AU2020104123A4 - An SNP Molecular Marker for Screening and/or Detecting Bovine Cell Viability - Google Patents
An SNP Molecular Marker for Screening and/or Detecting Bovine Cell Viability Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker for screening and/or detecting bovine
cell viability, in particular, SNPs sites are located at the 19 ,9 7 0th and 3 4 ,3 7 8th bases of the
INCENP gene, and two SNPs site have multiple genotypes combinations in the bovine
population, and the genomic DNA in breeding bull body fluid is extracted to detect the
genotypic combinations of the INCENP gene and predict bovine cell viability. The SNPs of
bovine INCENP gene are successfully screened, and the correlation between the single
nucleotide polymorphisms of bovine INCENP gene and cell viability is clarified for the first
time, a simple and efficient method for detecting bovine cell viability is provided, and a
corresponding detection kit is developed. The kit has the advantages of low cost, high
reliability, high efficiency and the like and can be used for screening early high-quality cattles.
Description
An SNP Molecular Marker for Screening and/or Detecting Bovine Cell Viability
[01] The invention relates to the field of molecular genetic biotechnology of domestic animals, in particular to an SNP molecular marker for screening and/or detecting bovine cell viability.
[02] Milk is of high nutritional value and is an indispensable food in our daily life. Holstein dairy cow in China is one of the most widely raised and milk-producing breeds in the country. In recent years, with the development of molecular genetic technology, Candidate Gene Approach has been widely used in cow breeding. By screening the polymorphic sites on candidate genes and analyzing the correlation between the polymorphic sites and cell quality, molecular markers related to the cell quality can be identified, which provides theoretical guidance for marker assisted selected (MAS). At present, several studies have identified the Single nucleotide polymorphisms (SNP) sites closely related to body fluid quality by genome-wide association analysis. It has been found that these SNP sites can affect cell viability, density and malformation rate.
[03] Changes in any component of a gene can affect gene expression and the functional activity thereof. The single nucleotide polymorphism (SNP) is the most common gene mutation, which mainly refers to the mutation of single base on a DNA sequence, including the transformation, subversion, insertion and deletion of a single base. The coding region SNP (cSNP) may have harmful effects on the structure and function of proteins, which is of great significance in the study of genetic diseases. Promoter region SNP, especially SNP site with transcription factor binding, affects promoter viability and regulates gene expression. In recent years, it has been found that SNPs in some gene promoter regions may change the binding sites of the major gene promoter region regulatory element which affects the quality of bull cells, and change the gene expression pattern, thus affecting the bull cell character. It is more meaningful to study haplotype combinations between multiple polymorphic sites than to explore the association between nucleotide polymorphisms at a single site of a gene and biological character, because this can take into account interactions between polymorphic sites simultaneously.
[04] As a centromere-specific protein, INCENP plays an important role in cell mitosis and meiosis and cytoplasmic division by interacting with microtubules and actin. Research shows that INCENP can guarantee the normal function of chromosome complex and is closely related to cell division, cancer occurrence and spontaneous abortion, and also plays an important role in biological processes. Hering et al. (2014) conducted a genome-wide association analysis of Holstein cows with high and low cell viability, screened all the genes in the 1MB region of a significant marker, and found several candidate genes related to cell quality, where INCENP is as one of the candidate genes, may be closely related to cell function and may play an important role in regulating cell quality. However, there are 3,376 known SNP sites of INCENP gene in Bos Taurus, and the related characters of different SNP sites are often different. How to screen out the SNP molecular markers which can be used to detect the bovine cell viability is a difficult problem in this field. The detection kits for SNP of INCENP gene and its genotypic combination, which affect bovine cells viability, are not reported in the existing technology.
[05] The present invention provides an SNP molecular marker for screening and/or detecting bovine cell viability to solve the problems existing in the above mentioned existing technology.
