Molecular marker for determining color of sheep wool and application thereof

Abstract

The invention discloses a molecular marker for sheep wool color judgment and application thereof, and belongs to the field of animal heredity and breeding. According to the invention, whole genome association analysis (GWAS) is carried out on sheep populations of different hair color types of a first filial generation and a first cross generation of a saffok ram and a Dumeng ewe, two key gene loci on chromosomes Chr14: 14251947 and Chr19: 31724207 in a sheep genome are identified, and a substance for detecting the two key gene loci is provided; the method can realize rapid and accurate identification of all-black, black-white and all-white hair color types, can stably distinguish three types of hair colors, has high consistency in mainstream sheep populations, can be used for early prediction of sheep hair colors, molecular-assisted selection and variety identification, and has important application values for accelerating a new variety breeding process and improving breeding efficiency.

Description

Technical Field

[0001] This invention relates to the field of animal genetics and breeding, and specifically to molecular markers for determining the color of sheep wool and their applications. Background Technology

[0002] Coat color, a key physical trait in sheep, directly affects breed identification and market value, and is closely related to breeding management efficiency and economic benefits. Currently, coat color determination mainly relies on manual observation, which is easily affected by environmental conditions and subjective judgment, resulting in low accuracy and instability. With the development of molecular genetics, research has confirmed that coat color is regulated by multiple genes, with key genes such as MC1R, ASIP, KIT, and MITF exhibiting significant phenotypic effects in different breeds. However, existing research is mostly limited to candidate gene analysis, lacking a stable and accurate molecular marker system for predicting coat color. Furthermore, traditional breeding methods are time-consuming and inefficient, making them unsuitable for the rapid improvement of coat traits required in modern meat sheep breeding. Therefore, developing molecular markers based on key genetic loci and their detection technologies to achieve early prediction, molecular-assisted selection, and breed identification of sheep coat color has significant research value and broad application prospects. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to quickly and accurately identify the coat color characteristics of sheep.

[0004] To address the aforementioned issues, this invention provides an application for detecting SNP genotypes or polymorphisms in the sheep genome, wherein the SNP can be SNP1 and / or SNP2 as follows: SNP1 is an SNP in the sheep genome, specifically nucleotide 14251947 of chromosome 14 in the reference genome ARS-UI_Ramb_V3.0, which is T or A; SNP2 is an SNP in the sheep genome, specifically nucleotide 31724207 of chromosome 19 in the reference genome ARS-UI_Ramb_V3.0, which is A or G; The application can be any of the following: A1) Identification or auxiliary identification of the color characteristics of sheep wool; A2) Prepare products for identifying or assisting in the identification of wool color traits; A3) Identify or assist in identifying that the wool color of the sheep is black, black and white, or white; A4) Prepare products for identifying or assisting in the identification of sheep wool that is black, black-white, or white; A5) Identify or assist in the identification of the wool color characteristics; A6) Prepare products for identifying or assisting in the identification of wool color traits; A7) Sheep breeding; A8) Prepare products for sheep breeding.

[0005] In the above applications, the substance may be a primer composition for amplifying sheep genomic DNA fragments including the SNP site, a PCR reagent containing the primer composition, and / or a kit containing the primer composition or the PCR reagent.

[0006] The present invention also provides a method for identifying or assisting in the identification of wool color traits, wherein the method may be any one of the following Q1-Q3: Q1 includes detecting the genotype of SNPs in the genome of the sheep to be tested, and identifying or assisting in identifying the sheep's wool color based on the genotypes. The SNPs are those described in claim 1, and the SNPs are SNP1 and SNP2. Q2 includes detecting the genotype of the SNP in the genome of the sheep to be tested, and identifying or assisting in the identification of sheep wool color based on the genotype, wherein the SNP is SNP1 as described in claim 1; Q3 includes detecting the genotype of the SNP in the genome of the sheep to be tested, and identifying or assisting in the identification of sheep wool color based on the genotype, wherein the SNP is SNP2 as described in claim 1.

[0007] In the above method, the sheep can be a sheep breed or a hybrid or crossbred offspring of a sheep breed.

