Molecular marker for screening and / or detecting retained fetal membranes resistance of dairy cow and application of molecular marker

Abstract

The invention belongs to the technical field of genetic breeding and molecular biology, and particularly relates to a molecular marker for screening and / or detecting retained fetal membranes resistance of dairy cows and application of the molecular marker. Specifically, aiming at the hereditary characteristic of the retained fetal membranes of the dairy cow, a large-group Holstein dairy cow blood sample is collected, a Bovine SNP chip (50K) is utilized to carry out genetic typing and whole gEBV evaluation on a cow individual, aiming at an estimated breeding value of the retained fetal membranes of the dairy cow, the estimated breeding value of the retained fetal membranes of the dairy cow and genome SNPs are subjected to correlation analysis, and the genetic typing result of the retained fetal membranes of the dairy cow is obtained. And the SNP site, namely rs42756603, which is remarkably related to the retained fetal membranes resistance of the dairy cow is screened out. According to the method, individuals with retained fetal membranes and high resistance can be effectively screened out, the reproductive disorder of retained fetal membranes of dairy cows can be improved from the genetic level, the population genetic improvement process of the dairy cows is accelerated, and therefore good economic and social benefits are generated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic breeding and molecular biology, and particularly relates to a molecular marker for screening and / or detecting dairy cow retained placenta resistance and application. BACKGROUND

[0002] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is publicly known.

[0003] Retained fetal membranes (RFM) is a common reproductive disorder in dairy cows, which refers to the inability to normally expel the fetal membranes within 24 hours after delivery, thereby causing endometritis. The incidence of retained fetal membranes in dairy cows is 10% to 30%, and it is reported that the incidence of retained fetal membranes is even as high as more than 50% in summer. The retained fetal membranes in the uterus will be corrupted, causing endometritis, slow recovery of the uterus, and even prolapse of the uterus, affecting re-pregnancy and milk production, and if the infection is serious, it can also lead to mastitis, septicemia, etc., endangering the life of the dairy cow. Therefore, retained fetal membranes in dairy cows has always been one of the problems that seriously affect the development of the dairy industry, causing huge economic losses to the dairy industry. There are many methods for treating RFM in dairy cows, such as antibiotic drug treatment and surgical stripping, but this is ultimately a temporary solution rather than a permanent solution.

[0004] The bovine placenta is also known as cotyledon placenta, and the placental cotyledon is disc-shaped and wrapped in the maternal cotyledon to form placental processes (80-120) in a mushroom structure. This type of placenta is closely inlaid and is not easy to fall off quickly due to uterine contraction. The characteristic of this placental structure is an intrinsic factor of retained placenta. Dairy cow retained placenta is closely related to excessive stretching of the myometrium and impaired muscle tension. The heritability of RFM is low, and it is difficult to bring about genetic improvement in the short term through conventional breeding methods. Therefore, through the means of molecular marker assisted breeding, and from the genetic point of view, the molecular regulation mechanism of dairy cow retained placenta is studied to provide a theoretical basis for accelerating the genetic improvement of dairy cow populations. However, the inventors have found that there is no GWAS association analysis research based on SNP chips or whole genome resequencing for the phenotype of dairy cow RFM, and the molecular regulation mechanism is still unclear, and the key functional genes still need to be excavated. SUMMARY

[0005] In view of the above deficiencies of the prior art, the inventors provide a molecular marker for screening and / or detecting dairy cow dystocia resistance and application thereof. Specifically, the inventors collect 2709 blood samples of Holstein dairy cows, genotype the bovine individuals by using a Bovine SNP50 chip, obtain the estimated breeding value of the dystocia trait of each bull, analyze the gEBV of the dairy cow dystocia and the genotype by using a general linear model of GEMMA software, correct the P value of multiple tests by using an FDR method, screen the gene and molecular marker of the healthy dairy cow, and establish a corresponding detection method. Based on the above research results, the present application is completed.

[0006] To achieve the above technical purposes, the present application adopts the following technical solutions. In a first aspect, the present application provides CPE The application of the gene as a molecular marker for screening and / or detecting dairy cow dystocia resistance.