[06] The present invention provides a set of SNP sites for screening and detecting bovine cell viability, and provides a method for detecting bovine cell viability by INCENP gene SNP, and develops a corresponding detection kit for bull growth. Firstly, the SNP sites of the INCENP gene were detected to determine the genotypic combinations of SNPs sites in individuals, and the correlation between the genotypic combinations and the quality of bull cells were analyzed to identify the genotypic combinations with high bovine cell viability.
[07] The technical scheme of the invention is as follows:
[08] A group of SNP sites: g.19,970 A>G and g.34,378 T>G, the two SNPs are located at the 11, 9 7 0 th 34, 3 7 8 th site bases of the INCENP gene (the first base C of the transcriptional initiation site of INCENP gene is +1).
[09] The above-mentioned SNP sites are used for screening and/or detecting bovine cell viability. Technical personnel in this field should be aware that the SNP site in the application is used for screening and/or detecting bovine cell viability. It is used for screening and/or detecting cattle genotypes with relatively high bovine cell viability. The purpose is to guide early screening of cattle. It is not a disease diagnosis and/or treatment method.
[010] A method for screening and/or detecting bovine cell activity. The DNA form breeding bull body fluid was used as a template, the primer was designed by amplifying DNA sequences containing two SNPs sites. The genotypic combinations were performed according to the SNP sites of the amplified sequences to screen and/or detect the bovine cell viability.
[011] Furthermore, the specific method is: the sequence of the genome DNA PCR amplification primer is as shown in sequence tables SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 6 and can specifically amplify the sequence containing the 1 9 , 9 7 0 th (as shown in SEQ ID NO. 1) and 3 4 , 3 7 8 th (as shown in SEQ ID NO. 4) nucleotides of the INCENP gene respectively;
[012] Furthermore, PCR products were digested with FastDigest restriction endonuclease NdeI and Alu I respectively. The digested products were detected by 2.5% agarose gel electrophoresis, and the genotypes were identified according to the results of electrophoresis. For 19 ,9 0 7 th SNP site of the INCENP gene: after enzyme digestion, the product fragments with the sizes of 606 bp and 110 bp were AA genotypes; the fragments with the sizes of 716 bp, 606 bp and 110 bp were AG genotypes; the fragments with the size of 716 bp were GG genotypes. For the 3 4 3 7 8th
SNP site of the INCENP gene: after enzyme digestion, the product fragments with the size of 205 bp were TT genotypes; the fragments with the sizes of 205 bp, 126 bp and 79 bp were TG genotypes; and the fragments with 126 bp and 79 bp were GG genotypes. Eight genotypic combinations were constructed according to two SNP genotypes, among which the breeding bull with H3H4 (GGTG) genotypic combination had the highest cell viability.
[013] The above-mentioned genomic DNA was extracted from the frozen body fluid of the bull by high-salt method, and the concentration and purity of genomic DNA were detected by a nucleic acid protein analyzer. The DNA was diluted to 50ng/L and stored at 4 °C for use.
[014] The FastDigest restriction endonuclease NdeI used above can identify the
1 9 , 9 7 0 th SNP site of the INCENP gene, and FastDigest restriction endonuclease AluII can identify the 3 4 , 3 7 8 th SNP site of the INCENP gene.
[015] These genotypic combinations were constructed as follows: three genotypes, Hi (AT), H2 (AG), H3 (GT) and H4 (GG), were first identified in the bull population according to the 1 9 , 9 7 0 th and 34,378th SNP sites of the detected INCENP gene in Chinese Holstein breeding bull. Eight genotypic combinations, HIH I(AATT), H1H2 (AATG), H1H3 (AGTT), H1H4 (AGTG), H2H2 (AAGG), H2H4 (AGGG), H3H3 (GGTT), H3H4 (GGTG), were detected in the population after the combination of three genotypes.
[016] Wherein, the cell quality of H3H4 genotype individual bull was significantly higher than that of H1H4 genotype individual bull, and was extremely significantly higher than that of H1H3, H2H4 and H3H3 genotype bulls; the cell activity of H3H4 genotype combination bull individual was significantly higher than that of the H1H3 genotype individual bull. Therefore, the GGTG genotype can be used as a molecular marker to determine bovine cell activity.
