Molecular marker for identifying sheep backfat thickness and application thereof

Abstract

The invention belongs to the technical field of genetic breeding, and particularly relates to a molecular marker for identifying sheep backfat thickness and application of the molecular marker, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and single nucleotide C-A mutation occurs at the 101bp site of the molecular marker. The molecular marker influencing the sheep backfat thickness is screened out through methods such as genome sequencing, variation site recognition, screening and annotation, whole genome association analysis and the like. By selecting sheep individuals with the genotype of CC as male parents or female parents, the backfat thickness of offspring can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding, and particularly relates to a molecular marker for identifying sheep backfat thickness and an application thereof. Background Art

[0002] The Suffolk, also known as the Suffolk, is a meat sheep breed originating in southeast England in the early 19th century. It is bred by crossing the South Downs sheep with the larger, leaner Black-faced Norfolk sheep as the female parent. This breed is characterized by its large size, early sexual maturity, rapid growth and development, excellent meat production, and strong adaptability. It has been introduced to several countries and is primarily used as a sire line for fattening lamb production. Since its introduction from Australia in the 1980s, the core population of the Suffolk has been primarily distributed in Inner Mongolia and Xinjiang. It is cultivated for purebred breeding and extensively crossbred with local coarse-wool and fine-wool sheep to improve meat quality. Backfat depth, a key growth trait of Suffolk sheep, directly influences carcass lean meat percentage and fat distribution. A moderate backfat thickness ensures juicy meat while preventing excessive fat accumulation that reduces economic value. In order to break through the bottleneck of growth rate and meat production performance of Suffolk sheep, it is urgent to analyze the molecular markers that regulate backfat thickness at the genomic level. Summary of the Invention

[0003] The invention provides a molecular marker for identifying sheep backfat thickness, which is used for identifying the sheep backfat thickness trait and genetic breeding.

[0004] The technical solution adopted in the present invention is: The present invention provides a molecular marker for identifying sheep backfat thickness. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1. A single nucleotide C to A mutation occurs at the 101 bp site of the molecular marker.

[0005] The second aspect of the present invention provides the use of the molecular marker, which is used to identify the backfat thickness trait of sheep.

[0006] Preferably, the method for identifying the backfat thickness trait of sheep comprises the following steps: Extraction of sheep genomic DNA; Using sheep genomic DNA as a template, detecting the genotype of the 101 bp site in the molecular marker; If the genotype is CC, the sheep is determined to have low backfat thickness; low backfat thickness means that the backfat thickness is less than 1.04 mm.

[0007] Preferably, the sheep genomic DNA is derived from sheep blood.

[0008] Preferably, the sheep genomic DNA is extracted using the phenol-chloroform method.

[0009] The third aspect of the present invention provides the application of the molecular marker, which is used for genetic breeding of sheep.

[0010] Preferably, the genetic breeding refers to reducing the backfat thickness of sheep offspring by selecting sheep individuals with the genotype CC of the molecular marker as parents.

[0011] Preferably, the sheep are Suffolk sheep.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a molecular marker for identifying sheep backfat thickness. The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1. A single nucleotide mutation from C to A occurs at the 101st bp position of the molecular marker. Through genome sequencing, identification, screening, and annotation of variant sites, and genome-wide association analysis, the present invention screens for molecular markers that influence sheep backfat thickness. By selecting sheep individuals with the CC genotype as sires or dams, backfat thickness in offspring can be reduced.

[0013] In addition, the present invention also achieves in-depth exploration of the genetic potential of sheep breeds by screening relevant genetic loci, establishing a breeding technology system, and precisely regulating fat metabolism pathways, providing a scientific basis for breeding new low-fat and high-yield meat sheep breeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the distribution diagram of SNPs in the 1Mb window of chromosome after quality control.

[0015] Figure 2 Principal component analysis diagram.

[0016] Figure 3 This is a visualization diagram of the G matrix.

[0017] Figure 4 The GWAS results of Suffolk sheep backfat thickness are reflected in the Manhattan plot.

[0018] Figure 5 The GWAS results of Suffolk sheep backfat thickness reflected in the QQ plot. DETAILED DESCRIPTION

[0019] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0020] The inventive concept of the present invention is as follows: The impact of backfat thickness on sheep is mainly reflected in the following aspects: (1) Impact on meat quality.

[0021] Moderate backfat thickness: It can make the lamb taste more tender and juicy. Backfat is a kind of fat storage in the sheep's body. The right amount of fat distribution can increase the fat content between muscles, making muscle fibers more tender, helping to enhance the tenderness and flavor of the meat, making the lamb taste more delicious.

