Methods and applications for identifying molecular markers related to sheep tail fat weight using multi-omics strategies

Abstract

This invention provides a method and application for identifying molecular markers associated with tail fat weight in sheep based on a multi-omics strategy. The method is based on a comprehensive analysis of whole-genome selection signal analysis, whole-genome association analysis of tail fat weight, and epigenomic and transcriptomic sequencing results of tail adipose tissue in sheep with different tail types. The marker is located at position 51760995 on chromosome 13 of the sheep reference genome Oar_rambouillet_v1.0, where an A / C base mutation exists, which is significantly correlated with tail fat weight. Amplification of the genomic region containing the SNP site statistically verified that the tail fat weight and relative tail fat weight of individuals with the AA genotype are significantly lower than those with the CC genotype. By detecting the genotype of the molecular marker, this invention allows for the selection of homozygous AA sheep for breeding in the core population, thereby reducing tail fat deposition and improving the economic benefits of sheep farming.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and molecular marker technology, specifically relating to a method and application for locking molecular markers related to sheep tail fat weight based on a multi-omics strategy. Background Technology

[0002] Tail fat deposition is an important economic trait in sheep, closely related to carcass quality, feed efficiency, and breeding costs. Currently, the main indicators for measuring tail fat deposition are tail type, tail size, and tail fat weight. However, tail type and tail size are difficult to accurately measure the amount of tail fat deposition among different individuals within a breed. Tail fat weight, on the other hand, refers to the weight of the tail fat separated directly from the carcass after slaughter and skinning, and can serve as a direct indicator of tail fat deposition in sheep. However, it loses its breeding value after slaughter; therefore, identifying the major genes affecting tail fat weight in sheep and their causal variations for early selective breeding is of great significance to the sheep farming industry.

[0003] In recent years, with the rapid development of sequencing technology and the implementation of projects such as ENCODE and FAANG, not only have abundant data resources been provided for analyzing complex economic traits, but also research ideas and methods have been provided for identifying molecular markers and major genes of important economic traits based on integrative omics strategies. Although domestic and foreign scholars have made significant progress in identifying candidate genes related to sheep tail fat deposition, there are few reports on tail fat weight as the research object, and even fewer reports on identifying major genes and genetic variations of sheep tail fat deposition based on integrative omics strategies. Therefore, this invention takes tail fat weight as the research object, and through integrating selection signal analysis of sheep with different tail types worldwide, genome-wide association analysis of tail fat weight traits, and epigenomic data of tail adipose tissue, aims to identify key target genes and genetic variations affecting sheep tail fat deposition, so as to accelerate the breeding process of new small-tailed sheep breeds or strains. Summary of the Invention

[0004] One objective of this invention is to identify molecular markers associated with sheep tail fat weight by integrating multi-omics data. Another objective is to provide a molecular marker associated with sheep tail fat weight and its application. This molecular marker is a SNP (Oar_rambouillet_v1.0Chr13:51760995A>C) site affecting sheep tail fat weight, obtained through integrated analysis of selection signal results from sheep with different tail types worldwide, genome-wide association studies of 2003 Hu sheep, and epigenetic and transcriptomic results from tail adipose tissue. Due to an A / C mutation at this site, A / C polymorphism occurs at this locus. This molecular marker is then applied to the breeding of sheep breeds that reduce tail fat weight.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a method for identifying molecular markers associated with the heavy trait of sheep tail fat based on a multi-omics strategy, comprising the following steps:

[0007] S1. Genome-wide selection signal analysis will be performed on sheep populations with different tail types to preliminarily screen candidate genomic regions related to tail fat deposition in sheep.

[0008] S2. Perform whole-genome resequencing on populations with accurate tail fat weight data. Then, perform genome-wide association analysis (GWAS) on the obtained high-quality SNP data and tail fat weight data, using –log10. (0.05 / total SNPs) As a standard screening standard, molecular marker sites that are significantly associated with sheep tail fat weight are selected; and overlap analysis is performed with the candidate genomic regions obtained in step S1 to screen out molecular marker sites located in the candidate genomic regions that are significantly associated with sheep tail fat weight.