[017] Furthermore, the above-mentioned process of genotyping using PCR products can use other conventional methods, such as direct sequencing of PCR products to obtain the genotypes corresponding to the SNP site.
[018] A kit for screening and/or detecting bovine cell activity, which comprises the primer for amplifying two SNPs: g.19,970A>G and g.34,378 T>G of the 1 9 ,9 7 0th and 3 4 , 3 7 8 th bases of the INCENP gene; PCR amplifying reagent.
[019] Furthermore, the above-mentioned primers are upstream primers (as shown in SEQ ID NO. 2) and downstream primers (as shown in SEQ ID NO. 3) used for amplification of genomic fragments containing the 1 9 ,9 7 0th nucleotide of the INCENP gene, and upstream primers (as shown in SEQ ID NO. 5) and downstream primers (as shown in SEQ ID NO. 6) used for amplification of genomic fragments containing the
3 4 , 3 7 8 th nucleotide of the INCENP gene.
[020] Furthermore, a kit for screening and/or detecting bovine cell viability, which is stored at-20 °C. The composition of the kit is shown below:
[021] FastDigest restriction endonuclease NdeI;
[022] FastDigest restriction endonuclease Alul I;
[023] 1OxFastDigest buffer;
[024] 2xTaq PCR MasterMix;
[025] ddH20;
[026] 2,000 bp DNA Ladder Marker.
[027] Upstream primers (as shown in SEQ ID NO. 2) and downstream primers (as shown in SEQ ID NO. 3) used amplification of genomic fragments containing
1 9 9 7 0 th nucleotide of the INCENP gene.
[028] Upstream primers (as shown in SEQ ID NO. 5) and downstream primers (as shown in SEQ ID NO. 6) used for amplification of genomic fragments containing the 3 4 , 3 7 8 th nucleotide of the INCENP gene.
[029] The invention discloses the following technical effects:
[030] (1) In the INCENP gene of Chinese Holstein Bull, a set of SNP molecular markers which can be used for screening and/or detecting bovine cell viability were obtained by screening. The correlation between SNP site g.19,970 A>G and the bull cell viability was first reported.
[031] (2) According to the invention, SNP molecular marker technique was used to assist breeding, and the screening method can avoid the limitations of long breeding cycle and low efficiency in conventional breeding, and has the advantages of high accuracy, high efficiency and low cost. According to the invention, an effective INCENP gene genotype combination GGTG was identified, bovine cell viability of the combination is the highest and the combination can be used for auxiliary growth.
[032] (3) The invention provides a novel applicable detection kit, which is used for screening bull cell individuals with high or low viability by using the 1 9 , 9 7 0th and 3 4 ,3 7 8 th SNP sites of the INCENP gene.
[033] (4) According to the invention, the method is broadly used in practice and can reduce the blindness of raising bulls, reduce the cost and increase the economic benefit of pasture by screening the bull at the early stage.
[034] In order to more clearly illustrate the embodiments of the present invention or the prior technical scheme, the drawings which are required to be used in the embodiments are briefly described below, and it is apparent that the following description are only some embodiments of the present invention, and ordinary technical personnel in the field can also obtain other drawing based on these drawings without any creative effort.
[035] Figure 1: The 1 9 , 9 7 0th and 3 4 , 3 7 8th SNP of INCENP gene of Chinese Holstein cattle.
[036] Figure 2: Agarose gel electrophoresis genotyping for the 1 9 ,9 7 0th and 3 4 ,3 7 8 th SNP sites of INCENP gene of Chinese Holstein cattle
[037] Various exemplary embodiments of the present invention are described in detail, detailed description shall not to be construed as limitation to the invention, but to be construed as a more detailed description of certain aspects, features and implementation schemes of the invention.
[038] It shall be understood that the terminology described in the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. In addition, for the numerical range in the present invention, it shall be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between intermediate values within any stated value or stated range and any other stated value or intermediate values within the stated range is also included within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range.
