DGAT1 gene SNPs marker for detecting sheep milk production character and application of DGAT1 gene SNPs marker

Abstract

The invention provides a DGAT1 gene SNPs marker for detecting sheep milk production traits and application thereof.Nucleotide sequence variation sites of the DGAT1 gene are detected with the first-filial generation of Dongfudrisheng sheep male * Hu sheep female as an object, a general linear mixing effect model is constructed, the correlation between nucleotide sequence variation and sheep milk production traits is researched, and the DGAT1 gene SNPs marker for detecting the sheep milk production traits is obtained. And digging a molecular genetic marker for regulating and controlling the lactation character of the sheep. Three SNPs (single nucleotide polymorphisms) sites are found in total; wherein two variation sites in the first intron are named as c.191 + 411 Cgt, and the second intron is named as c.191 + 411 Cgt; t (SNP1) and c.192-440 Cgt, and c. 292-440 Cgt; t (SNP2) and a variation site in the 17th exon are named as c.1461 Cgt; t (SNP3). For the SNP1 site, the average daily milk yield of the CT genotype ewe is increased by 8.2% and 17.04% compared with that of the CC genotype ewe and that of the TT genotype ewe respectively; for the SNP3 site, the milk fat percentage of the CC genotype ewe is improved by 0.314% compared with that of the CT genotype ewe. A new molecular marker can be provided for selection and improvement of lactation traits of the milk sheep, and the molecular marker has important application prospects in sheep screening or breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a DGAT1 gene SNPs marker for detecting the milk production trait of sheep and application thereof. BACKGROUND

[0002] Sheep milk is good in digestibility, strong in buffering capacity, rich in protein, amino acids and minerals, and has a higher nutritional value than goat milk and cow milk. In an insulin-resistant mouse model, dietary supplementation of sheep milk can enhance glucose metabolism in mice by regulating intestinal flora to improve type 2 diabetes. The unsaturated fatty acids such as conjugated linoleic acid, docosahexaenoic acid, linoleic acid and alpha-linolenic acid enriched in sheep milk can inhibit atherosclerosis and thrombosis, and have the functional properties of preventing cardiovascular diseases, anti-cancer and anti-diabetes. However, the milk sheep industry in China is still in its infancy, and there is a lack of excellent milk sheep breeds. Therefore, breeding new milk sheep breeds with high yield and high milk quality has become the primary task of breeders. East Friesian sheep, produced in Germany, is a world-renowned milk sheep breed. Adult ewes of East Friesian can produce 500-800 kg of milk in 260-300 days, and are widely used to improve the lactation performance of local breeds.

[0003] Economic traits including lactation traits are regulated by genetic and environmental factors. If functional genes associated with livestock quantitative traits can be identified, molecular marker-assisted selection techniques can be used to improve the target traits. So far, some single nucleotide polymorphism (SNP) sites have been used for molecular marker-assisted breeding to improve lactation performance. For example, Pegolo et al. found that SNPs identified on LEP, PRL, STAT5A, CCL3, ACACA, GHR, ADRB2, LPIN1, STAT1, FABP4 and CSN2 in dairy cows were associated with the fatty acid composition in cow milk. In addition, ACACA c.-87 G > T in sheep is associated with the milk fat percentage of sheep. However, compared with dairy cows and dairy goats, there are few studies on molecular genetic markers of lactation traits in sheep, and molecular genetic markers in different species are not mutually applicable.

[0004] Diacylglycerol O-acyltransferase 1 (DGAT1), a member of the acyl CoA: cholesterol acyltransferase (ACAT) gene family, is a key enzyme that catalyzes the final step in triglyceride synthesis. Knockout of DGAT1 in mice reduces triglyceride levels in mammary gland tissue and results in a loss of lactation. Numerous studies have identified quantitative trait loci (QTLs) associated with milk yield, milk fat percentage, and milk protein percentage in DGAT1. Furthermore, a C insertion (g.407_408) at the DGAT1 site was found to be significantly associated with milk yield and milk fat percentage in Xinong Saanen and Guanzhong dairy goats. However, no studies have examined SNPs in the DGAT1 gene and their association with lactation traits in East Friesen×Hu cross sheep. Summary of the Invention