[0009] S3. Epigenomic sequencing of the tail adipose tissue using ATAC-Seq, CUT&Tag, and Hi-C methods will be performed to identify regulatory elements in the tail adipose tissue.

[0010] S4. Integrate and analyze the results of the overlap analysis of candidate genomic regions, significantly related molecular marker sites and regulatory elements obtained in steps S1-3 above to identify key molecular marker sites; perform RNA-Seq sequencing on the tail adipose tissue of individuals with different genotypes of the identified key molecular marker sites, and count the expression levels of genes near the site to comprehensively identify key genes and causal variation sites affecting tail fat weight.

[0011] As described above, preferably, in step S1, the analysis uses Vcftools software to perform whole-genome selection signal scanning on different tail types or populations with a sliding window size of 150Kb and a step size of 75Kb, and the top 5‰ of the window is defined as the selected genomic region;

[0012] In step S2, genome-wide association analysis was performed using the MLM model in the rMVP software to analyze the tail fat weight and relative tail fat weight traits.

[0013] Further, in step S4, the nucleotide sequence of the causal variation site is obtained as shown in SEQ ID NO.1. The M at position 352bp of the sequence represents A or C. This mutation leads to the A / C polymorphism of sheep at this site. Among them, the tail fat weight and tail fat relative to carcass weight of AA genotype individuals are significantly lower than those of CC genotype individuals.

[0014] The application of a molecular marker associated with tail fat weight in sheep in screening for low-fat-weight sheep, wherein the molecular marker nucleotide sequence is shown in SEQ ID NO.1, wherein M at position 352 bp in sequence SEQ ID NO.1 is A or C, and this mutation leads to A / C polymorphism of the molecular marker, wherein individuals carrying the AA genotype have a significantly lower tail fat weight than individuals carrying the CC genotype.

[0015] The application of a primer pair for detecting the molecular markers associated with the above-mentioned tail fat weight trait in sheep in screening low-tail fat sheep, preferably, the sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0016] The application of an AQP primer pair for detecting the molecular markers associated with the above-mentioned tail fat weight trait in sheep in screening low-tail fat sheep, preferably, the sequence of the AQP primer is shown in SEQ ID NO.4-6.

[0017] The application of a kit for detecting the above-mentioned molecular markers associated with the tail fat weight trait in sheep in screening low-tail fat sheep, preferably, the kit includes ordinary PCR primer pairs or AQP sequence pairs, the sequences of the ordinary PCR primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; the sequences of the AQP primers are shown in SEQ ID NO.4-6.

[0018] An application of a method for detecting the aforementioned molecular markers associated with the tail fat weight trait in screening low-tail fat sheep includes the following steps:

[0019] 1) Amplify sheep genomic DNA using the above-mentioned ordinary PCR primer pairs, AQP primer pairs, or kits containing the above primer pairs;

[0020] 2) Identify the specific site at 352 bp of the nucleotide sequence of the amplification product obtained in step 1) as shown in SEQ ID NO.1, where M is A or C. Among them, the tail fat weight of individuals carrying the AA genotype is significantly lower than that of individuals carrying the CC genotype.

[0021] In step 2), the above-mentioned typing and identification methods include, but are not limited to, direct sequencing, fluorescent probe method, gene chip method, and high-resolution melting curve method.

[0022] Furthermore, preferably, when using ordinary PCR primer pairs for amplification, the polymorphic sites of the amplification products are identified by direct sequencing.

[0023] Furthermore, preferably, when using AQP primer pairs for amplification, the fluorescence signal is detected using an instrument and the genotyping results are viewed.

[0024] The above-described method was applied to sheep breeding, and sheep with low tail fat weight were screened by analyzing the types of polymorphic sites; sheep carrying the AA genotype had significantly lower tail fat weight than sheep carrying the CC genotype.