[039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technical personnel in the field of the present invention. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned in the specification are incorporated by reference for the purpose of disclosing and describing methods and/or materials related to said documents. The contents of the specification prevail in the case of conflict with any incorporated document.
[040] It is apparent to the technical in thefield that various modifications and variations can be made in the specific embodiments of the present specification without departing from the scope or spirit of the invention. Other embodiments obtained from the specification of the invention are apparent to the technical in the field. The specification and embodiments of the application are exemplary only.
[041] The terms "include", "comprise", "have", "contain" and the like, as used herein, are intended to be open terms, namely, referring to include but not limit to.
[042] Embodiment 1 Identification of SNP site and genotypic combinations in bovine INCENP gene
[043] According to the invention, direct sequencing and PCR-RFLP are adopted to identify the SNP of bovine INCENP.
[044] 1.1 Experimental materials
[045] According to the invention, the bulls are selected from three domestic bull stations: Beijing Dairy Cattle Center (83), Shanghai Bright Holstan co., Ltd. (56), and Shandong Ox Breeding Bull Station (125). The body fluids of the bulls are stored in liquid nitrogen for use.
[046] 1.2 Extraction of Genomic DNA from Frozen Body Fluid
[047] The genomic DNA of frozen body fluid was extracted by high salt method. The purity and concentration of DNA samples were detected by a nucleic acid protein analyzer. The DNA sample was finally diluted to 50 ng/ l, and stored at 4°C for use.
[048] 1.3 Design of primers
[049] Based on the bovine INCENP gene sequence (login number: AC_000186.1) provided by NCBI, Primer Premier 5.0 was used to design the primers. The sequences of two pairs of designed primers are as follows:
[050] The first pair of primers:
[051] 19970F: GGGCCGGGAAGTTACAGAAC
[052] 19970R: CAAGGCAGCAGCTTAGGTCT
[053] The second pair of primers:
[054] 34378F: GCCTACAGCCTGAAGAAG
[055] 34378R: AGTGCTGTCCACAGACCA
[056] 1.4 PCR amplification reaction
[057] The PCR reaction system of 25 L was: 2xPower Taq PCR MasterMix 12.5 t; the upstream primer and downstream primer were 0.5tl with a concentration of 10 pmol/1 respectively; the template was 1 1 with a concentration of 50 ng/ l; ddH20was supplemented to 25 d.
[058] PCR reaction procedure: pre-denaturation 95°C, 5 min; denaturation 95°C, sec, annealing 59°C, 30 sec, extension 72°C, 65 se, 35 denaturation-annealing extension cycles; final extension for 10 min at 72 °C.
[059] 1.5 Detection of SNP
[060] PCR products were detected by 1% agarose gel electrophoresis. After being identified as target bands, the PCR amplification products were sent to Beijing Liuhe Huada Gene Technology Company for two-way sequencing. The results of sequencing were compared with the reference sequence of INCENP gene on GenBank by DNAMAN, and by combining the base calling, the SNP sites on the target fragment were determined respectively as follows: g. 19,970 A>G and g. 34,378 T>G.
[061] 1.6 PCR-RFLP genotyping
[062] FastDigest restriction endonuclease NdeI can identify the 1 9 9 7 0th SNP site of the INCENP gene.
[063] FastDigest restriction endonuclease Alul I was used to identify the 34 ,3 7 8th
SNP site of the INCENP gene.
[064] After being detected as the target fragment, the PCR product was digested for one hour (37 °C) by corresponding restriction endonuclease. Enzyme digestion reaction system (30 l): 10 1 of PCR product; 1 1 of restriction endonuclease with a concentration of 10 U/ l; 2 1 of 10xbuffer; Supplementation to 30[ with ddH20. The enzyme-digested products were detected by 2.5% agarose gel electrophoresis. Different genotypes were identified according to the bands obtained by electrophoresis. After g. 19,970 A> G was identified by restriction endonuclease NdeI, the fragments with the sizes of 606 bp and 110 bp were AA genotypes; the fragments with the sizes of 716 bp, 6bp and 110 bp were AG genotypes; the fragments with the size of 716 bp were GG genotypes. After g. 34,378 T>G was identified by restriction endonuclease Alul, the fragments with the size of 205 bp were TT genotypes; the fragments with the sizes of 205 bp, 126 bp and 79 bp were TG genotypes; the fragments with the sizes of 126 bp and 79 bp were GG genotypes.