[0008] The hybrid offspring are F1 generation, and the cross-crossed offspring are the offspring of F1 generation after free mating.

[0009] This invention also provides the application of the above method in sheep breeding.

[0010] This invention also provides a method for sheep breeding, wherein the breeding index can be the sheep wool color trait, and the sheep wool color trait can be all black, black and white, or all white; the method is any one of the following M1-M3: M1 is a method for breeding sheep with all-white coat color, including selecting parent sheep for breeding to obtain sheep with all-white coat color, wherein the parent sheep is a sheep with SNP1 genotype TT; M2 is a method for breeding sheep with black and white coat color, which includes selecting parent sheep for breeding to obtain sheep with black and white coat color, wherein the parent sheep are sheep with SNP1 genotype TA or AA and SNP2 genotype GG. The M3 is a method for breeding sheep with all-black coat color, which includes selecting parent sheep for breeding to obtain sheep with all-black coat color, wherein the parent sheep are sheep with SNP1 genotype TA or AA and SNP2 genotype GA or GG.

[0011] In the above method, the parent sheep in M1 can be M11, M12 or M13: M11 is a sheep with SNP1 genotype TT and SNP2 genotype GG, M12 is a sheep with SNP1 genotype TT and SNP2 genotype GA, and M13 is a sheep with SNP1 genotype TT and SNP2 genotype AA; The parent sheep in M2 can be M21 or M22: M21 is a sheep with SNP1 genotype TA and SNP2 genotype GG, and M22 is a sheep with SNP1 genotype AA and SNP2 genotype GG. The parent sheep in M3 can be M31, M32, or M33: M31 is a sheep with SNP1 genotype TA and SNP2 genotype GA, M32 is a sheep with SNP1 genotype TA and SNP2 genotype AA, and M33 is a sheep with SNP1 genotype AA and SNP2 genotype GA.

[0012] In this invention, "all white" means that all the sheep wool is white and has no other color; "all black" means that all the sheep wool is black and has no other color; and "black and white" means that the sheep wool has both white and black.

[0013] This invention also provides a product for detecting polymorphisms or genotypes of SNP sites in the sheep genome, wherein the SNP sites may be the SNP sites described above, including SNP1 and / or SNP2, and the product contains the substances described above, and the product may be any of the following: C1) Products that detect or assist in the detection of single nucleotide polymorphisms or genotypes related to wool color traits. C2) Products used for identifying or assisting in the identification of wool color characteristics. C3) Products used in sheep breeding C4) Products selected or bred based on coat color; The product is as follows: D1), D2), or D3). D1) The product is a primer composition for amplifying sheep genomic DNA fragments including the SNP sites. D2) The product is a PCR reagent containing the primer composition described in D1). D3) The product is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0014] In this invention, the substance may be a primer composition, which may consist of SNP1 primer pairs and / or SNP2 primer pairs; The SNP1 primer pair consists of single-stranded DNA from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; specifically, SEQ ID NO: 3 can be 5′-GAAGGTGACCAAGTTCATGCTCCAGGCAGCAGATGAAGTAGTACA-3′, SEQ ID NO: 4 can be 5′-GAAGGTCGGAGTCAACGGATTCCAGGCAGCAGATGAAGTAGTACT-3′, and SEQ ID NO: 5 can be 5′-CCAAGAACCGCAACCTGCACTC-3′. The PCR product of the SNP1 primer pair is SEQ ID NO: 1. SNP1 is located at nucleotide 14251947 of chromosome 14 in the ARS-UI_Ramb_V3.0 genome. It is either T or A, and the polymorphism is TT / TA / AA, where TT is T.

[0015] The genotype TT for SNP1 means that SNP1 is homozygous for T; the genotype AA for SNP1 means that SNP1 is homozygous for A; and the genotype TA for SNP1 means that SNP1 is heterozygous for TA.