[0025] The application of the PCR primer pairs, AQP primers, or kits described above for detecting the aforementioned molecular markers in sheep breeding involves amplifying and detecting the genomic DNA of sheep using the primer pairs or kits described above. This allows for the determination of the genotype of the amplified product of the sample to be tested, as shown in SEQ ID NO.1 at position 352 bp. Sheep carrying the AA genotype have significantly lower tail fat weight than sheep carrying the CC genotype, thus enabling the selection of small-tailed sheep breeds with lower tail fat weight.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention provides a method for identifying molecular markers associated with tail fat weight in sheep based on a multi-omics strategy, and identifies the molecular markers influencing this trait. This method avoids errors caused by single-omics detection and accurately identifies molecular marker sites in non-coding regions, resulting in higher accuracy in identifying these marker sites. Furthermore, the method for genotyping SNP sites offers higher sensitivity, accuracy, and cost-effectiveness, enabling simultaneous detection of dozens to hundreds of SNP sites in hundreds to thousands of samples. By detecting these molecular markers and polymorphic sites, this invention can be used to select sheep with homozygous AA genes for breeding, thereby reducing tail fat deposition and demonstrating potential application value in large-scale molecular breeding of sheep.

[0028] In existing technologies, determining the degree of fat deposition in a sheep's tail requires waiting until the sheep is fully grown and then relying on phenotypes or slaughter traits. This process is time-consuming and uneconomical. The method provided by this invention for detecting molecular markers related to tail fat weight in sheep can extract DNA from sheep blood or tissues at any stage of life. By using the primer pairs provided by this invention for typing, the amount of tail fat deposition can be determined without waiting for the sheep to grow up. This method is easy to operate, fast, highly accurate, and low-cost. Furthermore, by detecting molecular markers and the genotype of these polymorphic sites, this invention can be used to select sheep with homozygous AA genes for breeding stock, thereby screening for sheep with low tail fat weight and improving the economic efficiency of sheep farming. Attached Figure Description

[0029] Figure 1Genome-wide selection signal analysis for sheep with different tail types; (A) photographs of sheep with different tail types, (B) selection signal analysis of fat-tailed and thin-tailed sheep populations, (C) Fst values ​​of sheep with different tail types in the 13 chromosome region, (D) Fst values ​​of sheep with different tail types in the 15 chromosome region, (E, F) distribution of allele frequencies in the selected regions on chromosomes 13 and 15 of sheep with different tail types.

[0030] Figure 2 Genome-wide association analysis of tail fat weight trait in sheep; (A) schematic diagram of carcass with tail fat, (B) Manhattan plot of genome-wide association analysis of tail fat weight and tail fat relative to carcass weight trait.

[0031] Figure 3 To integrate GWAS and epigenomic data to locate candidate variants significantly associated with fat deposition in sheep tails.

[0032] Figure 4 Validation of candidate loci. (A,B) Allelic frequency distribution of Chr13:51760995A>C and Chr13:51825895G>A loci in sheep with different tail types; (C,D) Tail fat weight and relative tail fat weight of individuals with different genotypes at Chr13:51760995A>C and Chr13:51825895G>A loci; (G,H) Expression levels of upstream and downstream genes at Chr13:51760995A>C and Chr13:51825895G>A loci in different genotypes.

[0033] Figure 5 These are the sequencing results of polymorphic sites in this invention. Detailed Implementation

[0034] This invention provides a method for identifying molecular markers associated with the trait of fat accumulation in sheep tails based on a multi-omics strategy. This method is applicable to any complex trait containing multi-omics data for identifying causal variations. The method includes the following steps:

[0035] S1. Global sheep breeds with available genomic data were classified into five categories based on their tail type: long fat tail, short fat tail, fat rump tail, long thin tail, and short thin tail. These were further divided into two groups: fat tail type and thin tail type. Genome-wide selection signal analysis was performed on these two groups and the groups with different tail types. The sliding window size was 150Kb, and the step size was 75Kb. The top 5‰ of the window was defined as the selected genomic region to preliminarily screen candidate genomic regions that affect fat deposition in sheep tails.

[0036] S2. Extract DNA from populations with accurate tail fat weight data records, perform whole-genome resequencing, and filter using vcftools software (-remove-indels, -minDP 5, -min-alleles 2, -max-alleles 2, -maf0.05, and -max-missing 0.8). Then, perform genome-wide association analysis (GWAS) using the MLM model in rMVP software with the obtained high-quality SNP data and tail fat weight data; using -log 10 (0.05 / total SNPs) Molecular markers associated with sheep tail fat weight were screened as a standard; and overlap analysis was performed with the candidate genomic regions obtained in step S1 to screen out molecular marker sites located in the candidate genomic regions that are significantly associated with sheep tail fat weight.

