less of the complementary sequences are substituted Primers as well as primers having additional sequences on the 5 ′ end side of these primers are included. That is, the reverse primer is a sequence complementary to the base sequence consisting of 15 or more consecutive bases in the region of the 2460th to 2974th bases in the base sequence shown in SEQ ID NO: 2 or the complementary sequence. Among them, the primer may contain a base sequence substituted with 10% or less of the base on the 3 ′ end side.

  The set of the primer consisting of the base sequence shown in SEQ ID NO: 6 and the primer consisting of the base sequence shown in SEQ ID NO: 7 used in the following example detects an AT insertion mutation in the CACNB2 gene region identified in the example However, the primer set for amplifying the CACNB2 gene region that can be used as a reagent for determining the conception rate of cattle is not limited to this.

  The primer set for amplifying a region containing a mutation site in the SETD6 gene region can be, for example, a primer set for amplifying a region on the bovine genome containing the 3002nd base in SEQ ID NO: 3. Such a primer set includes a forward primer that specifically hybridizes to a partial region within the region of the first to 3001 bases in the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 3. And a reverse primer that specifically hybridizes to a partial region in the region of the 3003rd to 3322nd bases.

  For the forward primer that specifically hybridizes to the partial region in the region of the first to 3001 bases in the base sequence shown in SEQ ID NO: 3, the first to the first in the base sequence shown in SEQ ID NO: 3 A primer having a base sequence of 15 bases or more, preferably 18 bases or more in the region of base 3001, a primer having a base sequence in which 10% or less of the base sequence is substituted, and a primer of these primers A primer having an additional sequence on the 5 ′ end side is included. That is, in the forward primer, 15 or more consecutive base sequences in the region of the first to 3001 bases in the base sequence shown in SEQ ID NO: 3 or 10% or less of the base sequences are replaced. It may be a primer containing the base sequence on the 3 ′ end side.

  The reverse primer that specifically hybridizes to the partial region in the region of the 3003rd to 3322nd bases in the base sequence shown in SEQ ID NO: 3 is the 3003rd to 3rd in the base sequence shown in SEQ ID NO: 3. It consists of a primer consisting of a sequence complementary to a base sequence of 15 bases or more, preferably 18 bases or more in the region of the 3322 base, and a base sequence in which 10% or less of the complementary sequence is substituted. Primers as well as primers having additional sequences on the 5 ′ end side of these primers are included. That is, the reverse primer is a sequence complementary to the base sequence consisting of 15 or more bases in the region of the 3003rd to 3322nd bases in the base sequence shown in SEQ ID NO: 3 or the complementary sequence. Among them, the primer may contain a base sequence substituted with 10% or less of the base on the 3 ′ end side.

  The set of the primer consisting of the base sequence shown in SEQ ID NO: 8 and the primer consisting of the base sequence shown in SEQ ID NO: 9 used in the following example is from C to A in the SETD6 gene region identified in the example. Although it is an example of the preferable primer set for detecting a substitution mutation, the primer set which amplifies the SETD6 gene area | region which can be used for the determination reagent of a bovine conception rate is not limited to this.

  The fertility determination reagent may be composed of only the primer set described above, or may be in a form in which the primer is dissolved in a buffer solution or the like, and a dried primer and a buffer solution or the like. It may be in the form of a set. Further, the reagent may contain various additives useful for preventing the decomposition of the primer.

  The above-described determination reagent for bovine conception can be appropriately combined with other reagents to provide a determination kit for bovine conception. Reagents other than the primers necessary for nucleic acid amplification methods such as PCR are well known. For example, the determination kit may further include a buffer solution for preparing a reaction solution for nucleic acid amplification reaction, a heat-resistant polymerase, and the like, in addition to the above-described conception rate determination reagent.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples. In the following description, unless otherwise specified, J. Sambrook & DW Russell (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001) This was carried out by the method described, or a method that was appropriately modified or modified. In addition, when using commercially available reagent kits and measuring devices, the procedures were performed according to the instructions of the attached protocol unless otherwise specified.

1. Identification of genes involved in conception rate
(1) Determination of the base sequences of bovine PKP2, CACNB2, and SETD6 genes First of all, holstein cattle populations with a conception breeding value of 51% or more and good fertility rates (192 heads) and a conception breeding value of 42% For a population of 192 cattle with poor conception rate, 43,924 SNPs covering all chromosomes were used to identify regions of the bovine chromosome related to conception rate. Of these, the base sequence of the region involved in the control of the expression control of the PKP2 gene in the fertility control region on chromosome 5 that was found to be strongly related was decoded, and a cow with a good conception rate was compared with a bad cow We found an insertion mutation specific to bad cattle. In addition, the base sequence of the region involved in the control of CACNB2 gene expression in the fertility control region on chromosome 13 where another strong association was seen was decoded, and cows with good fertility and bad cattle were compared. However, we found an insertion mutation specific to the bad cattle. Furthermore, the base sequence of the region encoding the protein of the SETD6 gene present in the fertility control region on chromosome 18 where another strong association was seen was decoded and compared with cattle with good fertility and bad cattle. We found amino acid mutations specific to bad cattle.