[0022] Insufficient backfat: The lamb may appear dry and lack flavor. Without sufficient fat for lubrication and protection, the muscle fibers become relatively coarse, and the meat tends to become tight and unpalatable after cooking, affecting the consumer's eating experience.

[0023] Excessive backfat reduces the lean meat percentage and increases the fat content, leading to a decrease in the economic value of the lamb. Excessive backfat means excessive fat deposition in the lamb, which not only results in an excessively high fat content in the lamb but also compromises its nutritional balance, failing to meet the lean meat demands of modern consumers and increasing breeding costs.

[0024] (2) Impact on reproductive performance.

[0025] Moderate backfat thickness is beneficial to the reproductive performance of ewes. Proper fat reserves provide the necessary energy support for reproductive activities, help maintain a normal estrus cycle, improve conception rates and embryo survival rates, and ensure sufficient energy supply during pregnancy and lactation, promoting the healthy growth of lambs.

[0026] Insufficient backfat can lead to decreased reproductive performance in ewes. When ewes are too thin and lack sufficient backfat, insufficient energy reserves can affect their normal estrus and ovulation, leading to irregular estrus cycles, decreased ovulation, and, consequently, reduced conception rates. During pregnancy, insufficient backfat and a lack of adequate nutritional supplementation can also affect fetal development, potentially leading to fetal stunting, premature birth, or stillbirth.

[0027] Excessive backfat also negatively impacts reproductive performance. Excessive backfat can lead to obesity, which can lead to various health problems, such as reproductive system disorders and dystocia. Obesity can also cause hormonal imbalances in ewes, affecting estrus and conception. It also increases risks during childbirth, hindering reproduction.

[0028] (3) Impact on growth and development.

[0029] Moderate backfat thickness is a sign of good growth and development of sheep, indicating that the sheep has a balanced nutritional intake during the growth process, which can not only ensure muscle growth but also store fat in an appropriate amount, which is beneficial to the overall health and growth performance of the sheep.

[0030] Insufficient backfat thickness may indicate insufficient nutrition or other health issues affecting normal growth and development. Sheep with chronically insufficient backfat thickness may experience slower growth, suboptimal weight gain, and restricted bone and muscle development, resulting in smaller size, weaker physique, and decreased disease resistance.

[0031] Excessive backfat can increase the burden on the sheep's body and negatively impact its growth and development. Excessive fat accumulation can make the sheep clumsy, increase the burden on joints and bones, and easily lead to muscular ailments such as arthritis. Excessive backfat can also impair heat dissipation and respiratory function, especially in high temperatures, causing heat stress and affecting normal growth and health.

[0032] (4) Impact on the ability to resist cold.

[0033] Backfat has a certain insulating effect, which can help sheep withstand cold environments. In cold seasons, backfat can act as an insulating layer, reducing heat loss from the sheep's body, making it easier for them to maintain their body temperature, thereby enhancing their ability to adapt to cold weather and reducing the stress response and incidence of diseases caused by low temperature environments.

[0034] Based on this, the present application proposes a molecular marker for identifying sheep backfat thickness, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and a single nucleotide C to A mutation occurs at the 101bp site of the molecular marker.

[0035] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0036] The abbreviations of the present invention are shown in Table 1.

[0037] Table 1 Abbreviations of the present invention

[0038] The experimental animals and phenotype sources of the present invention are as follows: The experimental sheep of the present invention were all from Siziwang Banner Sino Sheep Breeding Technology Co., Ltd. in Inner Mongolia Autonomous Region. The backfat thickness phenotype was measured and recorded from 2020 to 2024, as shown in Table 2.

[0039] Blood samples were collected from 300 Suffolk sheep, stored in anticoagulant tubes and placed in a -80°C refrigerator in the laboratory for DNA extraction.

[0040] Table 2 Description of backfat thickness traits of Suffolk sheep

[0041] Example A molecular marker for identifying sheep backfat thickness and its application are as follows: 1. Genomic DNA extraction and quality inspection.

[0042] DNA was extracted from blood samples using the phenol-chloroform method, and DNA concentration was measured using a NanoDrop 2000 spectrophotometer. The absorption wavelength ratio of the highest absorption peak at 260 nm / 280 nm was calculated to measure the content of DNA and protein phenolic substances. The absorption wavelength ratio of the highest absorption peak at 260 nm / 230 nm was calculated to measure the content of DNA and carbohydrates. DNA quality was evaluated using 1% agarose gel.

[0043] 2. Library construction and sequencing.

