SNP marker composition for predicting reproduction-related trait of Jeju black cattle and uses thereof

Abstract

The present invention relates to an SNP marker composition for predicting breeding-related traits of Jeju Black Cattle cows and the use thereof. Since the SNP marker composition of the present invention allows for the simultaneous analysis of various breeding-related traits of Jeju Black Cattle cows, it can be usefully utilized for selecting superior breeding cows, increasing breeding efficiency, and improving the breed.

Description

Technology Field

[0001] The present invention relates to an SNP marker composition for predicting breeding-related traits of Jeju Black Cattle and the use thereof. Background Technology

[0002] The Jeju Black Cattle is a native livestock unique to Jeju that has been raised in Jeju Special Self-Governing Province for a long time, and it was designated as Natural Monument No. 546 on July 22, 2013. Known to have been raised in the Jeju region since before the Common Era, the Jeju Black Cattle holds great historical and cultural value, evidenced by records in ancient documents such as the Annals of the Joseon Dynasty, the Tamna Circuit Map, and the Tamna Chronicle that they were requisitioned for ancestral rites and as tribute.

[0003] Jeju Black Cattle, which had long been raised as native livestock in Jeju, faced major crises due to extermination policies during the Japanese colonial period, the Jeju 4.3 Incident in 1948, and the Korean War in 1950. Not only were they exported as food resources by Japan, but with the enactment of the 'Hanwoo Standardization Act,' the nine domestic Hanwoo breeds (including Chikso, Hwangwoo, Baekwoo, and Heukwoo) were unified into a single breed called Hwangwoo. For this reason, many people think of Hwangwoo first when they hear the term "Korean native cattle," and there are many who believe that only yellow cattle constitute Hanwoo. Since the 1960s, thanks to the introduction of advanced foreign breeding techniques, the efforts of domestic researchers, and state-led improvement projects, Hwangwoo has grown larger and established itself as a delicacy and delicacy. However, Jeju Black Cattle have not undergone such improvement; their body size is only about 70% of that of Hwangwoo, and their growth is about 6 to 8 months slower than Hanwoo. Due to their smaller size, the adult form is rather sturdy.

[0004] The population of Jeju Black Cattle does not even amount to 1% of the total population of Korean native cattle, so Jeju Special Self-Governing Province is conducting various studies for the preservation and restoration of bloodlines to protect and breed Jeju Black Cattle. To increase the population, it is necessary to promote breeding improvement of Jeju Black Cattle by increasing genetic diversity, producing superior individuals, supplying high-quality semen, or selecting cows with excellent reproductive capabilities.

[0005] Livestock improvement has traditionally been carried out by selecting animals suitable for achieving improvement goals as breeding stock and producing offspring with superior capabilities through an appropriate mating system. The technology for selecting superior individuals has evolved from a method based simply on the phenotype of the individual to be selected to a method that estimates individual genetic potential by utilizing both the individual's own abilities and those of related individuals, and then selects based on this estimation. The improvement of Jeju Black Cattle has primarily focused on growth or meat quality traits, such as carcass characteristics; consequently, the importance of reproductive traits that influence calf production has not been highlighted, nor is their genetic potential being evaluated. If the importance of reproductive traits is emphasized, they can be utilized as key indicators for farm management, economic viability, calf productivity, and efficiency, thereby amplifying positive effects. To achieve this, a well-established breeding cow production system is required, and consistent calf production must be ensured through continuous management and monitoring.

[0006] Reproductive traits of cattle can be classified into those of cows and those of bulls; cow reproductive traits can be broadly divided into reproductive traits and calving traits. Reproductive traits are obtained through breeding records from the time a cow is born until culling, and include the age in days to the first calving, conception rate from the first insemination, calving date, calving interval, and the age in days from calving to the first insemination. Calving traits include calf size and difficulty of calving. Recent advancements in DNA analysis technology have made it possible to analyze variations at the genomic level and utilize this for individual selection and research on genetic characteristics within a population. If research is conducted through the systematic accumulation of data to calculate genetic potential more accurately and provide this information to farmers, it will help increase farm income and, at the national level, contribute to improving self-sufficiency by strengthening the competitiveness of the Jeju Black Cattle industry.

