JP6120448B2 - 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 application succeeded in newly identifying the UNC5C gene as a gene closely related to the conception rate, and completed the present invention.

That is, the present invention is a method for determining the fertility rate of a bovine, comprising detecting the presence or absence of a mutation in the UNC5C gene region of a nucleic acid sample isolated from a bovine, wherein the mutation is expressed by the expression of the UNC5C gene. Provided is a method that is a suppressed mutation, wherein the presence of a mutant allele is an indicator of low conception rate. The present invention is also a bovine conception rate determination reagent comprising a primer set for amplifying a region containing a mutation site in the UNC5C gene region, wherein the mutation site is the 432th base in SEQ ID NO: 16. A determination reagent is provided. 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. The conception rate is a quantitative trait involving multiple genes, but it is possible to evaluate the conception rate even when the UNC5C gene is used alone as an indicator. When a combination of UNC5C gene mutations and other gene mutations that are used as an indicator for conception rate determination, the accuracy of screening for cows with good conception rates is further increased. The present invention contributes to further speeding up genetic improvement for improving fertility.

It is a figure which shows the variation | mutation part of the UNC5C gene identified in the Example. The squares indicate the corresponding positions of the PCR primers UNC5C-F and UNC5C-R for detecting the mutated part, and the → indicates the 5 ′ to 3 ′ direction of the primer. It is the waveform data of the base sequence near the mutation site | part of the UNC5C gene identified in the Example. When the base sequence of the PCR-amplified fragment in the region shown in FIG. 1 is determined, the 169th base is adenine (A) in the amplified fragment using the high conception-type genomic DNA as a template, and the low-fertility type genomic DNA. In the amplified fragment using as a template, guanine (G) is seen at the 169th base.

  In the present invention, the `` gene region '' includes a region encoding a protein, an intron region, a promoter region, a region that can participate in the control of expression of the gene such as 5′- and 3′-UTR, A continuous region on the genome. Specifically, the “UNC5C gene region” is a region on the genome including the sequences shown in SEQ ID NOs: 1 to 16 in the sequence listing. SEQ ID NOs: 1 to 16 show the sequences of each exon of the UNC5C gene and introns in the vicinity thereof as shown in Table 1.

  In the present invention, gene mutation refers to a portion that differs from a base sequence serving as a reference. The base sequence used as a reference for the UNC5C gene region is the base sequence shown in SEQ ID NOs: 1 to 16 in the sequence listing. In SEQ ID NOs: 20 and 21, the cDNA base sequence of UNC5C gene and the amino acid sequence of the protein encoded by the gene are shown, respectively.

  The UNC5C (unc-5 homolog C) gene encodes a netrin receptor protein involved in nerve development. The mutation of the UNC5C gene region in the present invention is a mutation that decreases the function of the UNC5C gene in bovine. The decrease in the function of the UNC5C gene in the bovine body includes a decrease in the accumulated amount of wild-type UNC5C protein in the bovine body due to a decrease in the expression level of the UNC5C gene, and a mutant UNC5C protein with decreased physiological activity. It is included that it is produced in the bovine body. The mutation in the UNC5C gene region in the present invention can typically be a mutation that suppresses the expression of the UNC5C gene. A specific example of such a mutation is a single base substitution in which the 169th base of the 3'-UTR region (meaning the 169th base counting from the base codon next to the stop codon of the UNC5C gene) is replaced by adenine to guanine. Mutations. The 169th base is the 432th position in the base sequence shown in SEQ ID NO: 16 and the 2962nd position in SEQ ID NO: 20.

  Whether a mutation in the UNC5C gene region suppresses the expression of the gene can be examined by, for example, 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 in which a certain region including a mutation site in the UNC5C gene region is linked upstream or downstream of the luciferase gene is constructed, the construct is expressed in vitro, and the enzyme activity of the expressed luciferase is determined. Based on this, it may be evaluated whether the expression of the luciferase gene is suppressed. When 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 the expression of the UNC5C gene.