[0044] Qualified genomic DNA was randomly fragmented into 350bp fragments using a Covaris ultrasonic disruptor. The DNA fragments were then subjected to end-repair, poly(A) addition, sequencing adapters, purification, and PCR amplification to complete library preparation. After library construction, preliminary quantification was performed using Qubit 2.0, and the effective concentration of the library was accurately quantified using qPCR to ensure library quality. After passing the library quality test, sequencing was performed using the BGI-T7 sequencing platform in PE150 mode.

[0045] 3. Identification, screening and annotation of variant sites.

[0046] Fastp software version V0.20.0 was used to filter raw reads data into clean reads data, and a genome index was established for the reference genome. The quality-controlled clean reads data were aligned with the sheep reference genome using Burrows-Wheeler Aligner software version V0.7.17. The aligned sam files were converted into bam files using SAMtools software version V1.8-20, and the bam files were sorted. The MarkDuplicates program in the Genome Analysis Toolkit software version V3.8 was used to remove duplicate data from the sorted bam files to obtain the final bam files. The final bam files were indexed, and the HaplotypeCaller module in the GATK software was used to detect SNP variations. After obtaining the vcf file, it was filtered using the VariantFiltration module. The ANNOVAR software package is used to perform functional annotation on the detected genetic variants. Based on the location of the variant site on the reference genome and the gene location information on the reference genome, the region where the variant site occurs in the genome and the impact of the variant, such as synonymous mutations or non-synonymous mutations, can be obtained.

[0047] The reference genome of sheep used in the present invention is Oar_v4.0, GCF_000298735.2.

[0048] 4. Data quality control and population stratification correction.

[0049] Whole-genome resequencing was performed on 300 Suffolk sheep individuals to establish a genotype database, generating a total raw read size of 17,243.32 Gb and identifying 47,506,993 SNPs. Genotyping data were quality-controlled using Plink software version 1.90. Individuals with a genotype detection rate of less than 98%, SNPs with a detection rate of less than 98%, SNPs with a minimum allele frequency of less than 5%, and SNPs with a Hardy-Weinberg equilibrium test P value of less than 10 were excluded. -6 A total of 20,182,599 high-quality SNPs were identified in the Suffolk population. These sites were evenly distributed on the 26 pairs of autosomes in sheep, such as Figure 1 shown. Figure 1 The left Y-axis represents the chromosome name, and the upper X-axis represents the window size.

[0050] The first three principal components were calculated using the "--pca 3" parameter of Plink software. The PCA graph was drawn using R V3.6.0. The results are as follows Figure 2As shown in the figure, there is population stratification in the experimental samples and the genetic correlation between individuals is high. The first three principal components need to be used as covariates to correct the population stratification phenomenon of Suffolk sheep.

[0051] The genome kinship analysis of the population was performed based on the G matrix using Plink v1.90. The results are as follows Figure 3 As shown, this indicates that the average relatedness between Suffolk sheep individuals is relatively distant. Figure 3 In the figure, each small square represents the kinship value between samples. The smaller the value is, the closer it is to light green, that is, the more distant the kinship between the two individuals, and vice versa.

[0052] 5. Genome-wide association analysis.

[0053] The fastGWA-mlm model in GCTA V1.94.0beta software was used to perform association analysis between SNPs and backfat thickness traits. The formula is as follows: ; in, y is an n × 1 phenotype vector, where n is the number of individuals; X snp is a genotype vector whose effects are β snp ; X c is the correlation matrix of fixed covariates, sex, year of measurement and the first three PCAs, and its corresponding coefficients are β c ; g is the vector of total genetic effects captured by the SNP-derived genetic relationship matrix, g~N(0, ), π is the SNP-derived GRM vector with all off-diagonal elements set to 0, : genetic variance; e is the residual vector, e ~N(0, ), I is the identity matrix, is the residual variance.

[0054] Because the Bonferroni correction method of 0.05 / SNP number is too strict to determine the significance threshold of GWAS, independent SNPs are obtained through linkage disequilibrium (LD) screening to remove redundancy and use them to calculate the threshold. The parameters are: 50: window size, number of SNPs; 10: step length, number of SNPs; 0.2:r 2 value, remove one of the SNP pairs with LD greater than 0.2.

[0055] The present invention adjusts the threshold for genome-wide significant association to P = 1 / 620054, where 620054 is the number of independent SNPs that have been screened by LD. The genomic expansion factor λ of the test statistic is calculated by the slope of the linear regression between the observed quantile and the theoretical quantile in R V3.6.0. The calculated λ value for the backfat thickness trait is 0.926, indicating that there is no genomic expansion. Based on the resequencing data of 300 Suffolk sheep, two significant SNP sites associated with the backfat thickness trait were detected. These two sites are located on chromosome 1 and chromosome 6, respectively, as shown in Table 3. Figure 4 and Figure 5 shown.

