Method for locking molecular marker related to sheep tail fat weight based on multiple omics strategies and application

Abstract

The invention provides a method for locking a molecular marker related to sheep tail fat weight based on multiple omics strategies and application, and the method is obtained based on whole-genome selection signal analysis of sheep with different tail types, whole-genome association analysis of tail fat weight, epimics sequencing of tail adipose tissue and comprehensive analysis of transcriptome sequencing results. The invention relates to a sheep tail fat weight mutation site, which is located at a chromosome 51760995 of a sheep reference genome Orambouilletv1.0 version sequence 13, and A / C base mutation exists on the site and is obviously related to the sheep tail fat weight. Through amplification of a genome area where the SNP site is located, statistics verifies that the tail fat weight and the tail fat relative weight of an AA genotype individual are significantly lower than those of a CC type individual. By detecting the genotype of the molecular marker, AA homozygous sheep can be selected to enter a core group for breeding, so that the tail fat deposition is reduced, and the economic benefit of the sheep breeding industry is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology and molecular markers, and in particular relates to a method and application of locking molecular markers related to sheep tail fat weight based on a multi-omics strategy. Background Art

[0002] Tail fat deposition is one of the important economic traits of sheep, which is closely related to carcass quality, feed efficiency and breeding cost. At present, the main indicators for measuring tail fat deposition are tail shape, tail size and tail fat weight. However, it is difficult to accurately measure the amount of tail fat deposition between different individuals within the breed based on tail shape and tail size. Tail fat weight refers to the weight of tail fat separated directly from the carcass after the fur is removed after slaughter, which can be used as a direct indicator to measure tail fat deposition in sheep. However, it will lose its breeding value after slaughter. Therefore, it is of great significance to the breeding industry to identify the main effect genes and causal variants that affect the tail fat weight of sheep and use them for early breeding.

[0003] In recent years, with the rapid development of sequencing technology and the implementation of plans such as ENCODE and FAANG, not only a wealth of data resources are provided for analyzing complex economic traits, but also research ideas and methods are provided for locking molecular markers and major effect genes of important economic traits based on integrated omics strategies. Although scholars at home and abroad have made significant progress in the identification of candidate genes related to tail fat deposition in sheep, there are fewer reports on tail fat weight as the research object, and there are even fewer reports on the identification of major effect genes and genetic variations of sheep tail fat deposition based on integrated omics strategies. Therefore, the present invention takes tail fat weight as the research object, and through the integration of selection signal analysis of sheep with different tail types around the world, genome-wide association analysis of tail fat weight traits, and epigenomic data of tail fat tissue, it aims to lock the key target genes and genetic variations that affect sheep tail fat deposition, so as to accelerate the breeding process of new varieties or new strains of small-tailed sheep. Summary of the invention

[0004] One object of the present invention is to lock the molecular marker related to the fat weight of sheep tail by integrating multi-omics data, and another object of the present invention is to provide a molecular marker related to the fat weight trait of sheep tail and its application. The molecular marker is a SNP (Oar_rambouillet_v1.0Chr13:51760995A>C) site that affects the fat weight of sheep tail obtained by integrating and analyzing the selection signal results of sheep with different tail types around the world, the whole genome association analysis of 2003 Hu sheep, and the epigenetic and transcriptome results of tail adipose tissue. Because the base at this site has an A / C mutation, it leads to the A / C polymorphism at this site; and the molecular marker is applied to the breeding of varieties that reduce the fat weight of sheep tail.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for locking molecular markers related to sheep tail fat weight traits based on a multi-omics strategy, which comprises the following steps:

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

[0008] S2. Perform whole-genome resequencing on the population with accurate tail fat weight data records, and perform whole-genome association analysis on the obtained high-quality SNP data and tail fat weight data using –log10 (0.05 / total SNPs) The molecular marker sites significantly correlated with the sheep tail fat weight are screened as the standard; and overlap analysis is performed with the candidate genomic region obtained in step S1 to screen the molecular marker sites significantly correlated with the sheep tail fat weight in the candidate genomic region;

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

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

[0011] In the method described above, preferably, in step S1, the Vcftools software is used for analysis, and the whole genome selection signal scanning of different tail types or populations is performed with a sliding window size of 150 Kb and a step length of 75 Kb, and the top 5‰ window is defined as the selected genomic region;

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

[0013] Furthermore, the nucleotide sequence of the causal mutation site obtained in step S4 is shown in SEQ ID NO.1, and the M at the 352bp position of the sequence represents A or C, and the 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 individuals with AA genotype are significantly lower than those of CC type individuals.

