A molecular marker for identifying sheep backfat thickness and its application

Abstract

This invention belongs to the field of genetic breeding technology, specifically relating to a molecular marker for identifying backfat thickness in sheep and its application. 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 position 101 bp. This invention uses methods such as genome sequencing, identification and annotation of variant sites, and genome-wide association analysis to screen for molecular markers affecting sheep backfat thickness. By selecting sheep with the CC genotype as the paternal or maternal parent, the backfat thickness of the offspring can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding technology, specifically relating to a molecular marker for identifying the backfat thickness of sheep and its application. Background Technology

[0002] Suffolk sheep are a meat sheep breed originating in southeast England in the early 19th century. They were developed by crossing Southern Hills sheep as the sire with local Norfolk sheep (large in size and high lean meat percentage) as the dam. This breed is characterized by its large size, early sexual maturity, rapid growth, excellent meat production performance, and strong adaptability. It has been introduced to many countries and is primarily used as a sire breed in lamb production. In my country, since its introduction from Australia in the 1980s, its core population is mainly distributed in Inner Mongolia and Xinjiang, where purebred breeding is carried out, and it is also widely crossbred with local coarse-wool and fine-wool sheep to improve meat quality. Backfat thickness is a core indicator of Suffolk sheep growth traits, directly affecting the lean meat percentage and fat distribution. Appropriate backfat thickness ensures juicy meat while preventing excessive fat deposition that reduces economic value. To overcome the bottlenecks in 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] This invention provides a molecular marker for identifying backfat thickness in sheep, which can be used to identify the backfat thickness trait in sheep and for genetic breeding.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides a molecular marker for identifying the backfat thickness of sheep. 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 101 bp site of the molecular marker.

[0006] A second aspect of the invention provides the application of the molecular marker for identifying the backfat thickness trait in sheep.

[0007] Preferably, the method for identifying the backfat thickness trait of sheep includes the following steps:

[0008] Extracting genomic DNA from sheep;

[0009] Using sheep genomic DNA as a template, the genotype at the 101 bp site of the molecular marker was detected;

[0010] If the genotype is CC, the sheep is determined to have low backfat thickness; low backfat thickness refers to a backfat thickness of less than 1.04 mm.

[0011] Preferably, the sheep's genomic DNA is derived from the sheep's blood.

[0012] Preferably, the genomic DNA of the sheep is extracted using the phenol-chloroform method.

[0013] A third aspect of the present invention provides the application of the molecular marker for the genetic breeding of sheep.

[0014] 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.

[0015] Preferably, the sheep is a Suffolk sheep.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention provides a molecular marker for identifying backfat thickness in sheep. 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 position 101 bp. This invention uses methods such as genome sequencing, identification and annotation of variant sites, and genome-wide association analysis to screen for molecular markers affecting sheep backfat thickness. By selecting sheep with the CC genotype as paternal or maternal lines, backfat thickness in offspring can be reduced.

[0018] Furthermore, this invention also establishes a breeding technology system by screening relevant genetic loci and precisely regulating fat metabolism pathways, thereby achieving in-depth exploration of the genetic potential of sheep breeds and providing a scientific basis for breeding new low-fat, high-yield meat sheep breeds. Attached Figure Description

[0019] Figure 1 This is a distribution map of SNPs within a 1Mb window of the chromosome after quality control.

[0020] Figure 2 Principal component analysis plot.

[0021] Figure 3 A visualization of the G matrix.

[0022] Figure 4 GWAS results for back fat thickness in Suffolk sheep, as shown by the Manhattan plot.

[0023] Figure 5 The GWAS results for back fat thickness in Suffolk sheep are shown in the QQ graph. Detailed Implementation

[0024] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0025] The inventive concept of this invention is as follows:

[0026] The effects of thick backfat on sheep are mainly reflected in the following aspects:

[0027] (1) Effects on meat quality.

[0028] A moderate amount of back fat makes lamb more tender and juicy. Back fat is one of the fat storage areas in a lamb's body. An appropriate amount of fat distribution can increase the fat content between muscles, making the muscle fibers more tender and helping to improve the tenderness and flavor of the meat, making the lamb taste better.

[0029] Insufficient backfat: The lamb may be too dry and lack flavor. Without enough fat for lubrication and protection, the muscle fibers are relatively coarse, and the meat tends to shrink after cooking, resulting in a poor texture and negatively impacting the consumer's eating experience.