  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 of subjecting a nucleic acid sample to a nucleic acid amplification reaction such as PCR and examining the presence or absence of a mutation based on the base sequence of the amplified fragment can be mentioned. 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 of the UNC5C gene region, if the presence or absence of a single nucleotide substitution mutation in which the 169th base of the 3′-UTR region (the 432th base in SEQ ID NO: 16) substitutes adenine for guanine is detected, the relevant site A part of the UNC5C gene region (including a region of about 50 bp to 1000 bp is sufficient) is amplified from a genomic DNA sample by nucleic acid amplification, and the base sequence of the amplified fragment is determined, and whether there is a single base substitution You should confirm whether or not. When PCR is performed using the primer sets shown in SEQ ID NOs: 18 and 19, the region shown in FIG. 1 (SEQ ID NO: 17) in the UNC5C gene region is amplified. This primer is a primer that amplifies the 1st to 231st regions of the 3′-UTR region. When the 169th base (No. 432 in SEQ ID NO: 16) in this amplified fragment is compared, it is adenine when there is no single base substitution, and it is guanine when there is a single base substitution. Therefore, the presence or absence of the single nucleotide substitution mutation can be easily detected by confirming what is the 432nd base in SEQ ID NO: 16.

  In the method for determining the fertilization rate of the bovine of the present invention, the presence or absence of a mutation in the UNC5C gene is detected. You may investigate combining CTTNBP2NL gene (nonpatent literatures 1 and 2) known to become a fertility index. 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). Included).

  Since the mutation as defined above in the UNC5C gene is an indicator of the conception rate of cattle, a primer set that amplifies a region on the bovine genome including the mutation site, that is, a region including the mutation site in the UNC5C gene region is amplified. The primer set can be provided as a reagent for determining the conception rate of cattle. In addition, if this primer set is used for PCR using a wild-type sequence bovine-derived nucleic acid sample having no mutation as a template, an amplified fragment of a sequence without a mutation including a site corresponding to the mutation site can be obtained. .

  The primer set for amplifying a region containing a mutation site in the UNC5C gene region can be, for example, a primer set for amplifying a region on the bovine genome containing the 432th base in SEQ ID NO: 16. Such a primer set includes a forward primer that specifically hybridizes to a partial region in the region of the first to 431 th bases in the base sequence shown in SEQ ID NO: 16, and the base sequence shown in SEQ ID NO: 16 And a reverse primer that specifically hybridizes to a partial region within the region of the 433th to 2036th 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 MgCl _{2 at} an appropriate annealing temperature of about 54 ° C. to 60 ° C. For example, in the case of Northern hybridization Is to perform the reaction at an appropriate hybridization temperature of about 42 ° C. to 65 ° C. using a general hybridization solution such as 5 × SSPE, 50% formamide, 5 × Denhardt's solution, 0.1 to 0.5% SDS. . 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 first to 431th bases in the base sequence shown in SEQ ID NO: 16 is the first primer in the base sequence shown in SEQ ID NO: 16. To a primer consisting of a base sequence of 15 bases or more, preferably 18 bases or more in the region of No. 431 base, a primer consisting of a base sequence substituted with 10% or less of the base sequence, and these A primer having an additional sequence on the 5 ′ end side of the primer is included. That is, in the forward primer, 15 or more consecutive base sequences in the region of the 1st to 431rd bases in the base sequence shown in SEQ ID NO: 16 or 10% or less of the base sequences are substituted It may be a primer containing the base sequence on the 3 ′ end side.

  Similarly, the reverse primer that specifically hybridizes to the partial region in the region of the 433th to 2036th bases in the base sequence shown in SEQ ID NO: 16 is the 433 in the base sequence shown in SEQ ID NO: 16. A primer composed of a sequence complementary to a base sequence of 15 bases or more, preferably 18 bases or more in the region of No. 2036 bases, 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 433th to 2036th bases in the base sequence shown in SEQ ID NO: 16, 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: 18 and the primer consisting of the base sequence shown in SEQ ID NO: 19 used in the following example is a single base substitution mutation in the UNC5C gene region identified in the example. Although it is an example of the preferable primer set for detecting, the primer set which amplifies the UNC5C 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 sequence of bovine UNC5C gene First of all, cows with good conception rate (176 heads) with a conception breeding value of 50% or higher from the Holstein population and those with a conception rate breeding value of less than 42% For a population of bad cattle (646 heads), 43,851 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 regulation of UNC5C gene expression in the fertility control region on chromosome 6 that was found to be strongly related was decoded and compared with cattle with good fertility and bad cattle. We found a single base substitution mutation 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 single nucleotide substitution mutation on the expression level of UNC5C gene
The luciferase gene construct that connects the genomic fragments of the high and low conception types of UNC5C gene was introduced into human granulosa cell-derived strain COV434, and the expression of the luciferase gene was quantified by measuring the activity of the luciferase enzyme. . Note that a dual-luciferase reporter assay system (Promega, Madison, Michigan, USA) was used for construct production and luciferase assay. By PCR (described later) using primers UNC5C-F and UNC5C-R, the regions shown in FIG. The region of the 494th base, SEQ ID NO: 17) 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 UNC5C high fertility genotype had significantly increased activity compared to low conception genotype (Table 2). As a result of single nucleotide substitution mutations, suppression of UNC5C expression is thought to reduce the conception rate.