[0007] Meanwhile, Korean Published Patent No. 2009-0065412 discloses 'a cDNA chip capable of early diagnosis of genes related to growth, meat quality, and reproductive traits of pigs, a method for developing the same, and a method for selecting breeding pigs with excellent growth, meat quality, and reproductive traits using the chip,' and Korean Registered Patent No. 2173458 discloses 'a single nucleotide polymorphism marker composition for predicting the body condition index of domestic cattle and the use thereof,' but there is no description regarding the SNP marker composition for predicting reproductive traits of Jeju Black Cattle cows and the use thereof according to the present invention. The problem to be solved

[0008] The present invention was derived in response to the above requirements. The inventors extracted genomic DNA from Jeju Black Cattle cow samples and analyzed SNPs using the 160K Affymetrix SNP Axiom array. Genome-wide association analysis (GWAS) was performed on each SNP to analyze associations with reproductive traits (age at first calving, calving interval, gestation period, number of artificial inseminations per conception). As a result, 137 SNP markers with statistical significance for reproductive traits were identified, thereby completing the present invention. means of solving the problem

[0009] To solve the above problem, the present invention provides an SNP marker composition for predicting breeding-related traits of Jeju Black Cattle, comprising a polynucleotide composed of eight or more consecutive nucleotides including a single nucleotide polymorphism (SNP) located at the 36th position in each of the nucleotide sequences of SEQ ID NOs. 1 to 137, or a polynucleotide composed of the same or a polynucleotide complementary thereof.

[0010] In addition, the present invention provides a microarray for predicting breeding-related traits of Jeju Black Cattle, comprising a polynucleotide containing the above-mentioned SNP base or cDNA thereof.

[0011] In addition, the present invention provides a set of primers for predicting breeding-related traits of Jeju Black Cattle, for amplifying a polynucleotide containing the above SNP base.

[0012] In addition, the present invention provides a kit for predicting breeding-related traits of Jeju Black Cattle, comprising the primer set and a reagent for performing an amplification reaction.

[0013] In addition, the present invention provides a method for predicting reproductive traits of a Jeju Black Cattle cow, comprising the steps of: isolating genomic DNA from an individual Jeju Black Cattle cow; and determining the genotype of the base at each 36th SNP position of a polynucleotide composed of the base sequences of SEQ ID NOs 1 to 137 using the isolated genomic DNA as a template. Effects of the invention

[0014] Since the SNP marker composition of the present invention allows for the simultaneous analysis of various traits related to the reproduction of Jeju Black Cattle cows, it can be usefully utilized for selecting superior breeding cows, increasing breeding efficiency, and improving the breed. Specific details for implementing the invention

[0015] To achieve the objective of the present invention, the present invention provides an SNP marker composition for predicting breeding-related traits of Jeju Black Cattle, comprising a polynucleotide composed of eight or more consecutive nucleotides including a single nucleotide polymorphism (SNP) located at the 36th position in each of the nucleotide sequences of SEQ ID NOs 1 to 137, or a polynucleotide complementary thereof.

[0016] In the SNP marker composition of the present invention, the reproductive-related trait may be one or more traits selected from the group consisting of age at first birth, interval between births, gestation period, and number of artificial inseminations per conception, but is not limited thereto.

[0017] In one embodiment of the present invention, the consecutive nucleotides may be 8 to 100 consecutive nucleotides, but are not limited thereto.

[0018] In the present invention, the term “nucleotide” refers to a deoxyribonucleotide or ribonucleotide existing in a single-stranded or double-stranded form, and includes analogs of natural nucleotides unless specifically otherwise noted.

[0019] In an SNP marker composition according to one embodiment of the present invention, the SNP position base is the 36th of all base sequences of SEQ ID NOs 1 to 137, and polymorphic base information is indicated by [ / ] in the SNP base sequence information of Tables 6 to 14, and the base sequences of SEQ ID NOs 1 to 137 of the present invention refer to sequences including bases located before the diagonal line ( / ) in Tables 6 to 14.

[0020] The present invention relates to base variants at SNP locations in the base sequences of SEQ ID NOs. 1 to 137, but when such SNP base variants are found in double-stranded gDNA (genomic DNA), they are interpreted to include polynucleotide sequences complementary to the nucleotide sequences. Accordingly, the bases at the SNP locations in the complementary polynucleotide sequences also become complementary bases. In this regard, all sequences presented in this specification are based on sequences in the sense strand of genomic DNA, unless otherwise noted.

[0021] The present invention also provides a microarray for predicting breeding-related traits of Jeju Black Cattle, comprising a polynucleotide containing the SNP base or the cDNA thereof.

[0022] Preferably, the polynucleotide may be immobilized on a substrate coated with an active group of amino-silane, poly L-lysine, or aldehyde, but is not limited thereto. Additionally, the substrate may be a silicon wafer, glass, quartz, metal, or plastic, but is not limited thereto. Methods for immobilizing the polynucleotide on the substrate may include micropipetting using a piezoelectric method, a method using a pin-shaped spotter, etc.

[0023] The microarray of the present invention can be manufactured by conventional methods known to those skilled in the art using a polynucleotide according to the present invention or its complementary polynucleotide, a polypeptide encoded by the same, or its cDNA (complementary DNA).

[0024] The present invention also provides a set of primers for predicting breeding-related traits of Jeju Black Cattle, for amplifying a polynucleotide containing the SNP base.