[0007] The gene includes CPE The gene includes CPE The 7th intron of the gene further comprises a SNP site chr17:g.692341_T>C site, i.e., rs42756603, which is significantly related to the dairy cow dystocia resistance, and the reference genome is Bos taurus UMD3.1.1.

[0008] The rs42756603 is a SNP site on the gene of the dairy cow, and the genotype of the rs42756603 is TT, TC and CC. CPE The rs42756603 is a SNP site on the gene of the dairy cow, and the genotype of the rs42756603 is TT, TC and CC.

[0009] In a second aspect, the present application provides the use of a substance for detecting the above molecular marker in the preparation of a product for screening and / or detecting the level of dairy cow dystocia resistance.

[0010] Specifically, the present application proves that the gEBV of the TT homozygous dairy cow is significantly higher than that of the CC homozygous dairy cow, indicating that TT is a high-resistance genotype of the dairy cow dystocia.

[0011] In a third aspect, the present application provides a product for screening and / or detecting the level of dairy cow dystocia resistance, which comprises a substance for detecting CPE The gene.

[0012] In a fourth aspect, the present application provides a system for screening and / or detecting the level of dairy cow dystocia resistance, which comprises at least: An acquisition unit configured to acquire the SNP site information of the above CPE The gene. an evaluation unit configured to determine the dystocia resistance level of the test cow according to the SNP site information obtained by the acquisition unit; In the acquisition unit, the test cow sample is a blood sample of the test cow.

[0013] The evaluation unit specifically evaluates the process as follows: when the genotype of SNP site rs42756603 is TT, it indicates that the test cow is a high-dystocia-resistant cow; when the genotype of the SNP site rs42756603 is CC, it indicates that the test cow is a low-dystocia-resistant cow; and when the genotype of the SNP site rs42756603 is TC, it indicates that the test cow is of medium dystocia resistance.

[0014] In a fifth aspect of the present application, the above-mentioned application, product, and system are applied in any one or more of the following: (a1) screening high-dystocia-resistant cows; (a2) cow breeding.

[0015] In a sixth aspect of the present application, a method for screening high-dystocia-resistant cows is provided, which comprises judging the dystocia resistance of the cow by using the above-mentioned product or system, thereby screening high-dystocia-resistant cows.

[0016] In a seventh aspect of the present application, a method for cow breeding is provided, which comprises genetically breeding high-dystocia-resistant cows screened by the method.

[0017] Compared with the prior art, the above-mentioned one or more technical solutions have the following beneficial effects: The above-mentioned technical solution is aimed at the genetic characteristics of the occurrence of cow dystocia, collects blood samples of a large group of Holstein cows, uses a Bovine SNP chip (50K) to genotype and evaluate the full gEBV of the individual cow, uses a general linear model of GEMMA software to perform correlation analysis on the estimated breeding value of the cow dystocia resistance and the genomic SNPs, and screens out the SNP site rs42756603 significantly related to the cow dystocia resistance.

[0018] The above-mentioned method can effectively screen out individuals with high dystocia resistance, which is of great significance to the breeding and genetic improvement of high-dystocia-resistant cows. The above-mentioned solution is a good application of molecular breeding technology in production practice, which can improve the reproductive disorder of cow dystocia from the genetic level and accelerate the genetic improvement process of the cow population, thereby producing good economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the application, and their

[0020] Figure 1 Genes with the strongest signal in the genome-wide association analysis of the bovine GEMMA software in Example 1 of the present application CPE and its schematic diagram of genomic structure.

[0021] Figure 2 Genes with the strongest signal in the genome-wide association analysis of the bovine GEMMA software in Example 1 of the present application CPE SNP detection peak map of the 7th intron of the gene.

[0022] Figure 3 Genes with the strongest signal in the genome-wide association analysis of the bovine GEMMA software in Example 1 of the present application CPE ESE motif score of the TT genotype and the CC genotype of the gene.