(3) Effect of UNC5C gene mutation on fertility breeding value
To examine the effect of the UNC5C gene on the fertility breeding value, we examined the genotypes using the DNA of 2528 bulls and 1418 cows and compared the fertility breeding values. As a result, it was found that the individuals having a high conception genotype homozygously had a significantly higher conception breeding value than those individuals having a low conception genotype homozygous (Table 3). From this, it is considered that the production efficiency of cattle having a low conception rate genotype UNC5C gene is reduced compared to individuals having a high conception rate genotype UNC5C gene.

2. Confirmation of the presence or absence of mutations in the UNC5C gene on bovine genomic DNA Primers UNC5C-F and UNC5C-R were synthesized to amplify DNA fragments containing mutation sites found in cattle with poor conception rates. The nucleotide sequences of primers UNC5C-F and UNC5C-R are shown in SEQ ID NOs: 18 and 19, respectively.

  Genomic DNA was prepared from bovine blood (including EDTA and heparin as an anti-pseudo-solid agent) using an automatic nucleic acid separator NA-1000 (manufactured by Kurabo Industries). PCR using these genomic DNAs as templates and primers UNC5C-F and UNC5C-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 a DNA fragment amplified by PCR using the high fertility type UNC5C gene as a template (regions of the 264th to 494th bases in SEQ ID NO: 16, SEQ ID NO: 17 and FIG. 1), the 169th base is adenine (A) However, the amplified fragment derived from the low conception type was guanine (G). As shown in FIG. 2, it was confirmed that the high and low conception types of the UNC5C gene can be easily distinguished by examining the bases at the site.

  The conception rate is a quantitative trait involving multiple genes, but according to the present invention, the UNC5C gene is part of the gene involved in the control of conception rate. It was clarified that the conception rate of cattle can be determined by determining the presence or absence.

Claims (9)

A method for determining the conception rate of a bovine, comprising detecting the presence or absence of a mutation in the UNC5C gene region of a nucleic acid sample isolated from a bovine, wherein the mutation is a mutation that suppresses the expression of the UNC5C gene A method wherein the presence of a mutant allele is an indicator of low conception rate. Wherein said mutation is a mutation # 432 th base of the base sequence shown in SEQ ID NO: 16 is replaced with guanine adenine The method of claim 1, wherein. The method according to claim 1 or 2, wherein the nucleic acid sample is a genomic DNA sample. Detection of the presence or absence of mutations in UNC5C gene region comprises a region comprising SEQ ID NO: 16 in 432 th nucleotide (SEQ ID NO: 17 in the 169th base) amplified by nucleic acid amplification method from genomic DNA samples, according to claim 3 The method described. When the 432rd base in SEQ ID NO: 16 (the 169th base in SEQ ID NO: 17) is adenine, the conception rate is high, and when it is guanine, the conception rate is determined. 5. The method according to any one of claims 1 to 4 . A determination reagent for bovine conception rate, comprising a primer set for amplifying a region containing a mutation site in the UNC5C gene region, wherein the mutation site is the 432th base in SEQ ID NO: 16 . The primer set includes a forward primer that specifically hybridizes to a partial region in the region of the first to 431 th bases in the base sequence shown in SEQ ID NO: 16, and the first primer in the base sequence shown in SEQ ID NO: 16. The determination reagent according to claim 6 , which is a set with a reverse primer that specifically hybridizes to a partial region within the region of the 433rd to 2036th bases. The determination reagent according to claim 7 , wherein the primer set is a set of a primer consisting of the base sequence shown in SEQ ID NO: 18 and a primer consisting of the base sequence shown in SEQ ID NO: 19. 9. A bovine conception rate determination kit comprising the determination reagent according to any one of claims 6 to 8 .