[0056] Figure 4 This is a Manhattan plot of the present invention, showing the results of a GWAS for backfat thickness in Suffolk sheep. Genome-wide significant SNPs are shown in red. A Manhattan plot is a visualization tool commonly used in genome-wide association studies to display the significance level of individual genetic markers, such as SNPs, across the genome, typically using a p-value as a metric. Its name comes from the fact that the highly significant points in the plot are distributed across the chromosome like "skyscrapers," resembling the Manhattan skyline. Figure 5 The QQ plot is a statistical graphical tool used to test whether data follows a certain theoretical distribution or to compare the consistency of the quantiles of two sets of data. The core idea is to intuitively judge the degree of consistency between the data distribution and the theoretical hypothesis by comparing the positional relationship between the sample quantile and the theoretical quantile.

[0057] Table 3 Significant SNP loci associated with backfat thickness traits

[0058] 6. SNPs affecting backfat thickness in Suffolk sheep.

[0059] Further verification of the above SNPs that reached the genome-wide significance level found that the C→A mutation at position 89305500 on chromosome 6 of the Suffolk sheep genome can significantly affect the back fat thickness trait of Suffolk sheep.

[0060] The association analysis between the SNP locus at 89305500 on chromosome 6 of the Suffolk sheep genome and the backfat thickness trait is shown in Table 4.

[0061] Table 4 Polymorphism at position 89305500 on chromosome 6 of Suffolk sheep genome

[0062] Note: In Table 4, different letters in the backfat thickness column indicate significant differences, p < 0.05; the same letter indicates no significant differences, p > 0.05.

[0063] It can be seen from Table 4 that the back fat thickness of individuals with CC genotype is the smallest.

[0064] In a genome-wide association analysis using a mixed linear model, the SNP marker at position 89,305,500 on chromosome 6 of the Suffolk sheep genome reached genome-wide significance, indicating that this marker is significantly associated with the backfat thickness trait in Suffolk sheep. Furthermore, when the base of this marker is C, Suffolk sheep have the smallest backfat thickness. The gene frequency and genotype frequency of the SNP at position 89,305,500 on chromosome 6 of the Suffolk sheep genome were subsequently calculated (see Table 5).

[0065] Table 5 SNP gene frequency and genotype frequency at position 89305500 on chromosome 6 of Suffolk sheep genome

[0066] The nucleotide sequence of the molecular marker for identifying backfat thickness in Suffolk sheep according to the present invention is shown in SEQ ID NO.1.

[0067] SEQ ID NO.1: ATAAACTGGTATTTAATGGCATTGATATGGATCTGTTAGAACTGTATGAAAAGAAAAAAAGCTGCCTAAGCATACAATCCTGCAAAAAAAAAAAAAAAAACCAGTATCACAGAGGAGCAGAAAGAGACGAGCTCTCAAGGGATCAAGAAGGAACAGCCACAGAACTAAGAAGAGAAGTTTCCTAGAAGAATTTGATCAACT.

[0068] SEQ ID NO.1 shows the sequence of 100 bp upstream and downstream of the mutation site, the bold part is the mutation site, and the site in bold is C or A.

[0069] In summary, the A→C mutation at position 89305500 on chromosome 6 of the Suffolk sheep genome can be used to select the Suffolk sheep breed with the smallest backfat thickness. By selecting individuals with the CC genotype as the father or mother, the backfat thickness of Suffolk sheep offspring can be reduced.

[0070] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A molecular marker for identifying sheep backfat thickness, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, and a single nucleotide mutation from C to A occurs at the 101 bp position of the molecular marker.

2. The use of the molecular marker according to claim 1, characterized in that: The molecular marker is used to identify the backfat thickness trait of sheep.

3. The use according to claim 2, characterized in that The method for identifying the backfat thickness trait of sheep comprises the following steps: Extraction of sheep genomic DNA; Using sheep genomic DNA as a template, detecting the genotype of the 101 bp site in the molecular marker; If the genotype is CC, the sheep is determined to have low backfat thickness; low backfat thickness means that the backfat thickness is less than 1.04 mm.

4. The use according to claim 3, characterized in that The sheep genomic DNA is derived from sheep blood.

5. The use according to claim 3, characterized in that The sheep genomic DNA is extracted using the phenol-chloroform method.

6. The use of molecular markers according to claim 1, characterized in that: The molecular marker is used for genetic breeding of sheep.

7. The use according to claim 6, characterized in that The genetic breeding refers to reducing the backfat thickness of sheep offspring by selecting sheep individuals with a genotype of CC at the 101bp site of the molecular marker as parents.

8. The use according to claim 2 or claim 6, characterized in that The sheep are Suffolk sheep.

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