[0037] S3. Epigenomic sequencing of tail adipose tissue will be performed using ATAC-Seq, CUT&Tag, and Hi-C. Overlap analysis will be performed with the candidate genomic regions, significantly associated molecular marker sites and regulatory elements obtained in step S2 to identify molecular marker sites in regulatory elements that are significantly associated with sheep tail adipose weight.

[0038] S4. Analyze the allele frequencies of the key molecular marker sites identified in step S3 in populations with different tail types; and perform RNA-Seq sequencing on individuals with different genotypes to analyze the expression levels of genes near their molecular marker sites in order to screen out key causal variation sites that affect tail fat weight.

[0039] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0040] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, all reagents used in this invention are of analytical grade or higher.

[0041] Example 1

[0042] A method for identifying molecular markers associated with the fat weight trait in sheep tails based on a multi-omics strategy includes the following steps:

[0043] 1. Genome-wide selection signal analysis of sheep with different tail types

[0044] Based on sequencing and integration of public data, the inventors obtained whole-genome genetic variation information for five tail types of sheep: long-fat-tailed, short-fat-tailed, fat-rump-tailed, long-lean-tailed, and short-lean-tailed. These were then categorized into two groups: fat-tailed and lean-tailed. Using Vcftools software, whole-genome selection signal scanning was performed on different tail types or groups with a 150kb window and a 75kb step size. The top 5‰ of the selected genomic regions were chosen as candidate genomic regions influencing fat deposition in sheep tails. The results showed significant differences between fat-tailed and lean-tailed sheep in genomic regions on chromosomes 13 (51.15-52.05Mb) and 15 (3.67-4.50Mb). This indicates that these two genomic regions may be key candidate regions influencing fat deposition in sheep tails (e.g., [missing information]). Figure 1 ).

[0045] 2. Genome-wide association analysis of the weighted trait of tail fat in Hu sheep

[0046] The inventors collected tail fat weight and carcass weight data and individual DNA from 2003 Hu sheep. Whole-genome resequencing was performed on the DNA of these individuals using the Illumina NovaSeq 6000 platform to obtain whole-genome genetic variation information. The sheep genome sequence information version number used was Oar_rambouillet_v1.0. Genome-wide association analysis was performed on tail fat weight and relative tail fat weight (tail fat weight / carcass weight) using the MLM method in rMVP. Bonferroni correction was used to define a significance threshold, and -log10 was used. (0.05 / total snps) Defined as the genome-wide significance threshold. Based on this threshold, 981 SNP loci on sheep chromosome 13 were found to be significantly associated with tail fat weight and relative tail fat weight traits. These loci are all located on chromosome 13 and are highly linked in non-coding regions (see...). Figure 2 ).

[0047] 3. Identifying key causal variations influencing tail fat weight in sheep based on a multi-omics strategy.

[0048] The inventors obtained high-quality data by performing ATAC-Seq, CUT&Tag (H3K27ac and H3K4me3) and Hi-C sequencing on the tail adipose tissue of two 6-month-old half-sib male Hu sheep. Overlap analysis was then performed with the key candidate regions identified in step 1 and the SNP sites screened in step 2. Two SNPs (Chr13:51760995A>C and Chr13:51825895G>A) overlapped and were located in enhancer regions (see...). Figure 3 (As shown).

[0049] Further analysis of the allele frequency distribution of these two SNPs in different tail-type sheep populations in step 1 revealed significant differences between the two SNPs in fat-tailed and thin-tailed sheep populations, and significant differences in tail fat weight and relative tail fat weight among individuals with different genotypes.