[0016] The SNP2 primer pair consists of single-stranded DNA from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. Specifically, SEQ ID NO: 6 can be 5′-GAAGGTGACCAAGTTCATGCTGTTGGCACACTTTCTTTAAAGGGCC-3′, SEQ ID NO: 7 can be 5′-GAAGGTCGGAGTCAACGGATTGTTGGCACACTTTCTTTAAAGGGCT-3′, and SEQ ID NO: 8 can be 5′-GTAATAGACCTTACATCCTTCAAAGC-3′. The PCR product of the SNP2 primer pair is SEQ ID NO: 2. SNP2 is located at nucleotide 31724207 on chromosome 19 of the ARS-UI_Ramb_V3.0 genome. It is either A or G, and the polymorphism is GG / GA / AA.

[0017] The genotype GG for SNP2 means that SNP2 is homozygous for G; the genotype AA for SNP2 means that SNP2 is homozygous for A; and the genotype GA for SNP2 means that SNP2 is heterozygous for GA.

[0018] The present invention also provides PCR reagents and / or kits containing the above-described primer compositions.

[0019] The present invention also provides a DNA molecule, wherein the nucleotide sequence of the DNA molecule may be SEQ ID NO: 1 and / or SEQ ID NO: 2. In this invention, the sheep to be tested can be selected from: East Australian Lake, Texas, Australian Lake, Australian White sheep, Dorper sheep, East Frisian, Lake sheep, or black sheep, etc.

[0020] In this invention, the physical location on the sheep genome is based on the ARS-UI_Ramb_v3.0 version as the reference sequence.

[0021] This invention, based on genome-wide association analysis (GWAS) and combined with large-scale population genotyping data, screened two key SNP loci significantly associated with coat color: Chr14:14251947, T→A mutation, and Chr19:31724207, G→A mutation. KASP genotyping validation confirmed that the combination of these loci can stably and accurately distinguish between black, black-and-white, and white coat color phenotypes, providing an effective molecular tool for early prediction and molecularly assisted selection of sheep coat color. Attached Figure Description

[0022] Figure 1 This is a QQ graph of the 3-class GWAS results.

[0023] Figure 2 This is a Manhattan plot of the GWAS results for a three-class classification.

[0024] Figure 3 This is a LDblock map of three significant loci.

[0025] Figure 4 This is a genotype-phenotype correspondence diagram for three significant loci.

[0026] Figure 5 This is a QQ graph of the binary GWAS results.

[0027] Figure 6 This is a Manhattan plot of the binary GWAS results.

[0028] Figure 7 This is a genotype-phenotype correspondence diagram for significant binary loci.

[0029] Figure 8 This is the fluorescence typing cluster diagram of the OV900016_K01 and OV900017_K01 samples from Lake Audu.

[0030] Figure 9 It is a genotyping diagram of SNP1 and SNP2 for multiple varieties of samples. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] The sheep breeds involved in this invention—Australian White, Texel, East Friesian, Dorper, Suffolk, Hu sheep, and Dumont sheep—are all commercial breeds. The Dumont sheep breed approval certificate number is Agricultural 03 New Variety Certificate No. 27. The relevant biological materials are available to the public from China Agricultural University. These biological materials are only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0034] Example 1: Screening of SNP sites for sheep wool color trait I. Phenotypic Collection This invention selected 155 F1 crossbred sheep (sires) of Suffolk and Dormont sheep (dams) and 126 F1 crossbred sheep obtained through selective breeding from the F1 crossbred sheep as the research population, totaling 267 individuals. All individuals were raised under the same environmental conditions and fed a uniform diet to avoid environmental factors causing differences in coat color. Based on coat color phenotype, the F1 crossbred sheep were divided into three categories: 87 completely black, 20 black and white, and 19 completely white. Coat color phenotypes were double-determined by two professionals to ensure accurate classification. Simultaneously, based on the F1 and F1 crossbred populations, 85 complete pedigrees were constructed, with an average pedigree size of 3.14. Pedigree information was double-verified using ear tags and pedigree records to ensure the completeness and accuracy of the pedigree data.

[0035] II. Genomic DNA Extraction and Whole Genome Resequencing Five mL of blood was collected from the jugular vein of each sheep, and DNA was extracted using the TIANamp Blood Genomic DNA Extraction Kit (Tiangen Biotech) according to the manufacturer's instructions. DNA purity (A260 / A280 between 1.8 and 2.0) was measured using a NanoDrop 2000, and DNA concentration was measured using a Qubit 3.0 to ensure the samples met the requirements for library construction. DNA samples were stored at -20°C for later use.