[0014] A molecular marker related to the tail fat weight trait of sheep is used in screening low tail fat sheep. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the M at the 352 bp in the sequence SEQ ID NO.1 is A or C, and the 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.

[0015] A primer pair for detecting the molecular marker associated with the sheep tail fat weight trait is used in screening low tail fat sheep. Preferably, the sequence of the primer pair is as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0016] An application of an AQP primer pair for detecting the above-mentioned molecular marker related to the sheep tail fat weight trait in screening low tail fat sheep, preferably, the sequence of the AQP primer is shown as SEQ ID NO.4-6.

[0017] A kit for detecting the above-mentioned molecular markers related to the sheep tail fat weight trait in screening low tail fat sheep, preferably, the kit comprises a common PCR primer pair or an AQP sequence pair, the sequence of the common PCR primer pair is shown in SEQ ID NO.2 and SEQ ID NO.3; the sequence of the AQP primer is shown in SEQ ID NO.4-6.

[0018] A method for detecting the molecular markers related to the tail fat weight trait of sheep is used in screening low tail fat sheep, comprising the following steps:

[0019] 1) amplifying sheep genomic DNA using the above-mentioned common PCR primer pair, AQP primer pair or a kit containing the above-mentioned primer pair;

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

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

[0022] Furthermore, preferably, when a common PCR primer pair is used for amplification, the polymorphic sites of the amplified products are identified by direct sequencing.

[0023] Further, preferably, when the AQP primer pair is used for amplification, an instrument is used to detect the fluorescent signal and check the typing results.

[0024] The above method is applied to sheep breeding, and sheep with low tail fat weight are screened out by analyzing the types of polymorphic sites; the tail fat weight of sheep carrying the AA genotype is significantly lower than that of sheep carrying the CC genotype.

[0025] The detection method of the PCR primer pair, AQP primer or kit for detecting the above-mentioned molecular markers is used in sheep breeding, by using the above-mentioned primer pair or kit to amplify and detect the genomic DNA of the sheep, and determine the nucleotide sequence of the amplified product of the sample to be tested, such as the genotype at the 352bp shown in SEQ ID NO.1, wherein the tail fat weight of sheep carrying the AA genotype is significantly lower than that of sheep carrying the CC genotype, thereby breeding a small-tailed sheep breed with lower tail fat weight.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a method for locking molecular markers related to sheep tail fat weight traits based on a multi-omics strategy and molecular markers related to sheep tail fat weight traits. The method can avoid errors caused by single-omics detection, and can accurately lock molecular marker sites in non-coding regions. The identified molecular marker sites have higher accuracy. At the same time, the method of SNP site genotype has higher sensitivity, accuracy, and cost-effectiveness, and can simultaneously detect tens to hundreds of SNP sites in hundreds to thousands of samples. The present invention can be used to select sheep with AA homozygous genes as breeding sheep for breeding, thereby reducing fat deposition in the tail of sheep, and has potential application value for large-scale molecular breeding of sheep.

[0028] In the prior art, if one wants to determine the degree of fat deposition in a sheep's tail, one can only wait until the sheep grows up and then determine it based on the phenotype or slaughter traits, which is time-consuming and uneconomical. The method for detecting the above-mentioned molecular markers related to the sheep tail fat weight trait provided by the present invention can extract sheep blood or tissue DNA at any period, and use the primer pairs provided by the present invention for typing to determine the amount of fat deposition in the sheep's tail, without waiting for the sheep to grow up. The method is easy to operate, fast, high in accuracy, and low in cost. The present invention can be used to select sheep with homozygous AA genes as breeding sheep for breeding, and to screen sheep with low tail fat weight, through the detection of molecular markers and the genotype of the polymorphic site, to help improve the economic benefits of the sheep breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a genome-wide selection signal analysis of sheep with different tail types; including: (A) photos 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 chromosome 13 region, (D) Fst values ​​of sheep with different tail types in the chromosome 15 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 This is a genome-wide association analysis of the tail fat weight trait in sheep; (A) a schematic diagram of carcass tail fat, (B) a Manhattan plot of the genome-wide association analysis of tail fat weight and tail fat relative to carcass weight.

