Application of SNP molecular marker related to cholesterol content in longissimus dorsi muscle of Hu sheep in assisted breeding of Hu sheep

Abstract

The application discloses application of a SNP molecular marker related to cholesterol content of a back longissimus muscle of a Lake sheep in assisted breeding of the Lake sheep and belongs to the technical field of sheep genetic breeding. The SNP molecular marker related to the cholesterol content of the back longissimus muscle of the Lake sheep is located at 10932342 bp of chromosome 18 of a sheep reference genome ARS-UI_Ramb_v2.0, and a base mutation is T>C. Through correlation analysis of whole genome resequencing data and the cholesterol content of the back longissimus muscle of a 6-month-old Lake sheep, it is found that the SNP molecular marker has a significant influence on the muscle cholesterol content of the Lake sheep, and the muscle cholesterol content of TT homozygous type and TC heterozygous type Lake sheep individuals is extremely significantly lower than that of CC homozygous type individuals (P<0.001). P The method can be used for early screening of a molecular marker of the cholesterol content trait of the Lake sheep.

Description

Technical Field

[0001] This invention belongs to the field of sheep genetics and breeding technology, specifically involving the application of SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep in assisted breeding of Hu sheep. Background Technology

[0002] Cholesterol is a crucial indicator for evaluating the nutritional quality of meat. Long-term excessive consumption of cholesterol-rich animal products may be associated with an increased risk of various metabolic diseases, including obesity, hyperlipidemia, coronary heart disease, and atherosclerosis. This awareness of health risks has prompted the public to pay closer attention to dietary cholesterol and the intake of cholesterol-rich animal products. Therefore, developing low-cholesterol sheep breeds is of great significance in meeting consumer demand for healthy meat products.

[0003] The Hu sheep is an excellent local sheep breed in China, characterized by its high reproductive capacity, early sexual maturity, rapid early growth and development, and excellent meat quality, making it an ideal material for studying the genetic basis of meat quality traits. However, current research on the genetic regulatory mechanism of cholesterol content in the longissimus dorsi muscle of Hu sheep is still relatively weak, and there is a lack of molecular markers that can be used for low-cholesterol breeding, hindering the early breeding process of low-cholesterol Hu sheep.

[0004] Genome-wide association analysis (GWAS) enables high-throughput genotyping of large-scale population samples, scanning for single nucleotide polymorphisms (SNPs) across the entire genome, and statistically assessing the association strength between each locus and the target trait. This allows for the identification of SNP molecular markers significantly associated with cholesterol content, providing a scientific basis for molecular marker development. Traditionally, cholesterol content determination requires post-slaughter sampling, making it unsuitable for early selection of live breeding sheep, resulting in low breeding efficiency and high costs. Therefore, developing molecular markers significantly associated with cholesterol content in Hu sheep muscle to achieve early, rapid, and low-cost live genotyping is crucial for accelerating the genetic improvement of the nutritional quality of Hu sheep meat. This strategy not only shortens the breeding cycle and reduces testing costs but also provides technical support for the precise selection of high-quality meat sheep breeds, promoting the sustainable development of the meat sheep industry towards high efficiency, high quality, and health. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep in assisted breeding of Hu sheep.

[0006] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the invention discloses the application of SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep in assisted breeding of Hu sheep. The SNP molecular markers are located at 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, and the SNP site is either T or C.

[0007] Preferably, the SNP molecular marker has a significant correlation with the cholesterol content of the longissimus dorsi muscle of the Hu sheep.

[0008] Preferably, the cholesterol content in the longissimus dorsi muscle of Hu sheep individuals with the TT and TC genotypes of the SNP molecular marker is significantly lower than that of Hu sheep individuals with the CC genotype.

[0009] Preferably, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the SNP molecular marker is located at the 151st base from the 5' end of the sequence shown in SEQ ID NO.1, with the SNP site being T or C.

[0010] Preferably, the application is for breeding of meat quality traits in Hu sheep, identification of meat quality traits in Hu sheep, or prediction of cholesterol content in Hu sheep muscle.

[0011] In a second aspect, the present invention discloses the application of SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep in the preparation of products for predicting cholesterol content in the longissimus dorsi muscle of Hu sheep. The SNP molecular markers are located at 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, and the SNP site is T or C.

[0012] Preferably, the product is a primer pair for amplifying SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep, or a kit containing the primer pair.

[0013] A third aspect of the present invention discloses a primer set for detecting SNP molecular markers associated with cholesterol content in the longissimus dorsi muscle of Hu sheep, the primer set comprising an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0014] A fourth aspect of the present invention discloses a kit containing the primer set described above for detecting SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep.