[0030] Excessive backfat: This reduces the lean meat percentage of mutton and increases its fat content, leading to a decrease in the economic value of the mutton. Excessively thick backfat indicates excessive fat deposition in the sheep's body, not only making the mutton too high in fat but also potentially affecting its nutritional balance, failing to meet modern consumers' demand for lean mutton, and increasing breeding costs.

[0031] (2) Effects on reproductive performance.

[0032] Moderate backfat thickness is beneficial to the reproductive performance of ewes. Appropriate fat reserves provide the necessary energy support for the reproductive activities of ewes, helping to maintain normal estrous cycles, improve conception rates and embryo survival rates, and ensure sufficient energy supply during pregnancy and lactation, promoting the healthy growth of lambs.

[0033] Insufficient backfat thickness: This can lead to decreased reproductive performance in ewes. When ewes are too thin and lack sufficient backfat, their energy reserves are insufficient, which may affect their normal estrus and ovulation functions, leading to irregular estrus cycles, reduced ovulation, and ultimately lower conception rates. During pregnancy, if ewes lack sufficient backfat and cannot obtain adequate nutrition, it may also affect fetal development, potentially causing fetal maldevelopment, premature birth, or stillbirth.

[0034] Excessive backfat: This also negatively impacts reproductive performance. Excessive backfat can cause ewes to become obese, leading to various health problems such as reproductive system diseases and dystocia. Obesity can also cause hormonal imbalances in ewes, affecting estrus and conception, and increasing the risks during parturition, ultimately hindering offspring reproduction.

[0035] (3) Effects on growth and development.

[0036] Moderate backfat thickness is an indicator of good growth and development in sheep, indicating that the sheep has a balanced intake of nutrients during its growth process, which can ensure muscle growth and store an appropriate amount of fat, thus benefiting the sheep's overall health and growth performance.

[0037] Insufficient backfat thickness: This may indicate that the sheep is not getting enough nutrition or has other health problems, affecting its normal growth and development. Sheep with insufficient backfat thickness over a long period may experience slower growth, unsatisfactory weight gain, and restricted bone and muscle development, resulting in smaller size, weaker constitution, and decreased disease resistance.

[0038] Excessive backfat increases the burden on a sheep's body, negatively impacting its growth and development. Excessive fat accumulation makes sheep clumsy, increases the burden on joints and bones, and easily leads to musculoskeletal diseases such as arthritis. Furthermore, excessive backfat can impair a sheep's heat dissipation and respiratory function, especially in high-temperature environments, easily causing heat stress and affecting its normal growth and health.

[0039] (4) The effect on cold resistance.

[0040] Backfat has a certain insulating effect, helping sheep resist cold environments. In cold seasons, backfat can act as an insulation layer, reducing heat loss from the sheep's body and making it easier for them to maintain their body temperature. This enhances their ability to adapt to cold weather and reduces the likelihood of stress and disease caused by low temperatures.

[0041] Based on this, this application proposes a molecular marker for identifying sheep backfat thickness, the nucleotide sequence of which is shown in SEQ ID NO.1, and a single nucleotide C to A mutation occurs at position 101bp of the molecular marker.

[0042] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0043] The list of abbreviations for this invention is shown in Table 1.

[0044] Table 1. List of abbreviations for this invention

[0045]

[0046] The experimental animals and phenotypes used in this invention are as follows:

[0047] The sheep used in this invention were all from Sainuo Breeding Sheep Technology Co., Ltd., Siziwang Banner, Inner Mongolia Autonomous Region. The backfat thickness phenotype was measured and recorded from 2020 to 2024, as shown in Table 2.

[0048] Blood samples were collected from 300 Suffolk sheep, stored in anticoagulant tubes, and kept in a laboratory freezer at -80°C for DNA extraction.

[0049] Table 2. Description of the back fat thickness trait in Suffolk sheep.

[0050]

[0051] Example

[0052] A molecular marker for identifying sheep backfat thickness and its application are detailed below:

[0053] 1. Genomic DNA extraction and quality control.

[0054] DNA was extracted from blood samples using the phenol-chloroform method, and the DNA concentration was determined using a NanoDrop2000 spectrophotometer. The ratio of the absorption wavelengths of the highest absorption peaks at 260 nm and 280 nm was calculated to measure the content of DNA and protein phenolic substances. The ratio of the absorption wavelengths of the highest absorption peaks at 260 nm and 230 nm was calculated to measure the content of DNA and carbohydrates. DNA quality was assessed using a 1% (w / w) agarose gel.