[0025] In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be copied, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer must allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the complexity of the required DNA or RNA target, as well as primer usage conditions such as temperature and ionic strength.

[0026] In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate, or a peptide nucleic acid, or may include an intercalating agent.

[0027] The present invention also provides a kit for predicting breeding-related traits of Jeju Black Cattle, comprising the primer set and a reagent for performing an amplification reaction.

[0028] In the kit of the present invention, the reagent for performing the amplification reaction may include, but is not limited to, DNA polymerase, dNTPs, and a buffer.

[0029] The kit of the present invention may also additionally include a user guide describing optimal reaction performance conditions. The guide is a printed document explaining how to use the kit, for example, how to prepare PCR buffer, the presented reaction conditions, etc. The guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and on the surface of a package containing the kit. Additionally, the guide includes information disclosed or provided through electronic media, such as the Internet.

[0030] The present invention also,

[0031] Step of isolating genomic DNA from Jeju Black Cattle cow individuals; and

[0032] A method for predicting reproductive traits of Jeju Black Cattle cows is provided, comprising the step of determining the genotype of each 36th SNP position base of a polynucleotide composed of the base sequences of SEQ ID NOs 1 to 137 using the isolated genomic DNA as a template.

[0033] In a method according to one embodiment of the present invention, the polynucleotides are as described above, and the genotype information of the SNP position bases of each polynucleotide is as disclosed in Tables 6 to 14.

[0034] In a method according to one embodiment of the present invention, the reproductive-related trait may be one or more traits selected from the group consisting of age at first birth, interval between births, gestation period, and number of artificial inseminations per conception, but is not limited thereto.

[0035] The aforementioned age at first calving refers to the period from birth to the first calving; while a shorter age at first calving can be considered a superior trait, calving too early may affect future reproductive performance. The calving interval refers to the period from the previous calving date to the current calving date; since a long calving interval makes it difficult to ensure a smooth supply of breeding stock, a shorter calving interval is considered a superior trait. The gestation period refers to the time from when a cow becomes pregnant until she gives birth; since a shorter gestation period can increase calf production, a shorter gestation period is considered a superior trait. The number of artificial inseminations refers to the number of attempts made until pregnancy is achieved; since cows that fail to conceive despite multiple attempts are a major cause of economic loss for farms, a lower number of artificial inseminations is considered a superior trait.

[0036] In addition, in the SNPs of Tables 1 to 5 of the present invention, the SNP located before the diagonal line ( / ) represents a minor allele with low frequency, and the SNP located after the diagonal line ( / ) represents a major allele with high frequency. The Estimates in Tables 1 to 5 represent the regression coefficient (SNP effect) of the SNP for the corresponding trait, representing an additive effect, and represent the expected value when the first allele (minor allele) is replaced by the second allele (major allele).

[0037] Regarding the age at first birth in Table 1, based on an average of approximately 742 days, it can be predicted that the age at first birth is early if the Estimate value based on the minor allele genotype is negative (-), and late if the Estimate value is positive (+). Regarding the interval between births in Table 2, based on an average of approximately 377 days, it can be predicted that the interval between births is short if the Estimate value based on the minor allele genotype is negative (-), and long if the Estimate value is positive (+). Regarding the duration of pregnancy in Table 3, based on an average of approximately 287 days, it can be predicted that the duration of pregnancy is short if the Estimate value based on the minor allele genotype is negative (-), and long if the Estimate value is positive (+). Regarding the number of artificial inseminations in Tables 4 and 5, based on an average of about 1.4 times, it can be predicted that if the Estimate value is negative (-) for the genotype based on the minor allele, the number of artificial inseminations is low, and if the Estimate value is positive (+), the number of artificial inseminations is high.

[0038] In the identification method of the present invention, the method of isolating genomic DNA from a subject (a Jeju Black Cattle cow individual) can be carried out through conventional methods known in the art. For example, it can be carried out by directly purifying DNA from tissues or cells, or by using an amplification method such as PCR to specifically amplify a specific region and isolate it. In the present invention, DNA includes not only DNA but also cDNA synthesized from mRNA. The step of obtaining nucleic acids from a subject may use, for example, PCR amplification, ligase chain reaction, transcription amplification, self-sustained sequence replication system (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878), and nucleic acid sequence-based amplification, but is not limited thereto.

[0039] Determination of the genotype of isolated DNA, that is, analysis of the base sequence, can be performed by various methods known in the art. For example, methods such as determining the nucleotide sequence of the nucleic acid directly by the dideoxy method, or determining / analyzing the nucleotide sequence of the polymorphic site by hybridizing a probe containing the sequence of the SNP site or a probe complementary thereto with said DNA and measuring the degree of hybridization obtained therefrom may be used, but are not limited thereto. The degree of hybridization may be achieved, for example, by labeling the target DNA with a detectable label and specifically detecting only the hybridized target DNA, and other methods such as electrical signal detection may be used, but are not limited thereto.