[0005] The purpose of the present invention is to solve the above technical problems.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a single nucleotide polymorphism genetic molecular marker associated with sheep milk production traits, wherein the single nucleotide polymorphism genetic marker is located in the sheep DGAT1 gene, and the single nucleotide polymorphism genetic molecular marker is any one or more combinations of the following (a) to (c);

[0008] (a) The single nucleotide polymorphism genetic marker is located at the c.191 + 411 bp position of the DGAT1 gene on the positive strand of chromosome 9 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a C / T base mutation;

[0009] (b) The single nucleotide polymorphism genetic marker is located at the c.192-440 bp position of the DGAT1 gene on the positive strand of chromosome 9 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a C / T base mutation;

[0010] (c) The single nucleotide polymorphism genetic marker is located at the c.1461 bp position of the DGAT1 gene on the positive strand of chromosome 9 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a C / T base mutation.

[0011] Further, the milk production trait is milk fat percentage and / or average daily milk yield.

[0012] The application provides at least one of the following three SNP sites as a detection target for identifying or assisting in identifying a sheep milk production trait:

[0013] The SNP site is that a cytosine C at position c.191+411 of the DGAT1 gene on the positive strand of chromosome 9 is replaced by thymine T according to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version;

[0014] The SNP site is that a cytosine C at position c.192-440 of the DGAT1 gene on the positive strand of chromosome 9 is replaced by thymine T according to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version;

[0015] The SNP site is that a cytosine C at position c.1461 of the DGAT1 gene on the positive strand of chromosome 9 is replaced by thymine T according to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version.

[0016] The application provides a method for identifying or assisting in identifying a sheep milk production trait, which is any one of the following 1)~3):

[0017] 1) comprising the following steps: detecting the genotype of SNP site c.191+411 C / T in the DGAT1 gene of the test sheep; the genotype of the SNP site c.191+411 C / T is CC, CT or TT;

[0018] The average daily milk yield of the test sheep with the genotype of SNP site c.191+411 C / T in the DGAT1 gene being CT is better than that of the test sheep with the genotype of SNP site c.191+411 C / T in the DGAT1 gene being CC and TT;

[0019] 2) comprising the following steps: detecting the genotype of SNP site c.192-440 C / T in the DGAT1 gene of the test sheep; the genotype of the SNP site c.192-440 C / T is CC, CT or TT;

[0020] 3) comprising the following steps: detecting the genotype of SNP site c.1461 C / T in the DGAT1 gene of the test sheep; the genotype of the SNP site c.1461 C / T is CC or CT;

[0021] The milk fat rate of the test sheep with the SNP site c.1461 C / T genotype CC in the DGAT1 gene is better than that of the test sheep with the SNP site c.1461 C / T genotype CT.

[0022] The application provides application of the method in screening of milk sheep or breeding of milk sheep.

[0023] Further, in the application, the test sheep of any one of the following is selected for milk production or breeding:

[0024] The test sheep with the SNP site c.191 + 411 C / T genotype CT or CC or TT in the DGAT1 gene;

[0025] Or the test sheep with the SNP site c.192-440 C / T genotype CT or CC or TT in the DGAT1 gene;

[0026] Or the test sheep with the SNP site c.1461 C / T genotype CC or CT in the DGAT1 gene.

[0027] The application provides a primer pair for detecting a mutation in a DGAT1 gene, wherein the nucleotide sequences upstream and downstream of the primer pair are shown in SEQ ID NO. 1-2; or the nucleotide sequences upstream and downstream of the primer pair are shown in SEQ ID NO. 3-4; or the nucleotide sequences upstream and downstream of the primer pair are shown in SEQ ID NO. 5-6.