[0050] Finally, RNA-Seq sequencing was performed on the tail adipose tissue of 74 six-month-old Hu sheep (provided by Minqin County Defu Agricultural Technology Co., Ltd.) from individuals with different genotypes. The effects of different genotypes on the expression of upstream and downstream genes were analyzed. The results showed that the Chr13:51760995A>C mutation affected the expression of the BMP2 gene. Moreover, at the Chr13:51760995A>C site, the tail fat weight and relative tail fat weight of individuals with the AA genotype were significantly lower than those of individuals with the CC genotype (P<0.01) (see...). Figure 4 (Table 1) Therefore, it was determined that the Chr13:51760995A>C site can affect tail fat deposition by influencing the expression of the BMP2 gene, and can serve as a key molecular marker affecting tail fat deposition in sheep. This study provides a successful case for identifying key causal variations and major genes based on integrated multi-omics data, and provides a reliable molecular marker site for the breeding of small-tailed sheep.

[0051] Table 1. Association analysis between the Chr13:51760995A>C polymorphism site and tail fat weight trait in sheep.

[0052]

[0053] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0054] Example 2: Detection of the genomic region containing the Chr13:51760995A>C locus in sheep.

[0055] (1) Primer design

[0056] Primers MF and MR were designed based on the sequence of the region containing sheep Chr13:51760995A>C. The primer sequences are as follows.

[0057] MF (SEQ ID NO.2): 5′-TTGCCAACAAATCTGCAT-3′,

[0058] MR (SEQ ID NO. 3): 5′-TCCCCATTGTTTTAGGTCA-3′.

[0059] (2) Amplification and detection of the genome sequence of sheep Chr13:51760995A>C

[0060] DNA was extracted from the blood of 10 sheep and used as a template for amplification and sequencing. PCR amplification was performed using a 25 μL reaction system, containing 1 μL DNA template, 12.4 μL 2×PCR Master Mix, 0.8 μL forward primer, 0.8 μL reverse primer, and 10 μL ddH2O. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 60 s, for 35 cycles, followed by a final extension at 72℃ for 10 min.

[0061] The amplified PCR fragment was sequenced, and the sequencing results showed that the specific nucleotide sequence of the amplified fragment is shown in SEQ ID NO.1. An A / C polymorphism exists at the 352 bp site of the amplified fragment, meaning this site is the same as the site identified in Example 1 based on a multi-omics strategy. Figure 5 As shown.

[0062] SEQ ID NO. 1: TTGCCAACAAAATCTGCATAATCAAAGCTATGATT.

[0063] Example 3: Detection of the Chr13:51760995A>C site in sheep

[0064] 1. Primer design for AQP technology

[0065] Based on the SNP sites identified in Examples 1 and 2, a primer set for detecting these SNP sites using AQP technology was designed, including a first primer, a second primer, and a universal primer. The nucleotide sequence of the first primer is shown in SEQ ID No. 4; the nucleotide sequence of the second primer is shown in SEQ ID No. 5; and the nucleotide sequence of the universal primer is shown in SEQ ID No. 6. The regions containing the polymorphic sites were amplified using the first and second primers, and then extended using the universal primer. The extended products were analyzed to determine their genotype. The specific primer sequences are as follows:

[0066] First primer (SEQ ID NO.4): 5'-GAAGGTGACCAAGTTCATGCTTGGTTAGATAGCATCACTAACTCAATAAAA-3',

[0067] Second primer (SEQ ID NO.5): 5'-GAAGGTCGGAGTCAACGGATTTGGTTAGATAGCATCACTAACTCAATAAAC-3',

[0068] Universal primer (SEQ ID NO.6): 5'-GTCTTTCAGTATCTCCCAGAGTTT TCTA-3'.

[0069] The above primers were synthesized by Beijing Sangon Biotech Co., Ltd. Each primer pair in the AQP primer pair was diluted to 100 μmol / L and mixed with sterile water in a volume ratio of 12:12:30:46 (forward primer A1:forward primer A2:universal reverse primer C:sterile water) to prepare a primer mixture.

[0070] 2. Extracted genomic DNA and subjected to quality control.

[0071] Genomic DNA can be extracted from sheep blood using a DNA extraction kit. The extracted genomic DNA is then tested for quality using 1% agarose gel electrophoresis and Nanodrop 2100. The extracted DNA must meet the following requirements: (1) Agarose gel electrophoresis shows a single DNA band without significant diffusion. (2) Nanodrop 2100 shows A260 / 280 between 1.8 and 2.0; A260 / 230 between 1.8 and 2.0; and no significant light absorption at 270 nm. DNA that does not meet these requirements needs to be extracted again until it meets the requirements. Based on the AQPTM detection technology and genome size calculation from Beijing Jiacheng Biotechnology Co., Ltd., the required DNA dosage is 10–20 ng / sample. The extracted genomic DNA is then diluted to a concentration of 10–20 ng / μL as a DNA template.