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

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

[0033] Figure 5 It is the sequencing result of the polymorphic site in the present invention. DETAILED DESCRIPTION

[0034] The present invention provides a method for locking molecular markers related to sheep tail fat weight traits based on a multi-omics strategy. Any complex trait containing multi-omics data is suitable for locking causal variation by this method, comprising the following steps:

[0035] S1. The global sheep breeds with genomic data were divided into five categories according to their tail type: long fat tail, short fat tail, fat butt tail, long thin tail and short thin tail, and classified into two groups: fat tail type and thin tail type. The whole genome selection signal analysis was performed on these two groups and groups with different tail types, with a sliding window size of 150Kb and a step size of 75Kb; the top 5‰ window was defined as the selected genomic region to preliminarily screen the candidate genomic regions that affect fat deposition in the sheep tail;

[0036] S2. Extract DNA from the population with accurate tail fat weight data records, perform whole-genome resequencing, and filter with 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 with the obtained high-quality SNP data and tail fat weight data using the MLM model in rMVP software. 10 (0.05 / total SNPs) Using the above method as a standard to screen molecular markers related to sheep tail fat weight; and performing overlap analysis with the candidate genomic region obtained in step S1 to screen molecular marker sites that are significantly related to sheep tail fat weight in the candidate genomic region;

[0037] S3, performing epigenomic sequencing of tail adipose tissue using ATAC-Seq, CUT&Tag and Hi-C, and performing overlap analysis with the candidate genomic regions, molecular marker sites significantly associated with tail fat weight and regulatory elements obtained in step S2, to identify molecular marker sites located in regulatory elements that are significantly associated with sheep tail fat weight;

[0038] S4. Count 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 count the expression levels of genes near their molecular marker sites to screen out the key causal variation sites that affect tail fat weight.

[0039] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0040] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are of analytical grade or above.

[0041] Example 1

[0042] A method for locking molecular markers related to sheep tail fat weight traits based on a multi-omics strategy comprises the following steps:

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

[0044] Based on sequencing and integrating data from public data, the inventors obtained the whole genome genetic variation information of sheep with five tail types, namely long fat tail, short fat tail, fat hip tail, long thin tail and short thin tail, and classified them into two groups, fat tail type and thin tail type. Using Vcftools software, the whole genome selection signal scanning was performed on different tail types or groups with a window of 150kb and a step size of 75kb, and the top 5‰ window was taken as the selected genomic region as the candidate genomic region affecting fat deposition in the sheep's tail. The results showed that when fat-tailed sheep and thin-tailed sheep were compared, there were obvious differences in the genomic regions of chromosomes 13 (51.15-52.05Mb) and 15 (3.67-4.50Mb). This suggests that these two genomic regions may be key candidate regions affecting fat deposition in sheep tails (such as Figure 1 ).

[0045] 2. Genome-wide association analysis of traits related to tail fat weight in Hu sheep

[0046] The inventors collected tail fat weight and carcass weight data and individual DNA of 2003 Hu sheep, and used the IlluminaNovaSeq 6000 platform to perform whole genome resequencing of their individual DNA to obtain whole genome genetic variation information. The sheep genome sequence information version number used was Oar_rambouillet_v1.0. The MLM method in rMVP was used to perform whole genome association analysis on tail fat weight and tail fat relative weight (tail fat weight / carcass weight). The Bonferroni correction was used to define the significant threshold, and -log10 (0.05 / total snps) Based on this threshold, 981 SNPs were found to be significantly associated with tail fat weight and tail fat relative weight on chromosome 13 of sheep. These sites were all located on chromosome 13 and were highly linked to non-coding regions (see Figure 2 ).

[0047] 3. Identify key causal variants affecting sheep tail fat weight based on multi-omics strategy

[0048] The present inventors performed ATAC-Seq, CUT&Tag (H3K27ac and H3K4me3) and Hi-C sequencing on the tail fat tissue of two 6-month-old half-sibling Hu sheep lambs, obtained high-quality data, and performed overlap analysis with the key candidate regions identified in step 1 and the SNP sites screened in step 2. It was found that two SNPs (Chr13:51760995A>C and Chr13:51825895G>A) overlapped and were located in the enhancer region (see Figure 3 shown).

[0049] The frequency distribution of alleles of these two SNPs in sheep populations with different tail types in step 1 was further statistically analyzed, and it was found that these two SNPs were greatly different in fat-tailed and thin-tailed sheep populations, and there were significant differences in tail fat weight and relative tail fat weight of individuals with different genotypes.

