A SNP molecular marker related to the cholesterol content of longissimus dorsi muscle of tianhua mutton sheep and application thereof

Abstract

The application discloses a SNP molecular marker related to cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep and application thereof and belongs to the technical field of sheep genetic breeding. The SNP molecular marker is located at 41,862,843 bp of chromosome No. 2 of a sheep reference genome ARS-UI_Ramb_v2.0, and the base mutation is T>C. Whole genome resequencing and cholesterol content determination data show that the SNP marker has a significant influence on the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep, and the T allele is a low-cholesterol dominant allele. By detecting the SNP site and selecting and reserving TT homozygous individuals, early molecular assisted selection of the low-cholesterol trait of Tianhua mutton sheep can be realized, and an effective molecular tool is provided for mutton sheep meat quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of sheep genetics and breeding technology, specifically involving an SNP molecular marker related to the cholesterol content of the longissimus dorsi muscle of Tianhua meat sheep and its application. Background Technology

[0002] Cholesterol is one of the important indicators for evaluating the nutritional quality of meat. Long-term excessive intake of high-cholesterol foods is associated with an increased risk of various metabolic diseases such as obesity, hyperlipidemia, coronary heart disease, and atherosclerosis. Therefore, developing low-cholesterol sheep breeds is of great significance for meeting consumer demand for healthy meat products.

[0003] Tianhua sheep is a new breed of fine-wool meat sheep adapted to high-altitude, cold, and arid environments, suitable for grazing, semi-grazing / semi-grazing, and stall feeding. Through four generations of continuous selective breeding, the Tianhua sheep's sire is the South African Merino (62.5% bloodline), and its dam is the Gansu Alpine Fine-wool Sheep (37.5% bloodline). It is characterized by its large size, rapid growth, and high reproductive capacity. Furthermore, the Tianhua sheep combines the dual-purpose (meat and wool) characteristics of the South African Merino with the adaptability to cold regions and wool performance of the Gansu Alpine Fine-wool Sheep. However, research on the genetic regulation mechanism of cholesterol content in the longissimus dorsi muscle of the Tianhua sheep is still lacking, and molecular markers suitable for breeding low-cholesterol meat sheep are scarce.

[0004] Marker-assisted selection (MAG) is a selection process based on molecular markers that influence target traits. Single nucleotide polymorphisms (SNPs) are variations caused by changes in a single nucleotide in the genome and are the most common type of variation in the genome. Genome-wide association studies (GWAS) can scan for variant sites associated with complex traits across the entire genome and are an important technique for screening SNP molecular markers that affect meat texture traits. Traditionally, cholesterol content determination requires post-slaughter sampling and cannot be used for early selection of live breeding sheep, resulting in low breeding efficiency and high costs. Currently, there are some reported studies on SNP markers related to cholesterol content in sheep. For example, Muharram et al. (2025) studied the Olfactomedin-like 3 (Olfactomedin-like 3) markers in Indonesian native sheep. OLFML3 SNP typing of the gene revealed that the polymorphism at the g.90317673 C>T site was significantly associated with cholesterol content in the longissimus dorsi muscle. P <0.05%, with the TT genotype individuals having significantly lower cholesterol levels than the CC and CT genotype individuals. However, the sheep breeds involved in this study, and the genomic regions where the SNP loci are located ( OLFML3The genes involved are completely different from those of Tianhua mutton sheep and cannot be directly applied to the breeding practices of Tianhua mutton sheep. In addition, the patent application with publication number CN119979722A discloses the SNP locus combination for the identification of Tianhua mutton sheep breed, but these loci are mainly used to distinguish Tianhua mutton sheep from other breeds and do not involve the selection of meat nutritional quality traits such as cholesterol content.