[0072] 3. Perform genotyping

[0073] First, according to AQP TM The detection technology and genome size calculations determined the DNA usage to be 10–20 ng / sample. The extracted genomic DNA was then diluted to a concentration of 10–20 ng / μL to serve as a DNA template.

[0074] Each primer in the AQP primer pair is 100 μmol / L and is mixed with sterile water in a volume ratio of 12:12:30:46 (primer A1:primer A2:primer C:stere water) to prepare a primer mixture for later use.

[0075] Then, using a pipette, add 0.07 μL of primer mixture, 0.5 μL of sterile water, 2.5 μL of HiGeno 2×Probe Mix, and 2 μL of DNA to each well of a 384-well plate. After adding the components, seal the plate, vortex, centrifuge, and place it on a C1000Touch™ Thermal Cycler instrument for PCR amplification. The specific procedure is as follows:

[0076] Pre-denaturation at 95℃ for 10 minutes;

[0077] 95℃, 20 seconds (denaturation) — 61℃-55℃, 40 seconds (annealing & extension), amplification for 10 cycles, with a decrease of 0.6℃ per cycle;

[0078] 95℃, 20 seconds (denaturation) — 55℃, 40 seconds, continue amplification for 34 cycles.

[0079] After amplification, fluorescence signals were detected and genotyping results were observed using a C1000 Touch™ Thermal Cycler instrument at 37°C. Tail fat weight and tail fat relative to carcass weight for the same genotype were also statistically analyzed. The results of the tests on another batch of 281 six-month-old Hu sheep raised at Minqin County Defu Agricultural Technology Co., Ltd. are shown in Table 2.

[0080] Table 2. Validation of the sheep Chr13:51760995A>C polymorphism site.

[0081]

[0082] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0083] The results showed that individuals with the AA genotype at the 352bp site shown in SEQ ID NO.1 had significantly lower tail fat weight than those with the CC genotype. This is consistent with the results obtained from the site identified in Example 1 using a multi-omics strategy. Since tail fat weight is a direct indicator of tail fat deposition, selection based on tail fat deposition can improve economic efficiency. Therefore, the PCR primer pair of this invention was used to detect the Chr13:51760995A>C site polymorphism to screen AA genotype individuals to form a core population for breeding small-tailed sheep. Furthermore, this site and technical detection method can be used for early detection in sheep populations to eliminate individuals carrying the CC genotype, thereby improving the economic efficiency of sheep farming.

Claims

1. A method for identifying molecular markers associated with the heavy trait of sheep tail fat based on a multi-omics strategy, characterized in that, It includes the following steps: S1. Genome-wide selection signal analysis will be performed on sheep populations with different tail types to preliminarily screen candidate genomic regions related to tail fat deposition in sheep. S2. Perform whole-genome resequencing on populations with accurate tail fat weight data. Then, perform genome-wide association analysis (GWAS) on the obtained high-quality SNP data and tail fat weight data, using –log 10 (0.05 / total SNPs) As a standard screening site, molecular marker sites that are significantly associated with sheep tail fat weight; Overlap analysis was performed with the candidate genomic regions obtained in step S1 to screen out molecular marker sites located in the candidate genomic regions that are significantly associated with sheep tail fat weight; S3. Epigenomic sequencing of the tail adipose tissue using ATAC-Seq, CUT&Tag, and Hi-C methods will be performed to identify regulatory elements in the tail adipose tissue. S4. Integrate and analyze the results of the overlap analysis of candidate genomic regions, significantly related molecular marker sites and regulatory elements obtained in steps S1-3 above to identify key molecular marker sites; perform RNA-Seq sequencing on the tail adipose tissue of individuals with different genotypes of the identified key molecular marker sites, and count the expression levels of genes near the site to comprehensively identify key genes and causal variation sites affecting tail fat weight. In step S4, the nucleotide sequence of the causal variation site is obtained as shown in SEQ ID NO.