[065] 1.7 Construction of haplotypes
[066] Four haplotypes were identified in the experimental population: HI (AT), H2 (AG), H3 (GT) and H4 (GG). Eight genotypic combinations were found in the population: HIHI (AATT), H1H2 (AATG), H1H3 (AGTT), H1H4 (AGTG), 112H2 (AAGG), H2H4 (AGGG), H3H3 (GGTT), H3H4 (GGTG).
[067] Embodiment 2 Analysis of the association between the haplotype combination of bovine INCENP gene and bovine cell viability
[068] SAS 9.0 was used to compare the correlation between different genotypic combinations of bovine INCENP gene and cell viability. The model is:
[069] Yijk=t+Gi+Yj+Hk+ eijk
[070] Wherein, Yijk is the observed value of bovine cell viability character; p is the population average; Gi is the fixed effect of haplotype combination; Yj is the fixed effect of seasons; Hk is the fixed effect of the session; eijk is the random residual effect.
[071] The results of haplotype combination association showed that, in 8 genotypic combinations, the cell count of bull individuals of H3H4 genotypes was significantly higher than that of bull individuals of H1H4 genotypes (P<0.05), and extremely significantly higher than that of bull individuals of H1H3, H2H4 and H3H3 genotypes (P<0.01). The cell viability of bull individuals of H3H4 genotype combination was significantly higher than that of bull individuals of H1H3 genotype (P<0.05).
[072] Table 1 Analysis of correlation between different genotypic combinations of bovine INCENP gene and bovine cell viability
Haplotype combination Sample size Ejaculate volume Fresh sperm viability Haplotype combination Sample size Ejaculate volume/mL Fresh sperm viability(%) H11 3 6.51±1.56 70.10±7.77 111112 6 6.32±1.10 66.60±5.49 H1H3 12 4.95±0.90Aa 57.14±3.88Bb H1H4 114 6 .0 9 ±0.2 6b 67.75±1.28a 112112 78 6.67±0.32 65.76±1.55a H2H4 12 5 . 1 8 ±0. 7 8 B 66.06±3.88 H3H3 33 5.58±0.47B A 113114 6 8 . 8 8 ±1. 10 Aa 71.61±5.49a
[073] Note: H1=AT; H2=AG; H3=GT; H4=GG; There is a significant difference between the average values marked with different lowercase letters (a, b, c, d) in the same column (P <0.05). There is an extreme significant difference between the average values marked with different uppercase letters (A, B) (P<0.01).
[074] SAS software was used to analyze the correlation between different genotypic combinations of 1NCENP and body fluid quality character of Chinese Holstein bulls. The results showed that (table 1) the cell amount of H3H4 genotype bull individual was significantly higher than that of H1H4 genotype bull individual (P<0.05), and extremely significantly higher than that of H1H3, H2H4 and H3H3 genotype bull individuals (P<0.01). The cell viability of H3H4 genotype combination bull individual was significantly higher than that of HH3 genotype bull individual (P<0.05). Therefore, the GGTG genotype can be used as a molecular marker to determine the quantity and viability of bull cells. As a result, the technology can guide the early screening of bulls, reduce the blindness of dairy cattle feeding, reduce the breeding cost and increase the economic benefit of cattle farm. The technique of SNP molecular marker-assisted growth of bovine INCENP can be widely used in the future.
[075] Embodiment 3 detection kit
[076] As shown in embodiment 1, A>G at the 1 9 ,9 7 0 * site and T>G at the
3 4 , 3 7 8 th site of the bovine INCENP gene are both associated with bovine cell viability. Therefore, it is possible to develop an INCENP gene SNP molecular marker detection kit applicable for screening bovine cell viability. The specific primers of bovine INCENP gene can be designed based on the two SNP sites for amplification and detection.