[0036] Subsequently, qualified DNA samples were sent to the Illumina NovaSeq 6000 platform for whole-genome resequencing in PE150 (150 bp paired ends) sequencing mode, with an average sequencing depth of approximately 20×. After initial quality control, each sample yielded approximately 6.5 Gb of valid data.

[0037] III. SNP Variation Detection and Quality Control Raw sequencing data were quality controlled using FastP software (v0.23.2) to remove adapter sequences and low-quality reads. The quality-controlled data were then aligned to the reference genome ARS-UI_Ramb_v3.0 using BWA (v0.7.17), followed by variant detection using GATK (v4.2). All identified variant sites underwent rigorous quality control using Plink (v1.9), with sites having a deletion rate >0.1% being removed. Sites with a minor allele frequency (MAF) < 0.05 were removed; Sites with Hardy–Weinberg equilibrium test p < 1e-6 were removed.

[0038] Linkage disequilibrium (LD) r between sites 2 Redundant sites with a value >0.6 were removed to reduce false positive associations caused by population structure. Approximately 6.97 million high-quality SNP markers were ultimately obtained.

[0039] IV. Genome-wide association analysis 1. Three-category association analysis For the three coat color phenotypes (all black, black and white, and all white) of the crossover generation, genome-wide multi-category association analysis was performed using the PLOMM (Proportional Odds Logistic Mixed Model). Figure 1 , Figure 2 The physical location on the sheep genome was determined using ARS-UI_Ramb_v3.0 (Ovis aries Rambouillet GenomeAssembly (OAR_USU_Benz2616) v2.0, Date performed: 2023-07-20) as the reference sequence. The results showed that the Chr14:14248776 locus was significantly associated with coat color (P = 2.18 × 10⁻⁶). -15 Located in the region adjacent to the MC1R gene, false positives were excluded by LD block analysis. Figure 3 The genotypic effect is as follows: the TT / TC type corresponds to black or black and white, while the CC type is white, exhibiting a dominant inheritance pattern. Further investigation revealed that the tightly linked variant Chr14:14251947 at this locus is located in the MC1R functional region, showing a stronger association. Figure 4 (AA / AT is black or black and white, TT is white), but it is still impossible to distinguish between black and black and white individuals.

[0040] 2. Binary Association Analysis To further analyze the differences between black and black-and-white coat colors, a binary association analysis using the GACT model was conducted. The results identified a significant locus of Chr19:31724207 located in the MITF gene region (P = 1.88 × 10⁻⁶). -8 The genotypic effect is manifested as AA / AG being black and GG being black and white, showing a dominant effect. Figure 5 , Figure 6 , Figure 7 ).

[0041] 3. Family verification analysis Based on 267 individuals from the F1 and crossover generations, 85 families were constructed. Merlin-fastassoc method was used in Merlin v1.1.2 software for single-point family genetic association analysis, and ordered logistic regression (POLR) model was used to statistically validate the interaction effects between key SNPs. The results showed (Table 1): Chr14:14251947 had a LOD value of 9.125 and heritability of 0.42 in the three-category model; Chr19:31724207 had a LOD value of 15.212 and heritability of 0.62 in the two-category model. Chr14:14251947 alone explained 37.36% of the variation in coat color phenotype, and the interaction effect between Chr14:14251947 (SNP1) and Chr19:31724207 (SNP2) in the MITF region further explained 49.30% of the variation. When the main effect (SNP1) and the interaction effect (SNP1:SNP2) are combined in the model, the total explanatory power reaches 61.32%, which is significantly higher than the contribution level of a single locus or the interaction effect alone, indicating that these two loci have significant main and interaction effects in coat color formation.

[0042] To evaluate the model's rationality, a QQ diagram was drawn. Figure 1 , Figure 5 Most scatter points were distributed near the 45° diagonal, indicating that population stratification had a relatively small impact. Genome-wide significance was determined using the Bonferroni method with linkage disequilibrium correction, and the results are shown in the Manhattan plot. Figure 2 , Figure 6 ).