[0050] Finally, the tail fat tissue of individuals with different genotypes (74 6-month-old Hu sheep provided by Minqin County Defu Agricultural Technology Co., Ltd.) was commissioned to a sequencing company for RNA-Seq sequencing, and the effects of different genotypes on the expression of upstream and downstream genes were statistically analyzed. The results showed that Chr13:51760995A>C mutation affected the expression of BMP2 gene before and after, and at the Chr13:51760995A>C site, the tail fat weight and tail fat relative weight of AA genotype individuals were significantly lower than those of CC genotype individuals (P<0.01) (see Figure 4 , Table 1), thus determining that the Chr13:51760995A>C site can affect tail fat deposition by affecting the expression of the BMP2 gene, and can be used as a key molecular marker affecting sheep tail fat deposition. This study provides a successful case for identifying key causal variants and major effect 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 and tail fat weight in sheep

[0052]

[0053] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), while data with the same lowercase letters indicate no significant differences (P>0.05).

[0054] Example 2 Detection of the genomic region where the sheep Chr13:51760995A>C site is located

[0055] (1) Primer design

[0056] A pair of primers MF and MR were designed based on the sequence of the sheep Chr13:51760995A>C genomic region. 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 genomic sequence of sheep Chr13:51760995A>C

[0060] DNA extracted from the blood of 10 sheep was used as a template for amplification and sequencing. The PCR amplification used a 25 μL reaction system, including 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 reaction program was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 53°C annealing for 30 s, 72°C extension for 60 s, 35 cycles, and finally 72°C extension for 10 min.

[0061] The amplified PCR fragment was sequenced. The sequencing results showed that the specific nucleotide sequence of the amplified fragment was as shown in SEQ ID NO.1, wherein there was an A / C polymorphism at the 352 bp site of the amplified fragment, that is, the site was the same as the site locked based on the multi-omics strategy in Example 1. Figure 5 shown.

[0062] SEQ ID NO. 1: TTGCCAACAAAATCTGCATAATCAAAGCTATGATT.

[0063] Example 3 Detection of Sheep Chr13:51760995A>C Site

[0064] 1. Primer design of AQP technology

[0065] Based on the SNP sites locked in Examples 1 and 2, a primer set for detecting the SNP sites using AQP technology is 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; the nucleotide sequence of the universal primer is shown in SEQ ID No.6. The first and second primers are used to amplify the region where the polymorphic site is located, and then the universal primer is used to extend it to analyze the extension product and determine its genotype. The primer sequences are as follows:

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

[0067] The 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 Shenggong Bioengineering Co., Ltd. Each primer in the above 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. Extract genomic DNA and perform quality control

[0071] The extraction of genomic DNA from sheep blood can be performed using a DNA extraction kit. The quality of the extracted genomic DNA is tested by 1% agarose electrophoresis and Nanodrop2100, respectively. The extracted DNA is required to meet the following requirements: (1) Agarose electrophoresis shows a single DNA band without obvious diffusion. (2) Nanodrop2100 detection A260 / 280 is between 1.8-2.0; A260 / 230 is between 1.8-2.0; there is no obvious light absorption at 270nm. If it does not meet the above requirements, it needs to be re-extracted until it is qualified; and according to the AQPTM detection technology of Beijing Jiacheng Biotechnology Co., Ltd. and the size of the genome, the DNA dosage is calculated to be 10-20ng / sample, and the extracted genomic DNA is diluted to a concentration of 10-20ng / μL as a DNA template for standby use.

[0072] 3. Perform genotyping

[0073] First, according to AQP TM The detection technology and genome size calculated the DNA dosage to be 10-20 ng / sample, and the extracted genomic DNA was diluted to a concentration of 10-20 ng / μL as a DNA template.

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

[0075] Then use a pipette to add 0.07μL of primer mixture, 0.5μL of sterile water, 2.5μL of HiGeno 2×Probe Mix, and 2μL of DNA to each well of the 384-well plate. After adding, seal the membrane, shake and centrifuge, and place it on the C1000TouchTM Thermal Cycler instrument for PCR amplification. The specific procedure is:

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

[0077] 95°C, 20 seconds (denaturation) - 61°C - 55°C, 40 seconds (renaturation & extension), amplification for 10 cycles, decreasing 0.6°C per cycle;

[0078] Amplification was continued for 34 cycles from 95°C, 20 seconds (denaturation) to 55°C, 40 seconds.

[0079] After amplification, the C1000 TouchTM Thermal Cycler instrument was used to detect the fluorescent signal and check the typing at 37°C, and the tail fat weight and tail fat relative to carcass weight of the same genotype were counted. The test results of another batch of 281 6-month-old Hu sheep raised by Minqin County Defu Agricultural Technology Co., Ltd. are shown in Table 2.