[077] Preparation of a kit for detecting bovine cell viability. Detailed components are shown in Table 2:
[078] Table 2 Components of kit for detecting sperm viability of breeding bulls
Description Sequences Concentration
19970F: Primer used to amplify a GGGCCGGGAAGTTACAGAAC fragment containing the 1 9 ,9 7 0th nucleotide of the 19970R: INCENP gene. CAAGGCAGCAGCTTAGGTCT 10 pmol/L 34378F: GCCTACAGCCTGAAGAAG 10 pmol/L
Primer used to amplify a 34378R: AGTGCTGTCCACAGACCA fragment containing the 34,378* nucleotide of the INCENP gene. 2x Taq PCR MasterMix PCR reaction system ddH20 FastDigest restriction endonuclease NdeI 10U/pi FastDigest restriction endonuclease AlulI I1OU/pl 1OxFastDigest buffer Enzymic reaction system ddH20 DNA Marker 2000 bp DNA Ladder Marke
[079] Firstly, the body fluid of cattle was collected and stored it in liquid nitrogen for use, and the total DNA of frozen liquid was extracted by using a high-salt method. PCR amplification was carried out by using the components of the above-mentioned kit with the frozen liquid DNA as a template. Then the reaction was carried out according to the PCR system and conditions described in embodiment 1. The PCR reaction system of 25 pL: 12.5 pl of 2xPower Taq PCR MasterMix; 0.5 1 of upstream and downstream primers with a concentration of 10 tmol/l; 1 l of template with a concentration of50 ng/gl supplementation to 25pl with ddH20. PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec, extension at 72°C for 65 sec, 35 denaturation-annealing extension cycles; final extension at 72°C for 10 min. PCR-RFLP method was used to carry out genotyping on the PCR amplification products. Specific conditions and systems for enzyme digestion are the same as those described in embodiment 1. Enzyme digestion reaction system (30 pl): 1Opl of PCR product; 1pl of restriction endonuclease with a concentration of 10 U/pl; 2 pl of 10xbuffer; supplementation to 30 pl with ddH20, carrying out enzyme digestion reaction at 37°C with digestive enzyme digestion system for lh. The enzyme digestion products were detected by 2.5% agarose gel electrophoresis.
[080] As described in embodiment 1: after g.19,970 A> G was identified by restriction endonuclease NdeI, the fragments with the sizes of 606 bp and 100 bp were AA genotypes; the fragments with the sizes of 716 bp, 606 bp and 110 bp were AG genotypes; the fragments with the size of 716 bp were GG genotypes. After g. 34,378 T>G was identified by restriction endonuclease Alul, the fragments with the size of 205 bp were TT genotypes; the fragments with the sizes of 205 bp, 126 bp and 79 bp were TG genotypes; the fragments with the sizes of 126 bp and 79 bp were GG genotypes. Then, the cell viability of each genotypic combination individual was analyzed according to the method described in embodiment 1 to determine whether to keep the breed or not.
[081] Here, although direct sequencing can be used to detect two mutations of bovine INCENP gene g. 34,378 T>G and g. 34,378 T>G. However, direct sequencing is expensive and time-consuming, is long in period and is not suitable for detection of large batches of samples. The kit can well compensate for the defects of direct sequencing and provide technical and theoretical guidance for early breeding of breeding bulls.
[082] Examples with practicability of the invention:
[083] 1) The invention provides a method for indirect detection of bovine cell viability by identifying single nucleotide polymorphism of bovine INCENP gene. The method can be used to analyze the correlation between 2 SNP sites on bovine INCENP gene and bovine cell viability, and can be further used as a basis for early diagnosis of bovine cell viability.
[084] 2) The efficient method for detecting the sites related to association between bovine INCENP gene polymorphism and bovine cell viability can be similarly used in a kit for gene diagnosis of bovine cell viability.