[0043] Table 1. Results of GWAS Association Analysis and Family Analysis

[0044] Example 2: KASP detection and verification of multi-population sheep wool color SNPs Based on two key SNP loci for coat color (SNP1: Chr14:14251947; SNP2: Chr19:31724207) screened in the previous genome-wide association analysis, a group expansion validation was conducted on 634 sheep of different coat color types from seven breeds, including Australian-Dupu-Huzhou three-way crossbred sheep (F1 generation obtained by crossing Huzhou sheep as the maternal parent and Dorper sheep as the paternal parent, F1 generation obtained by crossing Dorper rams with Huzhou sheep as the maternal parent and then crossing Dorper rams with Dorper rams as the terminal paternal parent to produce F2 generation), East Australian-Huzhou three-way crossbred sheep (F1 generation obtained by crossing East Friesian rams with Huzhou sheep as the maternal parent, then crossing Dorper rams with East Friesian rams as the terminal paternal parent to produce F2 generation), Australian White sheep, Texel sheep, East Friesian sheep, Dumont sheep, and Dorper sheep. KASP technology was used for amplification and genotyping, and quantitative real-time PCR was used for detection. Sample genotypes were determined by clustering of fluorescence signals. Figure 8 ).

[0045] I. Sample DNA Extraction and Primer Design DNA was extracted from ear tissue or blood samples according to the instructions of the Tiangen Blood Genomics Extraction Kit. Primers containing SNP1 and SNP2 sites were designed using Primer 6.0 software and synthesized by Hunan Huazhi Biotechnology Co., Ltd. The primer sequences are listed in Table 3 and stored under standard conditions. The sequences of the products after PCR amplification are shown in Table 2.

[0046] Table 2. Sequences of KASP products detected at two mutation sites

[0047] Table 3. Sequences of primers for alleles at two mutation sites

[0048] II. KASP Amplification and Genotyping Refer to the PCR method in the template and use a real-time PCR instrument (such as Bio-Rad CFX96 or AppliedBiosystems QuantStudio) to complete the detection.

[0049] 1. Reaction system The total volume of the KASP amplification reaction was 10 μL, with the following components: 2 μL DNA template (approximately 20 ng), 5 μL KASPMaster Mix, 0.14 μL primer mixture, and sterile deionized water to bring the total volume to 10 μL. Primer sequences and design information are shown in Table 3. The primers were designed to include the target SNP sites and were labeled with different fluorescent groups at the 5′ end to distinguish alleles.

[0050] 2. Amplification program The amplification reaction was performed on a real-time PCR instrument. The reaction program was as follows: 94℃ pre-denaturation for 15 minutes, 94℃ denaturation for 20 seconds, 57℃ annealing for 1 minute (10–15 cycles in total), followed by 26–35 cycles of 94℃ denaturation for 20 seconds and 57℃ annealing extension for 1 minute, and finally holding at 57℃ for 1 minute.

[0051] 3. Genotype determination After amplification, the instrument automatically collects fluorescence signals and performs cluster analysis on the data. In the cluster diagram, the tested Audu Lake samples can be clearly divided into three genotypes: AA, Aa, and aa. The cluster points are clearly distributed with no significant overlap, indicating high accuracy in genotyping. Typical sample cluster diagrams are shown in Figure 8 and [Figure 9]. Figure 9 .

[0052] III. Population Validation and Genotype Frequency Analysis 1. Allele and genotype frequencies at SNP1 and SNP2 loci Allele and genotype frequencies of SNP1:Chr14:14251947 (Table 4) All-black (F1 sequencing, N = 163): T allele frequency was 0.488, A allele frequency was 0.512; T / T genotype was not detected, T / A accounted for 97.5% (159 / 163), and A / A accounted for 2.5% (4 / 163).

[0053] Black and white (KASP, N = 171): The frequency of the T allele was 0.250, and the frequency of the A allele was 0.750; the T / A and A / A ratios were close, at 49.7% (85 / 171) and 50.3% (86 / 171) respectively, and T / T was not detected.