[0023] Figure 4 Genes with the strongest signal in the genome-wide association analysis of the bovine GEMMA software in Example 1 of the present application CPE RFLP genotyping electropherogram of the SNP of the gene. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed description is merely exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0025] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. In addition, the molecular biology methods not specifically explained in the embodiments are the methods commonly used in the art, and the specific operations can be referred to the molecular biology guide or product manual. In the present application, the bull sperm motility refers to the fresh semen motility of the bull.

[0026] As described above, by means of molecular marker assisted breeding, and from the genetic point of view, the molecular regulation mechanism of the retained placenta of the dairy cow is studied, so as to provide a theoretical basis for accelerating the genetic improvement of the dairy cow population.

[0027] The inventors found through previous studies that CPECarboxypeptidases E (CPE) encoded by the gene can process hormone precursors into mature forms, such as CPE is involved in the processing of various peptide hormones by enzymatic cleavage, such as adrenocorticotropic hormone, insulinotropic and gonadotropin-releasing hormone. CPE is expressed in the placenta of normal people, which can process placental secreted steroid hormones and peptide hormones. CPE fat The fat mouse is a spontaneous ser202pro point mutation of the CPE protein, which reduces the stability of CPE and causes its degradation, and reduces the activity of CPE. CPE and carboxypeptidase D (CPD) - another very similar carboxypeptidase, are strictly co-localized in the mouse placenta from the middle, late and full-term of pregnancy, and the deletion of CPE leads to an occasional but significant placental phenotype characterized by an increase in the number of placental giant cells and glycogen cells and hypertrophy of giant cells. The above studies show that CPE is closely related to placental development during mammalian pregnancy.

[0028] Therefore, in one typical embodiment of the present application, there is provided CPE The application of the gene as a molecular marker for screening and / or detecting the resistance of dairy cows to retained placenta.

[0029] The application of the gene as a molecular marker for screening and / or detecting the resistance of dairy cows to retained placenta. CPE The gene includes CPE The 7th intron of the gene. Further comprising a SNP site, i.e., rs42756603, chr17:g.692341_T>C site, which is significantly related to the resistance of dairy cows to retained placenta, with reference to the Bos taurus UMD3.1.1 reference genome.

[0030] The rs42756603 is a SNP site on the gene of dairy cows, and the genotype of the rs42756603 is TT, TC and CC. CPE The rs42756603 is a SNP site on the gene of dairy cows, and the genotype of the rs42756603 is TT, TC and CC.

[0031] In another embodiment of the present application, there is provided the use of a substance for detecting the above-mentioned molecular marker in the preparation of a product for screening and / or detecting the resistance of dairy cows to retained placenta.

[0032] Specifically, the present application proves by research that the gEBV of dairy cows with TT homozygous genotype of the rs42756603 site is significantly higher than that of dairy cows with CC homozygous genotype, indicating that TT is a high-resistance genotype of dairy cows to retained placenta.

[0033] In another embodiment of the present application, there is provided a product for screening and / or detecting the resistance of dairy cows to retained placenta, which comprises a substance for detecting CPE The gene.

[0034] In one embodiment of the present application, the substance for detecting CPEThe genetic material includes, but is not limited to, detection CPE Primers and probes of the SNP site of the gene; The products include, but are not limited to, gene chips, nucleic acid preparations, detection kits, and related devices, equipment, etc.

[0035] Further, the primer can be a PCR primer for amplifying the DNA fragment of rs42756603.

[0036] Further, the PCR primer is shown as SEQ ID NO. 1-2.

[0037] Further, the product is a product obtained based on a restriction fragment length polymorphism (RFLP) method.

[0038] In another specific embodiment of the present application, a system for screening and / or detecting the resistance level of dairy cow dystocia is provided, and the system at least includes: An acquisition unit configured to acquire the SNP site information of the above-mentioned CPE Gene in the sample of the test dairy cow; An evaluation unit configured to determine the dystocia resistance level of the test dairy cow according to the SNP site information obtained by the acquisition unit; In the acquisition unit, the sample of the test dairy cow is a blood sample of the test dairy cow. The test dairy cow is a test cow, specifically a Holstein dairy cow.