[0028] The application provides a method for detecting a molecular marker related to a milk production trait of sheep, comprising the following steps:

[0029] S1. Extracting genomic DNA from sheep blood as a sample, and performing PCR amplification on a DGAT1 gene of the sheep by using the primer pair shown in SEQ ID NO. 1-6;

[0030] S2. Performing pool sequencing and sequence analysis on the PCR amplification product, obtaining a variation site and determining a variation type;

[0031] S3. Identifying a sheep individual by performing DGAT1 gene typing, and identifying a genotype of a SNP site in a detection region of a DGAT1 gene of the sheep according to a typing result.

[0032] The application provides a kit for detecting a molecular marker, and the kit comprises the primer pair.

[0033] Further, the primer pair or primer combination is applied to any one of the following (a)-(f):

[0034] (a) identifying or assisting in identifying sheep milk production traits;

[0035] (b) sheep screening;

[0036] (c) sheep breeding;

[0037] (d) preparing a kit for identifying or assisting in identifying sheep milk production traits;

[0038] (e) preparing a kit for sheep screening;

[0039] (f) preparing a kit for sheep breeding.

[0040] The beneficial effects of the present application are as follows:

[0041] The present application takes the first-generation crossbred sheep of Dongfuri sheep♂ and Hu sheep♀ as the research object, detects the nucleotide sequence variation sites of the DGAT1 gene by applying Sanger, Penta-primer amplification refractory mutation system (PARMS) and other technologies, constructs a general linear mixed effect model, studies the correlation between the nucleotide sequence variation and the lactation traits of sheep, and mines the molecular genetic markers for regulating the lactation traits of sheep. The present application has achieved some positive effects and has great advantages compared with the prior art, and the specific advantages are as follows:

[0042] (1) Three SNPs sites are found on the DGAT1 gene of sheep. Among them, two variation sites in the first intron are named as c.191 + 411 C > T (SNP1) and c.192-440 C > T (SNP2), and the variation site in the 17th exon is named as c.1461 C > T (SNP3).

[0043] (2) For SNP1 and SNP2 loci, all three genotypes found were analyzed for association with lactation traits. However, the association analysis between SNP3 and lactation traits was limited to genotypes CC and CT because the frequency of genotype TT was less than 1%. For SNP1 c.191+411 C > T locus, the daily average milk production of ewes with CT genotype was increased by 0.063 kg / d and 0.121 kg / d (P < 0.05) compared with CC and TT genotypes, respectively, with the proportion of increase being 8.20% and 17.04%, respectively. Notably, at the c.1461 locus, the milk fat percentage of ewes with CC genotype was increased by 0.314% (P = 0.039) compared with CT genotype; the new molecular marker can be provided for the selection and improvement of lactation traits of dairy sheep, and the sequence provided according to the molecular marker can be used for identifying or assisting in identifying the milk production traits of sheep; and has important application prospects for sheep screening or breeding. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Sequencing peak charts of SNP1 (A), SNP2 (B) and SNP3 (C) of the DGAT1 gene of sheep.

[0045] Figure 2 PARMS genotyping of SNP1 (A), SNP2 (B) and SNP3 (C) of the DGAT1 gene of sheep.

[0046] Figure 2 The X axis and the Y axis represent the fluorescence signal values of fluorescein phosphoramidite (FAM) and hexachlorofluorescein phosphoramidite (HEX), respectively. The orange, blue, purple and gray spots represent the fluorescence signal values of FAM, FAM / HEX, HEX and the negative control group, respectively, and the negative control group does not contain a DNA template. DETAILED DESCRIPTION

[0047] In order for those skilled in the art to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0048] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0049] Example 1. Sequencing and analysis of SNPs associated with daily milk yield and milk fat percentage in sheep

[0050] 1. Test method

[0051] 1.1 Test animals and sample collection

[0052] (1) Collection of blood

[0053] In Gansu Yuansheng Farming and Stockbreeding Technology Co., Ltd., 476 crossbred sheep of the first generation of East Fleece sheep♂×Huzhu sheep♀ with complete information records of parity, lambing number, age, etc. were selected. In the middle of lactation, 800 μL of blood was collected from the jugular vein and dropped on FTA cards for air drying, which was used for genomic DNA extraction.