[0054] All-white (KASP, N = 463): T allele frequency was 1.000, A allele was missing, and all were T / T genotype (100%).

[0055] Table 4. Allele and Genotype Frequency Table for Chr14:14251947

[0056] Allele and genotype frequencies of SNP2:Chr19:31724207 (Table 5) All-black (F1 sequencing, N = 163): G allele frequency was 0.490, A allele frequency was 0.509; G / A accounted for 96.9% (158 / 163), G / G and A / A were 0.6% (1 / 163) and 2.5% (4 / 163), respectively.

[0057] Black and white (KASP, N = 171): G / G genotype was detected only (100%), with a G allele frequency of 1.000.

[0058] All-white (KASP, N = 463): G allele frequency was 0.830, A allele frequency was 0.170; G / G accounted for 67.9% (314 / 463), G / A accounted for 30.5% (141 / 463), and A / A accounted for 1.7% (8 / 463).

[0059] Table 5. Allele and Genotype Frequency Table for Chr19:31724207

[0060] 2. SNP1 and SNP2 genotype combination frequencies and statistical analysis Table 6 summarizes the frequencies and expected number of individuals for different SNP1 and SNP2 genotype combinations in the three coat color groups. Based on the independence assumption, the chi-square test shows that: Pearson's x 2 = 1584.6, p-value = 9.999×10 -5 (Based on 10,000 simulations) the results indicate that there are significant differences in the distribution of SNP1 and SNP2 genotype combinations among different coat color types, which is statistically significant.

[0061] Table 6. Frequency and Statistical Results of SNP1 and SNP2 Genotype Combinations

[0062] 3. Association analysis between genotype combinations and sheep wool color type Based on different SNP1 and SNP2 genotype combinations, the number of observed individuals in three coat color groups (all black, black and white, and all white) was counted and compared with the expected number of individuals under the independence assumption. Standardized residuals were calculated to test significance (standardized residuals > 2). The results showed that there was a significant genotype combination preference among the different coat color groups (Table 7), and some samples are shown in Table 8: All-black group: SNP1-TA&SNP2-GA, SNP1-TA&SNP2-AA, and SNP1-AA&SNP2-GA were significantly enriched combinations with standardized residuals much greater than 2, indicating a high correlation with all-black coat color; other combinations were significantly missing.

[0063] In the black-and-white population: SNP1-TA & SNP2-GG and SNP1-AA & SNP2-GG were significantly enriched, showing strong correlation; most other combinations were significantly missing.

[0064] All-white population: The SNP1-TT genotype combination (especially SNP1-TT&SNP2-GG, SNP1-TT&SNP2-GA) was significantly enriched, with a standardized residual as high as 19.33, indicating that these combinations are highly associated with all-white coat color; while other combinations were generally significantly absent.

[0065] The results indicate a significant interaction between SNP1 and SNP2, which has a marked effect on the sheep wool color phenotype.

[0066] Table 7. Association between different SNP1 and SNP2 genotype combinations and coat color type

[0067] Table 8. Genotypes and corresponding coat color traits of sheep after testing (partial sample)

[0068] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An application for detecting SNP genotypes or polymorphisms in sheep genomes, characterized in that, The SNP is SNP1 and / or SNP2 as follows: SNP1 is an SNP in the sheep genome, which is nucleotide 14,251,947 of chromosome 14 in the reference genome ARS-UI_Ramb_V3.0, and is either T or A; SNP2 is an SNP in the sheep genome, which is nucleotide 31,724,207 of chromosome 19 in the reference genome ARS-UI_Ramb_V3.0, and is either A or G; The application is any one of the following: A1) Identification or auxiliary identification of the color characteristics of sheep wool; A2) Prepare products for identifying or assisting in the identification of wool color traits; A3) Identify or assist in identifying that the wool color is black, black and white, or white; A4) Prepare products for identifying or assisting in the identification of sheep wool as black, black-white, or white; A5) Identify or assist in the identification of the wool color characteristics; A6) Prepare products for identifying or assisting in the identification of wool color traits; A7) Sheep breeding; A8) Prepare products for sheep breeding.