[0055] 2. Library construction and sequencing.

[0056] The qualified genomic DNA was randomly fragmented into 350bp fragments using a Covaris ultrasonic disruptor. The DNA fragments underwent end repair, poly(A) addition, sequencing adapter addition, purification, and PCR amplification to complete the entire library preparation process. After library construction, preliminary quantification was performed using Qubit 2.0, and qPCR was used to accurately quantify the effective concentration of the library to ensure library quality. After passing quality checks, sequencing was performed using the BGI MGI-T7 sequencing platform in PE150 mode.

[0057] 3. Identification, screening, and annotation of variant sites.

[0058] Using FastP software version V0.20.0, raw reads data were filtered into clean reads data, and a genome index was built on the reference genome. Burrows-Wheeler Aligner software version V0.7.17 was used to align the quality-controlled clean reads data with the sheep reference genome. SAM tools software version V1.8-20 was used to convert the aligned SAM files into BAM files, and the BAM files were sorted. The MarkDuplicates program in Genome Analysis Toolkit software version V3.8 was used to remove duplicate data from the sorted BAM files, resulting in the final BAM file. An index was built on the final BAM file, and the HaplotypeCaller module in GATK software was used to detect SNP variations. The resulting VCF file was then filtered using the VariantFiltration module. The ANNOVAR software package is used to perform functional annotation on detected gene variants. Based on the location of the variant site on the reference genome and the gene location information on the reference genome, the region in which the variant site occurs in the genome and the impact of the variant, such as synonymous mutation or non-synonymous mutation, can be obtained.

[0059] The sheep reference genome used in this invention is Oar_v4.0, GCF_000298735.2.

[0060] 4. Data quality control and group stratification correction.

[0061] Whole-genome resequencing was performed on 300 Suffolk sheep individuals to establish a genotype database, generating a total of 17243.32 Gb of raw reads and identifying 47,506,993 SNPs. The genotyping data were quality controlled using Plink software version 1.90, removing individuals with a genotype detection rate <98%, SNPs with a detection rate <98%, SNPs with a minimum allele frequency <5%, and Hardy-Weinberg equilibrium test p-values ​​<10. -6 In the Suffolk population, a total of 20,182,599 high-quality SNPs were identified. These loci were evenly distributed across the 26 pairs of autosomes in sheep. Figure 1 As shown. Figure 1 The left-middle Y-axis represents the chromosome name, and the top X-axis represents the window size.

[0062] The first three principal components were calculated using the "--pca 3" parameter in the Plink software. The PCA plot was then drawn using R version 3.6.0, and the results are as follows. Figure 2As shown, the experimental sample exhibits population stratification and a high degree of genetic correlation among individuals. Therefore, the first three principal components need to be used as covariates to correct for the population stratification phenomenon in Suffolk sheep.

[0063] Genomic phylogenetic analysis based on the G matrix was performed on this population using Plink v1.90, and the results are as follows: Figure 3 As shown, this indicates that the average kinship among Suffolk sheep individuals is relatively distant. Figure 3 In the diagram, each small square represents the kinship value between samples. The smaller the value, the closer it is to light green, indicating that the two individuals are more distantly related, and vice versa.

[0064] 5. Genome-wide association analysis.

[0065] The association between SNPs and backfat thickness traits was analyzed using the fastGWA-mlm model in GCTA V1.94.0beta software, and the formula is as follows:

[0066] ;

[0067] in, y It is an n×1 phenotypic vector, where n is the number of individuals; X snp It is a genotype vector, and its effect is β snp ; X c It is a correlation matrix with fixed covariates: gender, year of determination, and the first three PCA analyses. The corresponding coefficients are... β c ; g It is a vector of total genetic effects captured by the genetic relationship matrix derived from SNPs, g~N(0, π is a GRM vector derived from SNP, where all off-diagonal elements are set to 0. Genetic variance; e It is the residual vector. e ~N(0, I is the identity matrix. This represents the residual variance.

[0068] Using the Bonferroni correction method with a value of 0.05 / number of SNPs to determine the significance threshold of GWAS is too stringent. Instead, redundant SNPs are removed using linkage disequilibrium LD screening to obtain independent SNPs, which are then used to calculate the threshold. The parameters are:

[0069] 50: Window size, number of SNPs;

[0070] 10: Step length, number of SNPs;

[0071] 0.2: r2 Delete one of the SNP pairs where LD is greater than 0.2.