[0054] (2) Collection of milk

[0055] For the above milk sheep ewes, when they were in the middle of lactation, they were milked using a DeLaval 9JP-2X24 side-by-side milking machine (DeLaval, Tianjin, China). The milk samples collected in the morning, noon, and evening were mixed in a 50 mL centrifuge tube, and immediately taken to the laboratory for detection of milk component indexes such as milk fat percentage, milk protein percentage, lactose percentage, ash content, non-fat milk solid content, and dry matter content using a UL40BC milk component analyzer (Youchuang, Hangzhou, China).

[0056] Unless otherwise specified, the quantitative test in the following examples was set up with three repeated experiments, and the average value was taken.

[0057] The following indexes were determined using a UL40BC milk component analyzer:

[0058] Milk fat percentage: the percentage of fat contained in milk.

[0059] Milk protein percentage: the percentage of protein contained in milk.

[0060] Ash content: the content of minerals in milk.

[0061] Non-fat milk solid content: the content of nutritional substances other than lipids and water in milk.

[0062] Dry matter content: the content of nutritional substances other than water in milk.

[0063] The daily milk yield of each test sheep was determined using a DeLaval 9JP-2X24 side-by-side milking machine.

[0064] 1.2 Extraction of blood genomic DNA

[0065] Two 1.2 mm diameter FTA card punches were taken from each FTA card using a puncher and were punched into 200 μL PCR tubes, respectively. First, 100 μL 20 mM NaOH was added, and the PCR tubes were placed in a 40°C water bath for 10 min, then removed and the NaOH was slowly aspirated with a pipette. Second, 100 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH=8.0) was added to the PCR tube and left at room temperature for 2 min, and the TE solution was aspirated. Finally, 30 μL of TE solution was added, and incubated at 95°C for 5 min to obtain gDNA.

[0066] 1.3 PCR and Sanger sequencing

[0067] Based on the sequence of sheep DGAT1 (GeneID: 100126245) in NCBI, three pairs of primers were designed to amplify the first intron and the 17th exon region of DGAT1 using Prime 3.0. The specific primer information is shown in Table 1.

[0068] Table 1 Primer sequence information

[0069] Sixty DNA samples were randomly selected as PCR amplification templates, and PCR amplification was performed on an Applied Biosystems SimpliAmp thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA), and the amplification products were detected by 1.5% agarose gel electrophoresis. The amplified three target gene fragments were mixed and sequenced, i.e. 3 μL of each primer amplification product was mixed, and then Sanger sequencing was performed. Finally, MEGA v7.0 was used for sequence alignment to screen SNPs sites.

[0070] 1.4 PARMS genotyping

[0071] For all 476 DNA samples, genotyping of the three SNPs sites screened was performed by Wuhan Jingpeibio Biotechnology Co., Ltd.

[0072] 1.5 Genetic diversity analysis

[0073] The allele frequency, genotype frequency and polymorphism information content (Polymorphism information content, PIC) of the SNPs sites were calculated according to the online website http: / / www.msrcall.com / Gdicall.aspx.

[0074] 2. Test results

[0075] 2.1 Identification results of sheep DGAT1 SNPs sites

[0076] The pool sequencing results of 60 DNA samples showed that there were 3 double peaks in the 1st intron and the 17th exon of DGAT1 in total Figure 1 Three SNPs were identified, including two mutation sites in the 1st intron, named c.191 + 411 C > T (SNP1) and c.192-440 C > T (SNP2), and a mutation site in the 17th exon, named SNP3 (c.1461 C > T), which encodes alanine and belongs to synonymous mutation.