[085] In conclusion, the polymorphic sites g. 34,378 T>G and g. 34,378 T>G of bovine INCENP gene are both significantly correlated with the bovine cell viability. Based on the correlation, the detection kit was developed and applied to gene diagnosis in practice.
[086] The invention provides a method for indirect detection of bovine cell viability by identifying single nucleotide polymorphism of bovine INCENP gene. According to the invention, only a very small amount of bovine genomic DNA was used to efficiently identify the SNP sites of bovine INCENP gene, the genotypic combinations were built, the effect of these genotypic combinations on the bovine cell viability was analyzed. The invention also provides a kit applicable for detecting the bovine cell viability. Finally, the invention provides a method and kit for detecting bovine cell viability by using the SNP molecular marker of the INCENP gene.
[087] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.
[088] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable
Claims (10)
1. A set of SNP sites, which are characterized in that the SNPs sites are g. 19,970 A>G of the 1 9 , 9 7 0 th base and g. 34,378 T>G of the 3 4 ,3 7 8 th base of the bovine INCENP gene.
2. Application of the SNP site according to claim 1 for screening and/or detecting bovine cell viability.
3. A method for screening and/or detecting bovine cell viability, which is characterized by identifying the genotypes of the 1 9 , 9 7 0 th and 3 4 ,3 7 8 th SNPs sites of bovine INCENP gene according to the SNP sites according to claim 1, and predicting the bovine cell viability according to the genotypic combinations of the two SNPs sites
4. A method according to claim 3, which is characterized in that the genotypic combinations are: two SNP sites include four haplotypes-AT, AG, GT, GG, and there are genotypic combinations in the bull population-AATT, AATG, AGTT, AGTG, AAGG, AGGG, GGTT, GGTG.
5. A method according to claim 4, which is characterized in that the cell viability of the breeding bull is highest when the genotype of the 1 9 ,9 7 0th and 3 4 , 3 78 th SNPs sites of breeding bull INCENP gene is GGTG
6. A method according to claim 3-5, which is characterized in that the primers were designed to amplify the DNA sequences containing two SNP sites using bull body fluid DNA as a template. The sequences of the primers were as follows: the sequence of the primer for amplification of the fragment containing the 1 9 ,9 7 0th nucleotide of INCENP gene is as shown in SEQ ID NO.2 and SEQ ID NO.3; the sequence of the primer for amplification of the fragment containing the 3 4 ,3 7 8th nucleotide of INCENP gene is as shown in SEQ ID NO.5 and SEQ ID NO.6
7. A primer group for screening and/or detecting the bovine cell viability, which is characterized in that the primer group includes: the primer for amplification of the fragment containing the 1 9 , 9 7 0 th nuleotide of INCENP gene, as shown in SEQ ID
NO.2 and SEQ ID NO.3; the primer for amplification of the fragment containing the 3 4 ,3 7 8 th nucleotide of INCENP gene, as shown in SEQ ID NO.5 and SEQ ID NO.6;
8. Application of the primer group according to claim 7 for the preparation of a kit for screening and/or detection bovine cell viability.
9. A detection kit for screening bull cell viability, which is characterized in that the kit comprises an upstream primer and a downstream primer used for amplification of a genome fragment containing the 1 9 , 9 7 0th nucleotide of INCENP gene; an upstream primer and a downstream primer used for amplification of a genome fragment containing the 34 ,3 7 8th nucleotide of INCENP gene; and a PCR amplification reagent.
10. A kit according to claim 9, which is characterized in that the kit includes: PCR amplification reagent; an upstream primer as shown in SEQ ID NO. 2 and a downstream primer as shown in SEQ ID NO. 3 used for amplification of a genome fragment containing the 1 9 , 9 7 0 th nucleotide of INCENP gene; an upstream primer as shown in SEQ ID NO. 5 and a downstream primer as shown in SEQ ID NO. 6 used for amplification of a genome fragment containing the 3 4 ,3 7 8th nucleotide of INCENP gene; restriction endonuclease NdeI; restriction endonuclease Alul; enzyme digestion buffer.
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