[0039] The SNP site is specifically rs42756603, and the SNP site information includes the polymorphism or genotype of the SNP.

[0040] The evaluation unit specifically evaluates the process, which includes: when the genotype of the SNP site rs42756603 is TT, it indicates that the test dairy cow is a high-resistance dairy cow; when the genotype of the SNP site rs42756603 is CC, it indicates that the test dairy cow is a low-resistance dairy cow; and when the genotype of the SNP site rs42756603 is TC, it indicates that the test dairy cow is of medium resistance.

[0041] In another specific embodiment of the present application, the above-mentioned application, product, and system are applied in any one or more of the following: (a1) screening high-resistance dairy cows for dystocia; (a2) dairy cow breeding.

[0042] In still another embodiment of the present application, a method for screening a dairy cow with high resistance to retained placenta is provided, which comprises judging the resistance of the dairy cow to retained placenta by using the product or system described above, thereby screening a dairy cow with high resistance to retained placenta.

[0043] In still another embodiment of the present application, a method for breeding dairy cows is provided, which comprises genetically breeding a dairy cow with high resistance to retained placenta by the screening method described above.

[0044] The present application is further described below in connection with specific examples. The following examples are intended to explain the present application and are not intended to limit the content thereof. If the specific conditions of the experiments are not indicated in the examples, the conventional conditions or the conditions recommended by the sales company are generally used; and if the present application is not particularly limited, it can be purchased through commercial channels.

[0045] Example 1 Screening of SNPs related to resistance to retained placenta in Holstein dairy cows Collection of blood samples and extraction of DNA from Holstein dairy cows Blood samples were collected from 2709 Holstein dairy cows in 11 large-scale farms. The locations of the farms and the number of samples collected from each farm are shown in Table 1.

[0046] Table 1. Locations of dairy farms and number of samples collected

[0047] 2. Genotypes of the collected samples were determined using Illumina BovineSNP50 chips. The genotypes were determined by NuGen, and a total of 47843 SNP marker sites were obtained. The gEBV of each individual for retained placenta was estimated based on the genotypes. The gEBV was provided by NuGen.

[0048] 3. Genome-wide association analysis using GEMMA software Genome-wide association analysis was performed on the whole genome SNP sites and the gEBV of retained placenta using GEMMA software. The mixed linear model was used in the analysis model, and the farm and the age of the cow were used as covariates. Multiple test correction was performed using the FDR method. The most significant SNP site was located on the 7th intron of the gene, with the most significant P value and FDR value (P value 2.21E-07, FDR value 0.002442958) CPE Figure 1 ).

[0049] ​Screening strategy based on candidate genes The SNPs with the most significant P value associated with the retained placenta trait of dairy cattle were screened, and the annotated Refseq genes within each selected region defined by the SNP were retrieved using the UCSC Genome Browser. The genes within 50Kbp upstream and downstream of each significant SNP were screened, and the SNP with the 5th P value of significance test (chr17:692341_T>C) was screened, with a significant P value of 2.21E-07 and an FDR value of 0.0024. The gene annotation result showed that the SNP was located in the 7th intron of the gene rs42756603. CPE rs42756603.

[0050] Further, the distribution of the rs42756603 site in 2709 Holstein dairy cows was analyzed, of which 680 individuals carried the TT homozygous genotype, the average gEBV of the dairy cows with retained placenta was 0.0143; 1434 individuals carried the TC heterozygous genotype, the average gEBV of the dairy cows with retained placenta was 0.0268; and 592 individuals carried the CC homozygous genotype, the average gEBV of the dairy cows with retained placenta was -0.0140.

[0051] Example 2 Verification CPE Correlation between the 7th intron SNP of the gene and the resistance to retained placenta of dairy cows 1. Blood sample collection and gEBV collection of the Holstein cow verification population The blood samples of Holstein cows from Shandong Dongying, Shandong Dezhou and Hebei Xingtai were re-collected, with 390, 289 and 321 heads respectively, a total of 1000 heads. These cows had been tested by SNP chip in Nuoyin Company and had gEBV of retained placenta, and were used as the verification population. 2. Extraction of blood genomic DNA The blood genomic DNA was extracted by phenol-chloroform method. By organic solvent extraction, the impurities such as protein, polysaccharide and phenol were removed, and then the nucleic acid was separated by adding ethanol precipitation.