[0005] Current technologies lack specific SNP molecular markers for cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep, and there are no reports of using such markers for early in vivo screening of individuals with low cholesterol. Therefore, developing molecular markers that are significantly associated with cholesterol content to achieve early, rapid, and low-cost in vivo genotyping is of great significance for accelerating the genetic improvement of the nutritional quality of Tianhua mutton sheep. Summary of the Invention

[0006] The existing research on the genetic regulation mechanism of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep lacks sufficient information, and there is a lack of specific SNP molecular markers that are significantly associated with this trait. This makes it impossible to conduct early assisted selection for low cholesterol traits in Tianhua mutton sheep at the live stage, resulting in technical problems such as long breeding cycles, high testing costs, and low breeding efficiency. This invention aims to provide an SNP molecular marker related to cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep and its application.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides the application of SNP molecular marker products related to cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep in the assisted breeding of Tianhua mutton sheep. The SNP molecular marker is a polymorphism located at the 41862843 bp site on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, and the allele at this site is T or C.

[0008] The nucleotide sequence containing the SNP marker is shown in SEQ ID NO.1, wherein the SNP molecular marker is located at position 151 from the 5' end of the nucleotide sequence shown in SEQ ID NO.1, and its polymorphism is T / C.

[0009] The product is a primer combination, reagent, or kit for amplifying the SNP molecular marker.

[0010] The SNP site polymorphism is T / C. The cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TT genotype is lower than that of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TC and CC genotypes.

[0011] The assisted breeding includes: detecting the genotype of the Tianhua mutton sheep to be tested at the SNP molecular marker, and selecting individuals with the TT homozygous genotype.

[0012] Secondly, the present invention provides a method for detecting the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep, comprising: detecting the genotype of the Tianhua mutton sheep to be tested at the 41862843 bp site on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, and predicting the cholesterol content trait of the longissimus dorsi muscle of the Tianhua mutton sheep to be tested based on the correspondence between the genotype and the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep; The relationship between the genotype and the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep is as follows: the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TT genotype is lower than that corresponding to the TC and CC genotypes.

[0013] The steps for detecting the genotype of the Tianhua mutton sheep to be tested include: extracting genomic DNA from the Tianhua mutton sheep to be tested, performing PCR amplification using primer pairs specifically amplifying the 41,862,843 bp sites, and performing genotype analysis on the amplification products.

[0014] The genotyping was performed using sequencing, KASP typing, or TaqMan probe methods.

[0015] Thirdly, the present invention provides a primer pair for amplifying SNP molecular markers located at the 41,862,843 bp site on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, where the alleles are T or C. The nucleotide sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0016] Fourthly, the present invention provides a method for assisting in the breeding of low-cholesterol Tianhua meat sheep, comprising: detecting the genotype of the Tianhua meat sheep to be tested at the 41,862,843 bp site on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, and selecting individuals with the TT homozygous genotype as breeding individuals for low-cholesterol Tianhua meat sheep; Among them, the SNP site polymorphism is T / C, and the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TT genotype is lower than that of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TC and CC genotypes.

[0017] The method involves detecting the genotype of the Tianhua mutton sheep at the 41,862,843 bp locus on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, using the primer pairs for PCR amplification, sequencing or genotyping analysis of the amplification products, and determining the genotype of the individual to be tested based on the analysis results.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The application of the SNP molecular marker product provided by this invention, which detects cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep, in the assisted breeding of Tianhua mutton sheep, reveals for the first time a highly significant association between the 41862843 bp locus (T>C) on chromosome 2 of Tianhua mutton sheep and the cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep. P = 3.88×10 -8 Applying this SNP molecular marker to the assisted breeding of Tianhua mutton sheep allows for early prediction of cholesterol content by detecting genotypes in lambs, eliminating the need to wait for post-slaughter phenotyping. This fundamentally solves the technical challenge of traditional breeding methods that only allow cholesterol testing after slaughter, providing the first specific molecular tool for the genetic improvement of the low-cholesterol trait in Tianhua mutton sheep.

[0019] The average cholesterol content of the SNP molecular marker genotype TT was 33.29 mg / 100g, TC was 37.88 mg / 100g, and CC was 40.29 mg / 100g. The cholesterol content of the TT genotype was significantly lower than that of the TC and CC genotypes. P <0.05), the T allele is the dominant allele for the low cholesterol trait. By detecting this SNP locus and selecting homozygous TT individuals, molecular marker-assisted breeding of low-cholesterol Tianhua sheep can be achieved in the early stages of breeding, without the need for slaughter phenotyping. This significantly shortens the breeding cycle and reduces breeding costs. After 2-3 generations of continuous breeding, a stable new low-cholesterol Tianhua sheep strain can be developed, which has extremely strong industrial application value.