[0015] The fifth aspect of the present invention discloses a method for screening low-cholesterol sheep, which involves detecting whether the sheep to be tested carry SNP molecular markers related to the cholesterol content of the longissimus dorsi muscle of the sheep, and selecting sheep individuals with the genotype of TT homozygous or TC heterozygous. The SNP molecular marker is located at 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, and the SNP site is either T or C.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to the application of SNP molecular markers associated with cholesterol content in the longissimus dorsi muscle of Hu sheep in Hu sheep assisted breeding. Based on the muscle cholesterol content phenotype and whole-genome resequencing data of a large sample population of Hu sheep, the SNP molecular marker chr18:10932342, which is significantly associated with cholesterol content in the longissimus dorsi muscle, was screened. The average cholesterol content of this SNP molecular marker genotype TT is 32.95 mg / 100 g, genotype TC is 35.66 mg / 100 g, and genotype CC is 39.00 mg / 100 g. This molecular marker can serve as a candidate marker for marker-assisted selection and trait prediction of muscle cholesterol content in Hu sheep, accelerating the breeding process of meat quality traits and improving the efficiency and economic benefits of breeding high-quality meat sheep. Attached Figure Description

[0017] Figure 1 Manhattan plot of GWAS for cholesterol content trait in the longissimus dorsi muscle of Hu sheep according to the present invention; Figure 2 QQ graph of cholesterol content trait in the longissimus dorsi muscle of Hu sheep according to the present invention; Figure 3 This is a graph showing the statistical analysis results of cholesterol content in the longissimus dorsi muscle of Hu sheep in each SNP genotype group according to the present invention; where *** indicates P <0.001. Detailed Implementation

[0018] To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0019] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0020] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0021] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0022] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0023] In this article, SNP (single nucleotide polymorphism) mainly refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level.

[0024] This invention provides the application of a SNP molecular marker associated with cholesterol content in the longissimus dorsi muscle of Hu sheep in assisted breeding of Hu sheep. The SNP molecular marker associated with cholesterol content in the longissimus dorsi muscle of Hu sheep is located at position 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, with a base mutation of T>C. Association analysis between whole-genome resequencing data and cholesterol content in the longissimus dorsi muscle of 6-month-old Hu sheep revealed that this SNP marker has a significant impact on muscle cholesterol content in Hu sheep, and that the muscle cholesterol content of homozygous TT and heterozygous TC Hu sheep individuals is significantly lower than that of homozygous CC individuals. P <0.001).

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0026] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] (i) Obtaining SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep 1. Experimental Animals and Phenotypic Determination: 416 healthy 6-month-old male Hu sheep lambs were selected, with consistent feeding environment and diet. After slaughter in two batches, the longissimus dorsi muscle tissue was harvested. The cholesterol content in the muscle of each male Hu sheep lamb was determined according to Method I (Gas Chromatography) of the national standard GB 5009.128-2016 "National Food Safety Standard - Determination of Cholesterol in Food". Phenotypic data for muscle cholesterol in each male Hu sheep lamb were recorded. Outliers were removed using the 3σ criterion to obtain valid phenotypic data. SPSS software was used to organize, statistically analyze, and perform ANOVA on the phenotypic data. The average cholesterol content in the longissimus dorsi muscle of 6-month-old male Hu sheep lambs was 37.46 mg / 100 g, the standard deviation was 6.43 mg / 100 g, and the coefficient of variation was 17.17% (Table 1).

[0029] Table 1. Descriptive statistics of cholesterol content in the longissimus dorsi muscle of Hu sheep (unit: mg / 100 g)

[0030] 2. Whole-genome resequencing and SNP detection: Blood from the jugular vein of male sheep was collected into EDTA anticoagulant tubes, and genomic DNA was extracted for whole-genome resequencing. The raw sequencing data underwent quality control using Trimmomatic software, filtering out low-quality bases and removing adapter sequences to obtain high-quality reads. The resequencing data was further aligned to the sheep reference genome ARS-UI_Ramb_v2.0 (NCBI version GCF_016772045.1) using BWA software. 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.

[0031] 3. Genome-wide association analysis: A kinship matrix was constructed based on genotype data as a random effect. Using rMVP software, a GWAS analysis was performed on cholesterol content in the longissimus dorsi muscle 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 of SNP, β This represents the effect vector of the SNP; W The correlation matrix is ​​the random effects. μ For predicted random individuals; e The residuals are random.

[0032] The significance of SNPs was tested using the Bonferroni test, with the resultant value expressed as -log(s). 10 P = 6 is the threshold line. As a result, a SNP molecular marker chr18:10932342 (T>C) significantly associated with cholesterol content in the longissimus dorsi muscle was identified at the 10932342 bp locus on chromosome 18 of the Hu sheep. P The value is 6.91 × 10 -7 ( Figure 1 and Figure 2 This can be used for early prediction of cholesterol content in the muscles of Hu sheep. The SNP sites in the test samples contained two base types, T and C, that is, the sequence shown in SEQ ID NO.1 in Table 2 has a T / C polymorphism at position 151 from the 5' end.

[0033] Table 2 Sequence List

[0034] (ii) Detection of the effect of SNP markers The genotyping results of SNP (chr18:10932342) in 416 individuals were extracted using VCFtools software. One-way ANOVA with multiple comparisons was performed using R package to correlate different genotypes of this SNP with cholesterol content in the longissimus dorsi muscle (Table 3).