[0080] Table 2 Verification of the sheep Chr13:51760995A>C polymorphism

[0081]

[0082] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), while data with the same lowercase letters indicate no significant differences (P>0.05).

[0083] The results show that the tail fat weight of individuals with AA genotype at the 352bp site shown in the amplified fragment SEQ ID NO.1 is significantly lower than that of CC individuals. That is, the site is the same as the site locked based on the multi-omics strategy in Example 1. Since tail fat weight is a direct indicator for measuring tail fat deposition, the selection of tail fat deposition is beneficial to improving economic benefits. Therefore, the PCR primer pair of the present invention is used to detect the Chr13:51760995A>C site polymorphism to screen AA genotype individuals to form a core group for the breeding of small-tailed sheep; in addition, the site and technical detection method can be used for early detection of sheep populations to eliminate individuals carrying the CC genotype and improve the economic benefits of the sheep industry.

Claims

1. A method for identifying molecular markers related to sheep tail fat weight based on a multi-omics strategy, characterized in that: It includes the following steps: S1. Genome-wide selection signal analysis will be conducted on sheep populations with different tail types to preliminarily screen candidate genomic regions associated with sheep tail fat deposition; S2. Perform whole genome resequencing on the population with accurate tail fat weight data records, and perform whole genome association analysis on the obtained high-quality SNP data and tail fat weight data to obtain a log-log 10 (0.05 / totalSNPs) As a standard, molecular marker loci significantly associated with sheep tail fat weight were screened; and performing overlap analysis with the candidate genomic region obtained in step S1 to screen out molecular marker sites located in the candidate genomic region that are significantly correlated with sheep tail fat weight; S3. Epigenomic sequencing of tail adipose tissue using ATAC-Seq, CUT&Tag, and Hi-C will be performed to identify regulatory elements in tail adipose tissue; S4. Integrate the results of overlapping analysis of the candidate genomic regions, tail fat weight-significantly correlated molecular marker sites and regulatory elements obtained in the above steps S1-3 to identify key molecular marker sites; perform RNA-Seq sequencing on the tail fat tissue of individuals with different genotypes of the key molecular marker sites identified above, and count the expression levels of genes near the site to comprehensively identify the key genes and causal variation sites that affect tail fat weight.

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

3. The method according to claim 1, characterized in that The nucleotide sequence of the causal mutation site obtained in step S4 is shown in SEQ ID NO.1, and the M at the 352bp position of the sequence represents A or C. The 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 individuals with AA genotype are significantly lower than those of individuals with CC genotype.

4. Use of a molecular marker associated with sheep 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 the M at the 352 bp position in the sequence SEQ ID NO.1 is A or C, and the 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.

5. Use of a primer pair for detecting a molecular marker associated with the fat weight trait of sheep tail in screening low tail fat sheep, characterized in that: The sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO.

3.

6. An application of an AQP primer pair for detecting a molecular marker associated with the fat weight trait of sheep tail in screening tail fat type sheep, characterized in that: The nucleotide sequences of the AQP primers are shown in SEQ ID NO.4-6.

7. A kit for detecting the molecular markers related to the tail fat weight trait of sheep for screening low tail fat sheep, characterized in that: The kit includes a common PCR primer pair or an AQP sequence pair. The sequences of the common 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.

8. A method for detecting molecular markers associated with sheep tail fat weight trait in screening low tail fat sheep, comprising the following steps: 1) Amplifying sheep genomic DNA using a common PCR primer pair with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, or an AQP primer pair with nucleotide sequences as shown in SEQ ID NO.4-6; 2) Identifying the specific site where M is A or C at the 352 bp position of the nucleotide sequence of the amplified product obtained in step 1) as shown in SEQ ID NO.1, wherein: The tail fat weight of individuals carrying the AA genotype was significantly lower than that of individuals carrying the CC genotype.

9. Application of a PCR primer pair, an AQP primer, and a kit for detecting molecular markers related to sheep tail fat weight traits in sheep breeding, characterized in that: The molecular marker nucleotide sequence is shown in SEQ ID NO.1, wherein the M at the 352bp position in the sequence SEQ ID NO.1 is A or C, and the 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 AC or CC genotype; the sequence of the PCR primer pair is shown in SEQ ID NO.2 and SEQ ID NO.3; the sequence of the AQP primer is shown in SEQ ID NO.4-6; the kit includes a PCR primer pair or an AQP primer.

10. The use according to claim 9, characterized in that Breeding is used to select low-fat sheep breeds or strains with lower tail fat weight.

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