  The fertility breeding value was determined as follows according to the method of Takayoshi Kawahara et al., The Japanese Journal of Zootechnical Science 81 (2), p.121-132, 2010-05-25.

For the period from January 1990 to July 2009, we used records 2,391,649 indicating the presence or absence of pregnancy (0 or 1) after the first parturition of 1,218,264 Holstein cows who received a bovine herd test in Hokkaido, The records used for analysis satisfy the following three conditions.
・ First delivery at the age of 18-42 months, fertilization at the age of 18-54 months ・ First insemination within 21-160 days after the delivery of the first delivery, record of insemination within 345 days In the birth record, the gestational age should be between 265 and 295 days

  The model used for the genetic evaluation is a monomorphic / threshold animal model. The population effect is the age of insemination, the month of insemination, the number of days from parturition to insemination, the cow group / year / season inseminated as a random effect, and the insemination Cattle / year, breeding value, repetition of insemination for each cow, and residual were used.

(2) Effect of insertion mutation on the expression level of PKP2 or CACNB2 gene
The luciferase gene construct that connects the PKP2 gene with high and low conception type genomic fragments is introduced into the bovine endometrial cell line BEnEpC, and the expression of the luciferase gene is quantified by measuring the activity of the luciferase enzyme. did. Note that a dual-luciferase reporter assay system (Promega, Madison, Michigan, USA) was used for construct production and luciferase assay. By PCR using primers PKP2-F and PKP2-R (described later), the regions shown in FIG. 1 from the bovine genomic DNA having a high conception type sequence and a low conception type sequence (No. 2959 in SEQ ID NO: 1) The region of the 3534th base, SEQ ID NO: 10) was amplified, and each amplified fragment was ligated downstream of the luciferase gene contained in the kit to obtain an assay construct.

  As a result, it was found that the activity of PKP2 high conception genotype was significantly increased compared to the low conception genotype (Table 1). As a result of the insertion mutation, suppression of PKP2 expression is thought to reduce the conception rate.

  Similarly, for the CACNB2 gene, regions shown in FIG. 2 are obtained from the genomic DNA of cattle having a high conception type sequence and a low conception type sequence by PCR (described later) using primers CACNB2-F and CACNB2-R. (A region from 2270 to 2867 bases in SEQ ID NO: 2, SEQ ID NO: 11) was amplified, and a construct was prepared by linking each amplified fragment downstream of the luciferase gene. The luciferase gene expression was quantified by measuring the activity of the luciferase enzyme.

  As a result, the CACNB2 high conception genotype was found to have significantly increased activity compared to the low conception genotype (Table 2). As a result of the insertion mutation, suppression of CACNB2 expression is thought to bring about a decrease in conception rate.

(3) Effect of amino acid mutation on SETD6In order to investigate the function of SETD6 gene encoding methyltransferase, the SETD6 gene was introduced into mouse hypothalamus-derived cell line GT1-7 and expressed according to the following procedure, The methyl group transfer activity was measured according to a conventional method.

  RNA was extracted from the cerebrum of cattle with high and low conception rates using TRIzol (Life Technologies, Carlsbad, California, USA) and ReverTra Ace (registered trademark) reverse transcriptase (Toyobo, Osaka, Japan) RT-PCR was used to amplify the SETD6 gene cDNA sequence (SEQ ID NO: 13). Primers shown in SEQ ID NOs: 15 and 16 were used for cDNA amplification. The obtained amplified fragment was incorporated into a His-tagged expression vector, introduced into the mouse hypothalamic cell line GT1-7 for expression, and immunoprecipitated using the His tag. The methyl group transfer activity of SETD6 protein derived from high conception type and SETD6 protein derived from low conception type concentrated by immunoprecipitation was determined using SET7 / 9 Histone Methltransferase Assay Kit (BPS Bioscience, San Diego, Calif., USA). Measured and compared.

  As a result, it was found that methyl transfer activity was significantly increased in the low conception genotype of SETD6 compared to the high conception genotype (Table 3). As a result of amino acid mutations, increased methyl group transfer activity of SETD6 is thought to reduce the conception rate.