[0052] 3. CPE Amplification of the 7th intron SNP site of the gene Primers were designed upstream and downstream of the rs42756603 site, and the upstream and downstream gene sequences of the SNP were amplified by polymerase chain reaction (PCR). The primer sequences are as follows: F: 5‘-GCTTGGGGACTTGAGAATATCCA-3’ (SEQ ID NO. 1) R: 5‘-TTATCATAAAGGACCTGACTTTGCT-3’ (SEQ ID NO. 2) The PCR reaction primarily used a high-purity, thermostable DNA polymerase—2×Taq Master Mix (Dye Plus) (P111, Novizan). SEQ ID NO.1 and SEQ ID NO.2 were used as F and R primers for PCR amplification. The reaction system (25 μL system) was as follows: 2×Taq Master Mix: 12.5 μL, primer F (10 μM): 0.5 μL, primer R (10 μM): 0.5 μL, genomic DNA: 1 μL, ddH2O: 10.5 μL.

[0053] PCR reaction conditions were as follows: 95℃ pre-denaturation: 5 min; cycles of 95℃ denaturation: 30 s, 59℃ annealing: 30 s, 72℃ extension: 52 s (849 bp, 1 Kbp / min), repeated 35 times; final extension at 72℃: 5 min. Amplification was performed using an Applied Biosystems PCR instrument. Electrophoresis was performed on 1% agarose gels to analyze the amplification results, which were then sent to a sequencing company for Sanger sequencing. The Sanger sequencing results and peak diagrams were analyzed to genotype each cow. Analysis yielded peak diagrams for the TT, TC, and CC genotypes. Figure 2 ).

[0054] 4. CPE Association analysis of gene SNP loci with gEBV in retained placenta in dairy cows Based on the genotypes of 1000 dairy cows, analysis of variance was performed on genotypes and gEBV using SAS (version 8.02) software. The differences in gEBV for retained placenta in dairy cows with TT, TC, and CC genotypes were statistically analyzed to screen for genotypes associated with high resistance to retained placenta. The results are shown in Table 2. The gEBV for retained placenta in homozygous TT dairy cows was significantly higher than that in homozygous CC dairy cows (p<0.05), indicating that TT is the genotype for high resistance to retained placenta in dairy cows.

[0055] Table 2. Genotype frequencies of the rs42756603_T>C locus in Holstein dairy cattle and their association with gEBV in retained placenta.

[0056] 5. rs42756603_T>C gene for resistance to placental rejection in dairy cows. CPE Effects of variable shear In the NCBI database, g.692341_T>C (rs42756603) was detected located in CPEat the junction of the 7th intron and the 8th exon, and with alternative splicing transcript skipping the 8th exon. This site is a mutation of T>C that changes the binding site of SRSF6 predicted by alternative splicing factor (ESEfinder) Figure 3 and Table 3). This indicates that this SNP site can affect the alternative splicing and expression of CPE , thereby affecting CPE to play a role in the process of fetal membrane discharge.

[0057] Table 3 CPE ESE motif score of TT genotype and CC genotype of gene

[0058] Example 3 Cattle fetal membrane non-discharge resistance gene CPE Method for detecting SNP A method for detecting SNP (rs42756603_T>C) of cattle CPE A restriction fragment length polymorphism (RFLP) method for SNP (rs42756603_T>C) of cattle was established. Cattle genomic DNA was extracted, and the upstream and downstream gene sequences of SNP (rs42756603_T>C) were amplified by PCR using primers of SEQ ID NO. 1 and SEQ ID NO. 2. The PCR amplification system was as described in Example 2. The PCR product was subjected to agarose gel electrophoresis, and then the gel was cut and recovered using a gel recovery kit (DP214, Tiangen). The concentration and quality of the gel recovery product were detected using a Nanodrop 2000 (Thermo).