2. The application of the substance for detecting SNPs in sheep genomes according to claim 1, characterized in that, The substance is a primer composition for amplifying sheep genomic DNA fragments including the SNP site, a PCR reagent containing the primer composition, and / or a kit containing the primer composition or the PCR reagent.

3. A method for identifying or assisting in the identification of the color characteristics of sheep wool, characterized in that, The method is any one of the following Q1-Q3: Q1 includes detecting the genotype of the SNP in the genome of the sheep to be tested, and identifying or assisting in the identification of sheep wool color based on the genotype, wherein the SNP is the SNP described in claim 1; Q2 includes detecting the genotype of the SNP in the genome of the sheep to be tested, and identifying or assisting in the identification of sheep wool color based on the genotype, wherein the SNP is SNP1 as described in claim 1; Q3 includes detecting the genotype of the SNP in the genome of the sheep to be tested, and identifying or assisting in the identification of sheep wool color based on the genotype, wherein the SNP is SNP2 as described in claim 1.

4. The application of methods for identifying or assisting in the identification of sheep wool color traits in sheep breeding, characterized in that, The method described is the method described in claim 3.

5. A method for sheep breeding, characterized in that, The breeding index is the sheep wool color trait, which is all black, black and white, or all white; the method is any one of the following M1-M3: M1 is a method for breeding sheep with all-white coat color, including selecting parent sheep for breeding to obtain sheep with all-white coat color, wherein the parent sheep is a sheep with SNP1 genotype TT; M2 is a method for breeding sheep with black and white coat color, which includes selecting parent sheep for breeding to obtain sheep with black and white coat color, wherein the parent sheep are sheep with SNP1 genotype TA or AA and SNP2 genotype GG. The M3 is a method for breeding sheep with all-black coat color, which includes selecting parent sheep for breeding to obtain sheep with all-black coat color, wherein the parent sheep are sheep with SNP1 genotype TA or AA and SNP2 genotype GA or GG.

6. The method according to claim 5, characterized in that, In M1, the parent sheep is M11, M12, or M13: M11 is a sheep with SNP1 genotype TT and SNP2 genotype GG, M12 is a sheep with SNP1 genotype TT and SNP2 genotype GA, and M13 is a sheep with SNP1 genotype TT and SNP2 genotype AA. In M2, the parent sheep are M21 and M22: M21 is a sheep with SNP1 genotype TA and SNP2 genotype GG, and M22 is a sheep with SNP1 genotype AA and SNP2 genotype GG. In M3, the parent sheep are M31, M32, and M33: M31 is a sheep with SNP1 genotype TA and SNP2 genotype GA, M32 is a sheep with SNP1 genotype TA and SNP2 genotype AA, and M33 is a sheep with SNP1 genotype AA and SNP2 genotype GA.

7. A product for detecting SNP polymorphisms or genotypes in sheep genomes, characterized in that, The SNP site is the SNP site of claim 1, the product contains the substance of claim 1, and the product is any one of the following: C1) Products that detect or assist in the detection of single nucleotide polymorphisms or genotypes related to wool color traits. C2) Products used for identifying or assisting in the identification of wool color characteristics. C3) Products used in sheep breeding C4) Products selected or bred based on coat color; The product is as follows: D1), D2), or D3). D1) The product is a primer composition for amplifying sheep genomic DNA fragments including the SNP sites. D2) The product is a PCR reagent containing the primer composition described in D1). D3) The product is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

8. The application according to claim 2 or the product according to claim 7, characterized in that, The product is a primer composition, which consists of SNP1 primer pairs and / or SNP2 primer pairs; The SNP1 primer pair consists of single-stranded DNA from SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; The SNP2 primer pair consists of single-stranded DNA from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

9. PCR reagents and / or kits containing the primer composition of claim 8 for detecting polymorphisms or genotypes of SNP sites in sheep genomes.

10. A DNA molecule, characterized by, The nucleotide sequence of the DNA molecule is SEQ ID NO: 1 and / or SEQ ID NO: 2.

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