(4) Effects of PKP2, CACNB2, and SETD6 gene mutations on fertility breeding value
In order to examine the effect of PKP2, CACNB2, and SETD6 genes on the fertility breeding value, we examined the genotype using the DNA of 2528 bulls and 1418 cows and compared the fertility breeding values. As a result, it was found that an individual having a high conception genotype was significantly higher in fertility breeding value than an individual having a low conception genotype (Tables 4, 5, and 6). From this, it is considered that the production efficiency of cattle having low conception genotypes PKP2, CACNB2, and SETD6 genes is reduced compared to individuals having high conception genotypes PKP2, CACNB2, and SETD6 genes.

2. Confirmation of mutations in PKP2, CACNB2, and SETD6 genes on bovine genomic DNA Primers PKP2-F, PKP2-R, CACNB2-F for amplifying DNA fragments containing mutation sites found in cattle with poor conception rate CACNB2-R, SETD6-F, and SETD6-R were synthesized. The nucleotide sequences of primers PKP2-F, PKP2-R, CACNB2-F, CACNB2-R, SETD6-F, and SETD6-R are shown in SEQ ID NOs: 4, 5, 6, 7, 8, and 9 in the sequence listing, respectively.

(1) Discrimination between high and low fertility types in the PKP2 gene Genomics using bovine blood (including EDTA and heparin as anti-sham-fixing agents) and an automatic nucleic acid separator NA-1000 (Kurabo) DNA was prepared. PCR using these genomic DNAs as templates and primers PKP2-F and PKP2-R (35-cycle reaction with a cycle consisting of 94 ° C for 20 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute) The base sequences of the PCR products were compared using a 3730 fluorescent DNA sequencer (Applied Biosystems). In the DNA fragment amplified by PCR using the high conception-type PKP2 gene as a template (regions 2959 to 3534 in SEQ ID NO: 1, SEQ ID NO: 10 and FIG. 1), the 519th base is guanine (G). It was thymine (T) in the amplified fragment derived from the low conception type having a 2-base insertion. As shown in FIG. 2, it was confirmed that the high and low conception types of the PKP2 gene can be easily distinguished by examining the bases in the site.

(2) Discrimination between high and low conception types in the CACNB2 gene Genomics using bovine blood (including EDTA and heparin as anti-immortal agents) and an automatic nucleic acid separator NA-1000 (manufactured by Kurabo) DNA was prepared. PCR using these genomic DNAs as templates and primers CACNB2-F and CACNB2-R (35-cycle reaction with a cycle of 94 ° C for 20 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute) The base sequences of the PCR products were compared using a 3730 fluorescent DNA sequencer (Applied Biosystems). In the DNA fragment amplified by PCR using the high conception type CACNB2 gene as a template (region 2270-2867 in SEQ ID NO: 2, SEQ ID NO: 11 and FIG. 3), the 189th base is cytosine (C). The amplified fragment derived from the low conception type having a 2-base insertion was adenine (A). As shown in FIG. 4, it was confirmed that the high and low conception types of the CACNB2 gene can be easily distinguished by examining the bases at the site.

(3) Discrimination between high and low fertility types in the SETD6 gene Genomics using bovine blood (including EDTA and heparin as an anti-pseudo-solid agent) and an automatic nucleic acid separator NA-1000 (manufactured by Kurabo) DNA was prepared. PCR using these genomic DNAs as templates and primers SETD6-F and SETD6-R (35-cycle reaction with a cycle consisting of 20 seconds at 94 ° C, 30 seconds at 60 ° C, 1 minute at 72 ° C) The base sequences of the PCR products were compared using a 3730 fluorescent DNA sequencer (Applied Biosystems). In the DNA fragment amplified by PCR using the high conception-type SETD6 gene as a template (region 2877 to 3322 in SEQ ID NO: 3, SEQ ID NO: 12 and FIG. 5), the 126th base is cytosine (C). The low conception type was adenine (A). As shown in FIG. 6, it was confirmed that the high conception type and the low conception type of the SETD6 gene can be easily distinguished by examining the base at the site.

  The conception rate is a quantitative trait involving multiple genes, but according to the present invention, the PKP2, CACNB2, and SETD6 genes are part of the genes involved in conception control, and the DNA diagnostic method described here ensures that PKP2 It was clarified that the conception rate of cattle can be determined by determining the presence or absence of mutations in the CACNB2 and SETD6 genes.