[0020] The method for detecting cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep provided by this invention can predict the cholesterol content level of an individual by detecting the genotype at a 41,862,843bp locus. This enables early assessment of cholesterol traits in Tianhua mutton sheep during the live stage, avoiding the high cost and time consumption of chemical determination after slaughter for each candidate breeding sheep in traditional methods. Attached Figure Description

[0021] Figure 1 Manhattan plot of genome-wide association analysis of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep in Example 1 of this invention; Figure 2 This is a QQ graph showing the genome-wide association analysis of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep in Example 1 of this invention.

[0022] Figure 3 A graph showing the difference in cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep among different genotypes of the target SNP. Different superscript letters in the graph indicate significant differences between groups. P <0.05). Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0024] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For clarity, some terms are defined as follows: SNP (single nucleotide polymorphism) is a type of molecular genetic marker proposed in 1996 by Lander, a researcher at the Human Genome Research Center of MIT. It primarily refers to DNA sequence polymorphism caused by variations in a single nucleotide at the genomic level. SNP polymorphism involves variations in only a single base, manifesting as transitions, transversions, insertions, and deletions. In this invention, SNP specifically refers to the T>C base substitution at position 41,862,843 bp on sheep chromosome 2.

[0025] Genome-wide association study (GWAS) is an analytical method that scans the entire genome for genetic variants (such as SNPs) that are significantly associated with a specific trait (such as cholesterol content). This invention uses GWAS technology to screen for SNP loci significantly associated with cholesterol content in the longissimus dorsi muscle of the Tianhua sheep population.

[0026] Reference genome: refers to the standard genome sequence map of a species, used as a reference for sequence alignment and variant detection. The sheep reference genome used in this invention is ARS-UI_Ramb_v2.0, NCBI database accession number GCF_016772045.1.

[0027] Marker-Assisted Selection (MAS) is a breeding technique that uses molecular markers (such as SNPs) closely linked to a target trait to detect the genotype of individuals, thereby enabling early and accurate selection of individuals with superior traits. This invention utilizes the association between the TT genotype and low cholesterol to achieve MAS-assisted selection of the low-cholesterol trait in Tianhua sheep.

[0028] The Tianhua mutton sheep experimental group used in this invention was provided by Minqin County Defu Agricultural Technology Co., Ltd. (Wuwei City, Gansu Province).

[0029] The raw materials and reagents used in this invention can all be obtained through publicly available commercial channels. The operating methods described in the embodiments are all conventional techniques in the field, and those skilled in the art can repeat this invention according to the description in this specification.

[0030] This invention provides a SNP marker associated with cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep. The nucleotide sequence is shown in SEQ ID NO.1 in Table 1. The polymorphic site of the SNP marker associated with cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep is located at position 151 from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism is T / C. That is, the SNP marker associated with cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep is located at position 41,862,843 bp on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0 (NCBI version number GCF_016772045.1). NKX2-6 , STC1 and NKX3-1 The SNP molecular marker was located near the gene. This SNP molecular marker was significantly correlated with cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep. P = 3.24×10 -8 It can be used for early prediction of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep and for molecular marker-assisted selection breeding.

[0031] Table 1: Sequence List

[0032] This invention also provides a breeding method for low-cholesterol Tianhua sheep, which involves detecting the above-mentioned SNPs in the Tianhua sheep genome, selecting individuals with the TT genotype as breeding sheep for the low-cholesterol, healthier Tianhua sheep breed, and culling individuals with the TC and CC genotypes.

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are merely illustrative of the invention and are not intended to limit the scope of protection of the invention. Example 1: Phenotypic determination of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep (1) Experimental animals and phenotyping 225 healthy and normally developed Tianhua sheep aged 6-10 months (131 rams and 94 ewes) were selected. The feeding environment, diet level and management conditions were kept consistent, and they had free access to feed and water.