[0059] The PCR gel recovery product was digested with restriction endonuclease SfcI. The enzyme digestion system is shown in Table 4. All components were added to a 200 μL PCR tube according to Table 4, mixed gently, centrifuged briefly, and then placed in a PCR instrument for incubation at 37°C for 1 h and at 65°C for 20 min. After incubation, the digested product was subjected to agarose gel electrophoresis, and 1% agarose gel was used for electrophoretic band detection. The SfcI cutting sequence is CTRYAG, wherein the SNP sequence of the TT genotype is C T ACAG (“_” is the SNP site), which can be cut; the SNP sequence of the CC genotype is C C ACAG (“_” is the SNP site), which cannot be cut. Therefore, the band interpretation rule is: TT genotype, two bands, 260 bp and 590 bp; CC genotype, only one band, 850 bp; TC genotype, three bands, 850 bp, 590 bp and 260 bp (see Figure 4 ).

[0060] Table 4 Restriction endonuclease SfcI digestion system

[0061] 2. The kit for detecting the resistance of dairy cow to dystocia is also disclosed in the embodiment.

[0062] The kit comprises sampling tools, a genomic DNA extraction kit, PCR reaction reagents, SfcI restriction endonuclease and the like. The kit also comprises PCR amplification reagents and enzyme digestion reagents.

[0063] Specifically, the PCR amplification reagents mainly comprise 2×Taq Master Mix, primers (SEQ ID NO. 1 and SEQ ID NO. 2), ddH2O and the like; the enzyme digestion reagents comprise SfcI enzyme (10 U / µl), SfcI enzyme buffer, ddH2O and the like.

[0064] The above merely describes the preferred embodiments of the present application but should not be used to limit the present application. Various modifications and changes can be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. CPE The application of genes as molecular markers for screening and / or detecting resistance to placental retention in dairy cows; among which... The CPE Genes include CPE The 7th intron of the gene; further containing the SNP site rs42756603.

2. The use of the molecular marker substance described in claim 1 in the preparation of products for screening and / or detecting the level of placental retention resistance in dairy cows.

3. A product for screening and / or detecting the level of resistance in dairy cows with retained placenta, said product comprising a detection... CPE Genetic material; Furthermore, the detection CPE Genetic material includes detection CPE Primers and probes for gene SNP sites; The products include gene chips, nucleic acid preparations, test kits, and related devices and equipment.

4. The product as described in claim 3, characterized in that, The primers are PCR primers for amplifying DNA fragments including rs42756603; Furthermore, the PCR primers are shown in SEQ ID NO.1-2; Furthermore, the product is obtained based on the restricted fragment length polymorphism method.

5. A system for screening and / or detecting the level of resistance to retained placenta in dairy cows, characterized in that, The system includes at least: The acquisition unit is configured to: acquire samples from the tested dairy cows. CPE SNP site information of genes; An evaluation unit is configured to determine the level of placental retention resistance in the tested dairy cows based on the SNP locus information obtained by the acquisition unit.

6. The system as described in claim 5, characterized in that, In the acquisition unit, the test cow sample is a blood sample from the test cow; The SNP locus is specifically rs42756603, and the SNP locus information includes SNP polymorphism or genotype.

7. The system as described in claim 5, characterized in that, The specific evaluation process of the evaluation unit includes: when the genotype of SNP site rs42756603 is TT, it indicates that the tested dairy cow is a high-resistance dairy cow with retained placenta.

8. The application of claim 1 or 2, the product of claim 3, or the system of any one of claims 5-7, in any one or more of the following: (a1) Screening for cows with high resistance and retained placenta; (a2) Dairy cow breeding.

9. A method for screening highly resistant dairy cows with retained placenta, characterized in that, The method includes using the product of claim 3 or 4 or the system of any one of claims 5-7 to determine the resistance status of cows with retained placenta, thereby screening cows with high resistance to retained placenta.

10. A method for breeding dairy cows, characterized in that, The method includes screening for highly resistant dairy cows with retained placenta using the method described in claim 9 for genetic breeding.

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