Claims (8)

A method for determining the conception rate of a bovine, comprising detecting the presence or absence of a mutation in at least one gene region of a PKP2 gene, a CACNB2 gene and a SETD6 gene for a nucleic acid sample isolated from a bovine, in gene, Ri presence of a mutant allele is Do indicative of low conception rates, mutations in PKP2 gene region, TA is inserted between the first 3476 th nucleotide and the 3477 th base of the base sequence shown in SEQ ID NO: 1 The mutation in the CACNB2 gene region is a mutation in which AT is inserted between the 2457th base and the 2458th base in the base sequence shown in SEQ ID NO: 2, and the mutation in the SETD6 gene region is the sequence A method, wherein the 3002nd base in the nucleotide sequence shown in No. 3 is a mutation that replaces cytosine with adenine . The method of claim 1, wherein said nucleic acid sample is a genomic DNA sample. Detection of the presence or absence of a mutation in the PKP2 gene region includes amplifying a region containing the 3477th base in SEQ ID NO: 1 (the 519th base in SEQ ID NO: 10) from a genomic DNA sample by a nucleic acid amplification method, and the CACNB2 gene region The detection of the mutation in A includes amplification of a region containing the 2458th base in SEQ ID NO: 2 (the 189th base in SEQ ID NO: 11) from a genomic DNA sample by nucleic acid amplification, and detection of a mutation in the SETD6 gene region The method according to claim 2 , comprising amplifying a region containing the 3002 base in SEQ ID NO: 3 (126 th base in SEQ ID NO: 12) from a genomic DNA sample by a nucleic acid amplification method. The method according to any one of claims 1 to 3 , wherein the conception rate is determined based on the following criteria:
For the PKP2 gene, if the 3477th base in SEQ ID NO: 1 (519th base in SEQ ID NO: 10) is guanine, the conception rate is high, and if it is thymine, the conception rate is low;
For the CACNB2 gene, the 2458th base in SEQ ID NO: 2 (the 189th base in SEQ ID NO: 11) is cytosine, the conception rate is high, and if it is adenine, the conception rate is low;
Regarding the SETD6 gene, the fertility rate is high when the 3002nd base in SEQ ID NO: 3 (the 126th base in SEQ ID NO: 12) is cytosine, and the fertility rate is low when it is adenine. Of the primer set that amplifies the region including the mutation site in the PKP2 gene region, the primer set that amplifies the region including the mutation site in the CACNB2 gene region, and the primer set that amplifies the region including the mutation site in the SETD6 gene region A primer set for determining the conception rate of a bovine comprising at least one of the above, and a primer set for amplifying a region containing a mutation site in the PKP2 gene region is a bovine genome comprising the 3477th base in SEQ ID NO: 1. The primer set that amplifies the region, and the primer set that amplifies the region including the mutation site in the CACNB2 gene region is a primer set that amplifies the region on the bovine genome containing the 2458th base in SEQ ID NO: 2, and the SETD6 gene The primer set that amplifies the region containing the mutation site in the region is the 3002nd base in SEQ ID NO: 3. Is a primer set for amplifying a region of the bovine genome, including the determination reagent.
. The primer set that amplifies the region including the mutation site in the PKP2 gene region is a forward primer that specifically hybridizes to a partial region in the region of the 1st to 3476th bases in the base sequence shown in SEQ ID NO: 1. And a reverse primer that specifically hybridizes to a partial region in the region of the 3479th to 3985th bases in the base sequence shown in SEQ ID NO: 1, and includes a mutation site in the CACNB2 gene region The primer set for amplifying the region includes a forward primer that specifically hybridizes to a partial region in the region of the 1st to 2457th bases in the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO: 2. A set with a reverse primer that specifically hybridizes to a partial region in the region of the 2460th to 2974th base in the region including the mutation site in the SETD6 gene region The primer set to be wide is composed of a forward primer that specifically hybridizes to a partial region in the region of the first to 3001 bases in the base sequence shown in SEQ ID NO: 3, and a base sequence shown in SEQ ID NO: 3 The determination reagent according to claim 5 , which is a set with a reverse primer that specifically hybridizes to a partial region in the region of the 3003rd to 3322nd bases. A primer set for amplifying a region including a mutation site in the PKP2 gene region is a set of a primer consisting of the base sequence shown in SEQ ID NO: 4 and a primer consisting of the base sequence shown in SEQ ID NO: 5, and in the CACNB2 gene region A primer set for amplifying a region containing a mutation site is a set of a primer consisting of the base sequence shown in SEQ ID NO: 6 and a primer consisting of the base sequence shown in SEQ ID NO: 7, and a region containing a mutation site in the SETD6 gene region The determination reagent according to claim 6 , wherein the primer set for amplifying is a set of a primer consisting of the base sequence shown in SEQ ID NO: 8 and a primer consisting of the base sequence shown in SEQ ID NO: 9. A determination kit for the conception rate of a cow, comprising the determination reagent according to any one of claims 5 to 7 .