[0034] Longissimus dorsi muscle sample collection: After slaughtering the experimental sheep, the longissimus dorsi muscle tissue was taken, the visible fascia and fat were removed, the tissue was placed in a sterile sampling bag, marked and stored in an ultra-low temperature freezer at -80℃ for later use.

[0035] Cholesterol content determination: Cholesterol content was determined by gas chromatography according to the national standard "National Food Safety Standard - Determination of Cholesterol in Food" (GB5009.128-2016). Cholesterol phenotypic data for each sheep were recorded, and outliers were removed to obtain valid phenotypic data. SPSS software was used to organize, statistically analyze, and perform variance analysis on the phenotypic data (Table 2).

[0036] Table 2: Cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep of different ages and sexes

[0037] Note: Unit: mg / 100 g; different capital letters in the same row indicate extremely significant differences. P <0.001).

[0038] As shown in Table 2, the cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep aged 7 months and 8 months was significantly lower than that of 6-month-old sheep. P (<0.001), therefore, age in months needs to be corrected as a fixed effect in subsequent GWAS analysis.

[0039] (2) Whole genome resequencing and SNP detection Genomic DNA extraction: Blood was collected from the jugular vein of each experimental sheep, anticoagulated with EDTA, and stored at -20°C. Genomic DNA was extracted from the blood using the EasyPureBlood Genomic DNA Kit (Beijing TransGen Biotech Co., Ltd., catalog number: EE121-01).

[0040] Library construction and whole-genome resequencing: Genomic DNA was fragmented to approximately 350 bp using sonication. A paired-end library with an insert size of 350 bp was constructed using the TruSeq DNA Library Prep Kit (Illumina, USA). After the library passed quantification using a Qubit 2.0 fluorometer and Agilent 2100 Bioanalyzer, paired-end sequencing (150 bp reads) was performed on the BGI MGI DNBSEQ-T7 sequencing platform. The target sequencing depth was 10× coverage.

[0041] Sequencing data processing and SNP calling: BWA software was used to align the resequencing data to the sheep reference genome ARS-UI_Ramb_v2.0 (NCBI version number: GCF_016772045.1). Picard software was used for sorting and deduplication; GATK software was used for SNP detection, quality control, and annotation to obtain a high-quality SNP dataset.

[0042] (3) Estimation of genetic parameters The heritability of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep was estimated using ASReml software based on the following mixed linear model:

[0043] in, Y Observations representing individual traits; β This is a fixed effect; α It is a random additive genetic effect; X and Z These are the correlation matrices for fixed effects and random additive genetic effects, respectively. e This is a random residual effect.

[0044] The variance components were estimated using the restricted maximum likelihood (REML) method, and the heritability was calculated using the following formula:

[0045] in, h 2 Narrowly defined heritability. It represents the proportion of phenotypic variation of a trait that can be stably inherited by offspring (determined by additive inheritance effects), and is also a core indicator for measuring selection potential in breeding. Additive genetic variance. The variation caused by the cumulative effect (additive effect) of genes is the variance of the breeding value. This is the genetic component that can be stably inherited by offspring. Phenotypic variance. The total phenotypic variation of individual trait observations (YY) is equal to the sum of additive genetic variance and residual variance; Residual variance. The sum of all random factors except additive genetic effects, including environmental effects from random residual effects (e), non-additive genetic effects (such as dominance and epistasis), and measurement errors. The results are shown in Table 3.

[0046] Table 3: Variance components and genetic parameter estimation of cholesterol traits

[0047] As shown in Table 3, the heritability of cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep is 0.31±0.09, which is a trait with medium heritability (0.2~0.4). This indicates that the trait has good potential for genetic improvement and is suitable for molecular marker-assisted selection breeding.

[0048] (4) Genome-wide association analysis A kinship matrix was constructed based on genotype data as a random effect. Using rMVP software, a GWAS analysis of cholesterol content in the longissimus dorsi muscle was performed based on a mixed linear model. The specific calculation model is as follows: y = Xa + Zβ + Wμ + e In the formula: y This is a vector of cholesterol phenotype values; X The indicator matrix for fixed effects, a This is the fixed effects vector; Z The indicator matrix for SNPs, β This represents the effect vector of the SNP; W The correlation matrix is ​​the random effects. μ denoted as , where is the predicted random individual; e is the random residual.