[0077] SNP1: represents single nucleotide polymorphism site 1, which is c.191 + 411 C > T, indicating that there is a base C > T mutation at c.191 + 411 bp in the sequence of DGAT1 gene on sheep chromosome 9;

[0078] SNP2: represents single nucleotide polymorphism site 2, which is c.192-440 C > T, indicating that there is a base C > T mutation at c.192-440 bp in the sequence of DGAT1 gene on sheep chromosome 9;

[0079] SNP3: represents single nucleotide polymorphism site 3, which is c.318 + 126 C / T, indicating that there is a base C > T mutation at c.1461 bp in the sequence of DGAT1 gene on sheep chromosome 9;

[0080] Based on the three SNPs found, primers for amplifying the above SNPs can be designed, including forward primers and reverse primers located upstream and downstream of the above three single nucleotide polymorphism sites. Based on the primers of the above SNPs, kits for detecting the above SNPs can also be designed using existing technologies.

[0081] 2.2 Genotyping results of sheep DGAT1 SNPs

[0082] CC, CT and TT three genotypes were detected at c.191 + 411 C > T, c.192-440 C > T and c.1461 C > T sites Figure 2 A, B and C).

[0083] 2.3 Analysis of genetic diversity of sheep DGAT1 SNPs

[0084] In SNP1, 242, 196 and 38 ewes were CC, CT and TT genotypes, and the frequencies of the three genotypes were 0.508, 0.412 and 0.080, respectively. The frequencies of the two alleles C and T were 0.714 and 0.286, respectively. The polymorphic information content (PIC) of the population was 0.325.

[0085] In SNP2, there were 295, 161 and 20 CC, CT and TT genotypes, and the frequencies of the three genotypes were 0.620, 0.338 and 0.042, respectively. The frequencies of the two alleles C and T were 0.789 and 0.211, respectively. The polymorphic information content (PIC) of the population was 0.278.

[0086] In SNP3, a total of 378 CC genotypes, 95 CT genotypes and 3 TT genotypes were detected, and the frequencies of the three genotypes were 0.794, 0.200 and 0.006, respectively. The frequencies of the two alleles C and T were 0.894 and 0.106, respectively. The polymorphic information content (PIC) of the population was 0.172.

[0087] Example 2 Correlation analysis of lactation traits

[0088] 1. Test method

[0089] Based on the test results in Example 1, the effects of genotypes on the daily average milk yield and milk composition of sheep were analyzed by General Liner Mixed-effect Models (GLMMs) of SPSS 27.0 software. The analysis results were expressed as mean ± standard error. In order to determine the fixed effects and random effects included in the model establishment, the effects of age, lambing number and parity on the lactation traits of sheep were analyzed by one-way ANOVA test, and the results showed that the lambing number had no significant effect on the lactation traits, but the age and parity had extremely significant effects on the lactation traits

[0090] Therefore, we added genotypes, age and parity to the general linear mixed effect model.

[0091] The model is Y = µ + Genotype (Allele) + Age + Parity + e, where Y represents the lactation trait, µ is the population mean, Genotype is the genotype, Allele is the allele, Age is the age, Parity is the parity, and e is the random residual error.

[0092] 2. Test results

[0093] The results of the effect of genotypes on the average daily milk yield and milk components of sheep are shown in Table 2. As shown in Table 2, for SNP1 and SNP2 loci, all three genotypes found were subjected to association analysis with the milk yield traits. However, the association analysis between SNP3 and the milk yield traits was limited to genotypes CC and CT because the frequency of genotype TT was less than 1%. For SNP1 c.191+411C>T locus, the average daily milk yield of the ewes with CT genotype was increased by 0.063 kg / d and 0.121 kg / d (P < 0.05) compared to the ewes with CC and TT genotypes, respectively. Notably, at c.1461 locus, the milk fat percentage of the ewes with CC genotype was increased by 0.314% (P = 0.039) compared to the ewes with CT genotype. In addition, no association was found between the genotypes of SNP2 c.192-440 C>T locus and the milk yield traits (P > 0.05).