[0041] The present invention will be explained in detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0043] Example 1. Collection of samples from Jeju Black Cattle cows

[0044] From January 2015 to 2023, samples (blood or hair roots) from Jeju Black Cattle cows were collected from Jeju Black Cattle farms, the Jeju City Livestock Cooperative, the Jeju Livestock Promotion Institute, the National Institute of Animal Science, the Nanjichuksan Experimental Station, and the Jeju Black Cattle Hanwoo Farming Association. Genomic DNA was extracted and a 160K Affymetrix SNP Axiom array was used. In addition, reproductive traits such as age at first calving, gestation period, calving interval, and number of artificial inseminations per conception were collected.

[0046] Example 2. Genome-wide association study (GWAS) and genotyping

[0047] For each SNP obtained through 160K Affymetrix SNP Axiom array analysis, whole-genome association analysis was performed to analyze the association with reproductive traits such as age at first birth, interval between births, gestation period, and number of artificial inseminations per conception.

[0048] For the analysis, single trait analysis was performed on the analyzed traits by running the commercial MTG2 program (https: / / sites.google.com / view / s-hong-lee-homepage / mtg2), which executes a Linear animal mixed model. A G matrix between individuals utilizing SNP information from the animal model (GCTA software) was used, and the following general linear model (animal model) was applied.

[0049]

[0050] [Y: Phenotype, α: Intercept, Covariate (Fixed Effect), β: SNP Effect Size, u: Animal Additive Effects, e: Residual Vector]

[0052] Through the association analysis described above, for each trait, SNPs with genotypes N(AA), N(AB), and N(BB) ≥ 10, the FDR (false discovery rate) value was set to a threshold at a significance level α=0.0001, and -log 10 P SNPs with values ​​greater than the threshold were selected, as they were determined to have a statistically significant association with the corresponding trait.

[0053] As a result, as shown in Tables 1 to 5 below, a total of 138 significant SNPs were identified in four reproductive-related traits, including 10 in the age at first birth, 25 in the interval between births, 12 in the duration of pregnancy, and 91 in the number of artificial inseminations per conception.

[0054]

[0055]

[0056]

[0057]

[0058]

[0060] Among the 138 SNPs mentioned above, 'AX-169395194' appeared identically in the SNPs related to the age at first conception and the SNPs related to the number of artificial inseminations per conception, and the nucleotide sequence information for a total of 137 SNP markers, excluding one duplicate SNP, is shown in Tables 6 to 14 below.

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

Claims

Claim 1 An SNP marker composition for predicting breeding-related traits of Jeju Black Cattle, comprising a total of 137 polynucleotides or complementary polynucleotides composed of at least 8 consecutive nucleotides including a single nucleotide polymorphism (SNP) base located at the 36th position of each of the 137 polynucleotides formed by the nucleotide sequences of SEQ ID NOs 1 to 137. Claim 2 An SNP marker composition for predicting breeding-related traits of Jeju Black Cattle, characterized in that, in claim 1, the consecutive nucleotides are 8 to 100 consecutive nucleotides. Claim 3 An SNP marker composition for predicting reproductive-related traits of Jeju Black Cattle, characterized in that, in claim 1, the reproductive-related trait is one or more traits selected from a group consisting of age at first calving, calving interval, gestation period, and number of artificial inseminations per conception. Claim 4 A microarray for predicting breeding-related traits of Jeju Black Cattle, comprising a total of 137 polynucleotides or their cDNA, each comprising 8 or more consecutive nucleotides including a single nucleotide polymorphism (SNP) located at the 36th position of the 137 polynucleotides formed by the nucleotide sequences of SEQ ID NOs 1 to 137. Claim 5 A primer set composition for predicting breeding-related traits of Jeju Black Cattle, comprising 137 primer sets for amplifying each of a total of 137 polynucleotides, each composed of 8 or more consecutive nucleotides including a single nucleotide polymorphism (SNP) base located at the 36th position of each of the 137 polynucleotides formed by the nucleotide sequences of SEQ ID NOs 1 to 137. Claim 6 A kit for predicting breeding-related traits of Jeju Black Cattle, comprising the primer set composition of claim 5 and a reagent for performing an amplification reaction. Claim 7 A method for predicting reproductive traits of a Jeju Black Cattle cow, comprising: a step of isolating genomic DNA from an individual Jeju Black Cattle cow; and a step of determining the genotype of the base at the 36th SNP (single nucleotide polymorphism) of each of the 137 polynucleotides, each consisting of the base sequences of SEQ ID NOs 1 to 137, using the isolated genomic DNA as a template.

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