1. The M at position 352 bp of this sequence represents A or C. This mutation leads to the A / C polymorphism of sheep at this site. Among them, the tail fat weight and tail fat relative to carcass weight of AA genotype individuals are significantly lower than those of CC genotype individuals.

2. The method as described in claim 1, characterized in that, In step S1, the Vcftools software was used to perform whole-genome selection signal scanning on different tail types or populations with a sliding window size of 150Kb and a step size of 75Kb. The top 5‰ of the window was defined as the selected genomic region. In step S2, genome-wide association analysis was performed using the MLM model in the rMVP software to analyze the tail fat weight and relative tail fat weight traits.

3. The application of a molecular marker associated with the tail fat weight trait in screening low-tail fat sheep, characterized in that, The molecular marker nucleotide sequence is shown in SEQ ID NO.1, wherein M at position 352 bp in sequence SEQ ID NO.1 is A or C. This mutation leads to the A / C polymorphism of the molecular marker, wherein the tail fat weight of individuals carrying the AA genotype is significantly lower than that of individuals carrying the CC genotype.

4. The application of a primer pair for detecting molecular markers associated with the tail fat weight trait in sheep in screening for low-tail fat sheep, characterized in that, The sequences of the primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; the molecular marker nucleotide sequence is shown in SEQ ID NO.1, wherein M at position 352 bp in sequence SEQ ID NO.1 is A or C. This mutation leads to A / C polymorphism of the molecular marker, wherein the tail fat weight of individuals carrying the AA genotype is significantly lower than that of individuals carrying the CC genotype.

5. The application of an AQP primer pair for detecting molecular markers associated with tail fat weight traits in sheep in the screening of tail fat-type sheep, characterized in that, The nucleotide sequences of the AQP primers are shown in SEQ ID NO.4-6; the molecular marker nucleotide sequences are shown in SEQ ID NO.1, wherein M at position 352 bp in sequence SEQ ID NO.1 is A or C. This mutation leads to A / C polymorphism of the molecular marker, wherein the tail fat weight of individuals carrying the AA genotype is significantly lower than that of individuals carrying the CC genotype.

6. The application of a kit for detecting the above-mentioned molecular markers associated with the tail fat weight trait in sheep in screening low-tail fat sheep, characterized in that, The kit includes a standard PCR primer pair or an AQP sequence pair. The sequences of the standard PCR primer pair are shown in SEQ ID NO.2 and SEQ ID NO.

3. The sequences of the AQP primers are shown in SEQ ID NO.4-6. The molecular marker nucleotide sequence is shown in SEQ ID NO.1, wherein M at position 352 bp in sequence SEQ ID NO.1 is either A or C. This mutation leads to A / C polymorphism of the molecular marker, wherein individuals carrying the AA genotype have significantly lower tail fat weight than individuals carrying the CC genotype.

7. An application of a method for detecting molecular markers associated with tail fat weight in sheep in screening for low-tail fat sheep, comprising the following steps: 1) Amplify sheep genomic DNA using the ordinary PCR primer pairs with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, or the AQP primer pairs with nucleotide sequences as shown in SEQ ID NO.4-6; 2) Identify the specific site at position 352 bp (M is A or C) of the nucleotide sequence of the amplification product obtained in step 1), as shown in SEQ ID NO.

1. Individuals carrying the AA genotype had significantly lower tail fat weight than individuals carrying the CC genotype.

8. The application of PCR primer pairs, AQP primers, and kits for detecting molecular markers associated with tail fat weight traits in sheep in sheep breeding, characterized in that... The molecular marker nucleotide sequence is shown in SEQ ID NO.1, wherein M at position 352 bp in sequence SEQ ID NO.1 is A or C. This mutation leads to the A / C polymorphism of the molecular marker, wherein individuals carrying the AA genotype have significantly lower tail fat weight than individuals carrying the AC or CC genotypes; the sequences of the PCR primer pair are shown in SEQ ID NO.2 and SEQ ID NO.3; the sequences of the AQP primers are shown in SEQ ID NO.4-6; the kit includes PCR primer pairs or AQP primers; the breeding is for selecting low-fat sheep breeds or strains with lower tail fat weight.

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