[0094] Table 2 Association analysis of SNPs genotypes of DGAT1 of sheep with milk yield traits

[0095] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application but not to limit the solutions. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the present application on the basis of understanding the solutions, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A single nucleotide polymorphism genetic molecular marker associated with sheep milk production traits, characterized in that: The single nucleotide polymorphism genetic marker is located in sheep DGAT1 In the gene, the single nucleotide polymorphism genetic molecular marker is any one or more combinations of the following (a) to (c); (a) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 9 of the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information DGAT1 The gene c.191 + 411 bp position, and the presence of C / T base mutation; (b) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 9 of the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information DGAT1 The C / T base mutation exists at the c.192-440 bp position of the gene; (c) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 9 of the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information DGAT1 The c.1461 bp position of the gene contains a C / T base mutation.

2. The single nucleotide polymorphism genetic molecular marker related to sheep milk production traits according to claim 1, characterized in that: The milk production trait is milk fat percentage and / or average daily milk production.

3. Use of at least one of the following three SNP loci as a detection target in identifying or assisting in identifying milk production traits in sheep; The SNP site is the positive strand of chromosome 9 corresponding to the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information DGAT1 The cytosine C at position c.191 + 411 of the gene was replaced by thymine T; The SNP site is the positive strand of chromosome 9 corresponding to the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information DGAT1 The cytosine C at positions c.192-440 of the gene was replaced by thymine T; The SNP site is the positive strand of chromosome 9 corresponding to the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information DGAT1 The cytosine C at position c.1461 of the gene was replaced by thymine T.

4. A method for identifying or assisting in identifying sheep milk production traits, comprising any one of the following 1) to 3): 1) The following steps are included: Testing the test sheep DGAT1 The genotype of the SNP site c.191 + 411 C / T in the gene; the genotype of the SNP site c.191 + 411 C / T is CC, CT or TT ; described DGAT1 The genotype of the SNP site c.191 + 411 C / T in the gene is CT The average daily milk production of the tested sheep was better than that of the SNP site c.191 + 411 C / T genotype CC and TT of the test sheep; 2) The following steps are included: Testing the test sheep DGAT1 The genotype of the SNP site c.192-440 C / T in the gene; the genotype of the SNP site c.192-440 C / T is CC, CT or TT ; 3) The following steps are included: Testing the test sheep DGAT1 The genotype of the SNP site c.1461 C / T in the gene; the genotype of the SNP site c.1461 C / T is CC or CT ; described DGAT1 The genotype of the SNP c.1461 C / T in the gene is CC The milk fat rate of the tested sheep is better than that of the genotype of the SNP site c.1461 C / T. CT of the test sheep.

5. Application of the method according to claim 4 in dairy sheep screening or dairy sheep breeding.

6. The use according to claim 5, characterized in that In the application, any of the following sheep are selected for milk production or breeding: described DGAT1 The genotype of the SNP site c.191 + 411 C / T in the gene is CT or CC or TT of the test sheep; or DGAT1 The genotype of the SNP site c.192-440 C / T in the gene is CT or CC or TT of the test sheep; or DGAT1 The genotype of the SNP c.1461 C / T in the gene is CC or CT of the test sheep.

7. A test DGAT1 A primer pair for detecting a mutation in a gene, characterized in that The upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 1 to 2, respectively; or the upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 3 to 4, respectively; or the upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 5 to 6, respectively.

8. A method for detecting molecular markers related to sheep milk production traits, characterized in that: The steps include: S1. Genomic DNA was extracted from sheep blood and the primers shown in SEQ ID NO. 1 to 6 were used to identify the sheep. DGAT1 PCR amplification of the gene; S2. Perform mixed sequencing and sequence analysis on the PCR amplification products to identify the variant sites and determine the variant types; S3. By identifying individual sheep DGAT1 Genotyping, identification of sheep based on typing results DGAT1 The genotype of the SNP site in the gene detection region.

9. A kit for detecting molecular markers, characterized in that The kit comprises the primer pair according to claim 7.

10. Use of the primer pair or primer combination according to claim 7, wherein the use is any one of the following (a) to (f): (a) Identifying or assisting in the identification of milk production traits in sheep; (b) Sheep screening; (c) sheep breeding; (d) preparing a test kit for identifying or assisting in identifying milk production traits in sheep; (e) preparing a kit for sheep screening; (f) Preparation of a kit for sheep breeding.

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