[0049] Determining the significance threshold using the Bonferroni correction method: Setting a significance threshold P <1×10 -6 Screen for significantly associated SNPs.

[0050] GWAS analysis results are as follows Figures 1-2 As shown in the Manhattan plot and QQ plot, 41 SNP loci with highly significant associations to cholesterol content were screened within the interval 41,860,772–41,870,953 bp (approximately 10.2 kb) on chromosome 2 of Tianhua mutton sheep. Figure 1 (Table 4).

[0051] Table 4: Significant SNPs in Tianhua Meat Sheep GWAS

[0052] Table 5: Significant SNPs in Tianhua Meat Sheep GWAS

[0053] As shown in Tables 4 and 5, the most significant SNP locus is located on chromosome 2 at 41,862,843 bp, with a P-value of 3.88 × 10⁻⁶. -8 (-log10P ≈ 7.41), far exceeding the significance threshold at the genomic level. This site is located in the intergenic region, with neighboring candidate genes being STC1 (approximately 85 kb), NKX2-6 (approximately 12 kb), and NKX3-1 (approximately 210 kb).

[0054] (5) Effects of significantly associated SNP sites To verify the effectiveness of the SNP molecular markers obtained by GWAS screening, the most significant SNP locus (41862843 bp on chromosome 2, reference allele T, variant allele C) was used as a representative (Table 6). The genotyping results of SNP (chr2:41862843) in 225 individuals were extracted using VCFtools software. One-way ANOVA and multiple comparisons were performed using the R package to correlate different genotypes of this SNP with cholesterol content in the longissimus dorsi muscle (Table 7).

[0055] Table 6: SNP Information

[0056] Table 7: Genotype frequencies of SNP marker (chr2:41862843) and their association with cholesterol levels

[0057] Note: Different superscript letters (a, b) in the same column indicate significant differences (P<0.05).

[0058] From Table 7 and Figure 3 Data shows that the 41862843 locus (T>C) on chromosome 2 significantly affects the cholesterol content of the longissimus dorsi muscle in Tianhua sheep. Specifically, the cholesterol content of the TT type is significantly lower than that of the TC and CC types. P <0.05), while there was no significant difference between the TC and CC types ( P >0.05). Therefore, the T allele is the dominant allele for the low cholesterol trait. In actual breeding production, individuals with the TT allele can be selected as breeding sheep for the Tianhua meat sheep breed, which has lower cholesterol content and healthier meat quality.

[0059] Example 2: Assisted selection using SNP molecular markers associated with cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep. (1) Extraction of genomic DNA from Tianhua sheep: Genomic DNA was extracted from ear tissue, blood, or muscle tissue, and the DNA concentration and integrity were tested. Genomic DNA was extracted from the Tianhua sheep using the conventional phenol-chloroform extraction method or a commercial genomic DNA extraction kit (TAKARA, Dalian). The integrity of the extracted DNA was tested by agarose gel electrophoresis, and its purity and concentration were tested using a UV spectrophotometer.

[0060] (2) Primer design: Using the upstream and downstream sequences of the 41862843 bp site on chromosome 2 of the sheep reference genome ARS-UI-Ramb-v2.0 (as shown in Table 1 SEQ ID NO.1) as templates, specific PCR primers were designed using Primer Premier 5.0 software to amplify the DNA fragment containing this SNP site. The primer sequences are as follows: Upstream primer (F): 5'-TCCAAGTCTATGCCCCAACT-3' (SEQ ID NO.2) Downstream primer (R): 5'-CGAAGACCAAATCTGCCTGT-3' (SEQ ID NO.3) The expected amplified fragment length is 178 bp, and this fragment contains the SNP site.

[0061] (3) PCR amplification Using extracted Tianhua sheep genomic DNA as a template, PCR amplification was performed using the primers described above. The reaction system was as follows (20 μL): 10 μL 2×Taq PCR Master Mix, 0.5 μL each of forward and reverse primers (10 μM), 1 μL genomic DNA template (50 ng / μL), and ddH2O to a final volume of 20 μL. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 5 min; and storage at 4℃.