[0035] Table 3. Association analysis between SNPs and cholesterol levels

[0036] Note: Different superscript letters (a, b) in the same column of Table 3 indicate significant differences. P <0.001).

[0037] The above results indicate that SNP (chr18:10932342) significantly affected the cholesterol content of the longissimus dorsi muscle in Hu sheep, with the cholesterol content of the TT and TC types being significantly lower than that of the CC type. P <0.001)( Figure 3 In breeding practice, TT or TC type individuals can be selected as breeds of sheep with low cholesterol content.

[0038] (III) Assisted selection using SNP molecular markers associated with cholesterol content in the longissimus dorsi muscle of Hu sheep. 1. Extraction of sheep genomic DNA: Collect ear tissue, blood or muscle tissue from Hu sheep, and extract genomic DNA using the phenol-chloroform method or a DNA extraction kit (TAKARA, Dalian). Detect DNA concentration using a UV spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA), and check DNA integrity by agarose gel electrophoresis.

[0039] 2. Amplification primer design Using the upstream and downstream sequences of the 10932342 bp site on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0 (as shown in SEQ ID NO.1 in Table 2) as templates, PCR amplification primers were designed using Primer 5.0 software. The nucleotide sequences of the upstream and downstream primers are shown in SEQ ID NO.2 and SEQ ID NO.3 in Table 2, respectively.

[0040] 3. PCR amplification and product purification Using the extracted Hu sheep genomic DNA as a template, PCR amplification was performed using the primers described above. The amplification system is shown in Table 4. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and 72℃ final extension for 5 min.

[0041] Table 4 PCR amplification system

[0042] 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.

[0043] 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 target sheep at position 151 (5' end) of the nucleotide 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.

[0044] 5. Molecular marker-assisted selection Based on the genotyping results at the 10932342 bp locus, molecular marker-assisted selection was performed. Referring to the association analysis results (Table 3), the cholesterol content of the longissimus dorsi muscle in individuals with the TT and TC genotypes was significantly lower than that in individuals with the CC genotype. Therefore, individuals with the TT or TC genotype were selected as breeding sheep for the low-cholesterol Hu sheep breed, while individuals with the CC genotype were culled, thus achieving selective breeding of Hu sheep in terms of muscle cholesterol content.

[0045] 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 this invention.

Claims

1. The application of SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep in assisted breeding of Hu sheep, characterized by, The SNP molecular marker is located at 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, and the SNP site is either T or C.

2. The application of the SNP molecular marker related to cholesterol content in the longissimus dorsi muscle of Hu sheep as described in claim 1 in assisted breeding of Hu sheep, characterized in that, The SNP molecular markers showed a significant correlation with cholesterol content in the longissimus dorsi muscle of Hu sheep.

3. The application of the SNP molecular marker related to cholesterol content in the longissimus dorsi muscle of Hu sheep as described in claim 1 in assisted breeding of Hu sheep, characterized in that, The cholesterol content in the longissimus dorsi muscle of Hu sheep individuals with the TT and TC genotypes of the SNP molecular marker was lower than that of Hu sheep individuals with the CC genotype.

4. The application of the SNP molecular marker related to cholesterol content in the longissimus dorsi muscle of Hu sheep as described in claim 1 in assisted breeding of Hu sheep, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.

1. The SNP molecular marker is located at the 151st base from the 5' end of the sequence shown in SEQ ID NO.1, and the SNP site is T or C.

5. The application of the SNP molecular marker related to cholesterol content in the longissimus dorsi muscle of Hu sheep as described in claim 1 in assisted breeding of Hu sheep, characterized in that, The applications are for breeding of meat quality traits in Hu sheep, identification of meat quality traits in Hu sheep, or prediction of cholesterol content in Hu sheep muscle.

6. The application of SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep in the preparation of products for predicting cholesterol content in the longissimus dorsi muscle of Hu sheep, characterized in that, The SNP molecular marker is located at 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, and the SNP site is either T or C.

7. The application of the SNP molecular marker related to cholesterol content in the longissimus dorsi muscle of Hu sheep according to claim 6 in the preparation of products for predicting cholesterol content in the longissimus dorsi muscle of Hu sheep, characterized in that, The product is a primer pair for amplifying SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep, or a kit containing the primer pair.

8. A primer set for detecting SNP molecular markers related to cholesterol content in the longissimus dorsi muscle of Hu sheep, characterized in that, The primer set includes an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

9. A kit containing the primer set of claim 8 for detecting SNP molecular markers associated with cholesterol content in the longissimus dorsi muscle of Hu sheep.

10. A method for screening Hu sheep with low cholesterol content, characterized in that, The test was conducted to determine whether the Hu sheep to be tested carried SNP molecular markers related to the cholesterol content of the longissimus dorsi muscle of Hu sheep, and Hu sheep individuals with the genotype of TT homozygous or TC heterozygous were selected. The SNP molecular marker is located at 10932342 bp on chromosome 18 of the sheep reference genome ARS-UI_Ramb_v2.0, and the SNP site is either T or C.

Images