[0062] After PCR amplification, agarose gel electrophoresis was performed to confirm that the amplified fragment size was consistent with the expected size. After confirming the target fragment, the amplified product was recovered and purified using a PCR recovery kit (TaKaRa, Dalian), following the kit's instructions.

[0063] (4) Sequencing and analysis The purified PCR amplification products were sent to a sequencing company for first-generation sequencing. The sequencing results were compared with a reference sequence using DNASTAR software to determine the genotype of the Tianhua sheep sample at position 151 (5' end) of the sequence shown in SEQ ID NO.1. If the sequencing peak at this site was a single T peak, it was identified as the TT genotype; if it was a single C peak, it was identified as the CC genotype; and if it was a double peak of both T and C, it was identified as the TC genotype.

[0064] (5) Molecular marker-assisted selection Based on the genotyping results at the 41862843 bp locus, molecular marker-assisted selection was performed. Referring to the association analysis results (Table 7), the cholesterol content of the longissimus dorsi muscle in individuals with the TT genotype was significantly lower than that in individuals with the CC and TC genotypes. Therefore, Tianhua sheep individuals with the TT genotype were selected as breeding sheep for the low-cholesterol Tianhua sheep breed, while individuals with the TC and CC genotypes were eliminated, thus achieving selective breeding of Tianhua sheep in terms of muscle cholesterol content.

[0065] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The application of SNP molecular marker products related to cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep in the assisted breeding of Tianhua mutton sheep, characterized in that, The SNP molecular marker is a polymorphism located at the 41862843 bp site on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, where the allele is T or C.

2. The application according to claim 1, characterized in that, The nucleotide sequence containing the SNP marker is shown in SEQ ID NO.1, wherein the SNP molecular marker is located at position 151 from the 5' end of the nucleotide sequence shown in SEQ ID NO.1, and its polymorphism is T / C.

3. The application according to claim 1, characterized in that, The product is a primer combination, reagent, or kit for amplifying the SNP molecular marker.

4. The application according to claim 1, characterized in that, The SNP site polymorphism is T / C. The cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TT genotype is lower than that of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TC and CC genotypes.

5. A method for detecting the cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep, characterized in that, include: The genotype of the Tianhua mutton sheep to be tested was detected at the 41862843 bp locus on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.

0. Based on the correspondence between genotype and cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep, the cholesterol content trait of the longissimus dorsi muscle of the Tianhua mutton sheep to be tested was predicted. The relationship between the genotype and the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep is as follows: the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TT genotype is lower than that corresponding to the TC and CC genotypes.

6. The method for detecting cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep according to claim 5, characterized in that, The steps for detecting the genotype of the Tianhua mutton sheep to be tested include: extracting genomic DNA from the Tianhua mutton sheep to be tested, performing PCR amplification using primer pairs specifically amplifying the 41,862,843 bp sites, and performing genotype analysis on the amplification products.

7. The method for detecting cholesterol content in the longissimus dorsi muscle of Tianhua mutton sheep according to claim 5, characterized in that, The genotyping was performed using sequencing, KASP typing, or TaqMan probe methods.

8. A primer pair for amplifying SNP molecular markers, characterized in that, The SNP molecular marker is located at position 41,862,843 bp on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0, with alleles of T or C. The nucleotide sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO.

3.

9. A method for assisting in the breeding of low-cholesterol Tianhua meat sheep, characterized in that, include: The genotype of the Tianhua mutton sheep to be tested was detected at the 41,862,843 bp locus on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.

0. Individuals with the TT homozygous genotype were selected as breeding individuals for low-cholesterol Tianhua mutton sheep. Among them, the SNP site polymorphism is T / C, and the cholesterol content of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TT genotype is lower than that of the longissimus dorsi muscle of Tianhua mutton sheep corresponding to the TC and CC genotypes.

10. The method for assisted breeding of low-cholesterol Tianhua meat sheep according to claim 9, characterized in that, The method for detecting the genotype of the Tianhua sheep to be tested at the 41,862,843 bp locus on chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0 is to perform PCR amplification using the primer pair described in claim 8, and to perform sequencing or genotyping analysis on the amplification products, and to determine the genotype of the individual to be tested based on the analysis results.

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