[0072] This invention adjusts the threshold for genome-wide significant association to P=1 / 620054, where 620054 is the number of independent SNPs screened by LD. The genome expansion factor λ, used for testing statistics, is calculated by the slope of a linear regression between the observed quantiles and the theoretical quantiles in R V3.6.0. The calculated λ value for the backfat thickness trait is 0.926, indicating no genome expansion. Based on resequencing data from 300 Suffolk sheep, two significant SNP loci associated with the backfat thickness trait were detected. These two loci are located on chromosomes 1 and 6, respectively, as shown in Table 3. Figure 4 and Figure 5 As shown.

[0073] Figure 4 This is a Manhattan plot illustrating the GWAS results for backfat thickness in Suffolk sheep, with significant SNPs across the entire genome highlighted in red. A Manhattan plot is a visualization tool commonly used in genome-wide association studies (GWAS) to show the significance level of various genetic markers, such as SNPs, across the entire genome, typically expressed as a p-value. Its name derives from the fact that highly significant points in the plot resemble "skyscrapers" distributed across chromosomes, similar to the Manhattan skyline. Figure 5 A QQ plot is a statistical graphing tool used to test whether data follows a theoretical distribution or to compare the consistency of quantiles between two sets of data. Its core idea is to visually determine the degree of agreement between the data distribution and theoretical assumptions by comparing the relative positions of sample quantiles and theoretical quantiles.

[0074] Table 3 Significant SNP sites associated with backfat thickness trait.

[0075]

[0076] 6. SNPs affecting the back fat thickness trait of Suffolk sheep.

[0077] Further validation of the SNPs that reached genome-wide significance revealed that the C→A mutation at position 89305500 on chromosome 6 of the Suffolk sheep genome can significantly affect the backfat thickness trait of Suffolk sheep.

[0078] The association analysis between the SNP locus at position 89305500 on chromosome 6 of the Suffolk sheep genome and the backfat thickness trait is shown in Table 4.

[0079] Table 4. Polymorphism at position 89305500 on chromosome 6 of the Suffolk sheep genome.

[0080]

[0081] Note: In Table 4, different letters in the column for back fat thickness indicate significant differences (p < 0.05); the same letter indicates no significant differences (p > 0.05).

[0082] As shown in Table 4, individuals with the CC genotype have the smallest backfat thickness.

[0083] 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, it is beneficial for Suffolk sheep to have the minimum backfat thickness. Subsequently, the gene frequency and genotype frequency of the SNP at position 89,305,500 on chromosome 6 of the Suffolk sheep genome were calculated, as shown in Table 5.

[0084] Table 5. Frequency of SNP gene and genotype at position 89305500 on chromosome 6 of the Suffolk sheep genome.

[0085]

[0086] The nucleotide sequence of the molecular marker for identifying the backfat thickness of Suffolk sheep described in this invention is shown in SEQ ID NO.1.

[0087] SEQ ID NO.1:

[0088] ATAAACTGGTATTTAATGGCATTGATATGGATCTGTTAGAACTGTATGAAAAGAAAAAAAGCTGCCTAAGCATACAATCCTGCAAAAAAAAAAAAAAAAACCAGTATCACAGAGGAGCAGAAAGAGACGAGCTCTCAAGGGATCAAGAAGGAACAGCCACAGAACTAAGAAGAGAAGTTTCCTAGAAGAATTTGATCAACT.

[0089] SEQ ID NO.1 shows the sequence 100 bp upstream and downstream of the mutation site. The bolded part is the mutation site, and the bolded site is C or A.

[0090] In summary, the Suffolk sheep breed with the smallest backfat thickness can be bred by using the A→C mutation at position 89305500 on chromosome 6 of the Suffolk sheep genome. Individuals with the CC genotype can be selected as the paternal or maternal line to reduce the backfat thickness of Suffolk sheep offspring.

[0091] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0092] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of a molecular marker in sheep genetic breeding, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and a single nucleotide C to A mutation occurs at position 101 bp of the molecular marker; The genetic breeding refers to at least one of the following: 1) Identify the low backfat thickness trait in sheep; the low backfat thickness refers to a backfat thickness of less than 1.04 mm; 2) Reduce the backfat thickness of sheep offspring; The sheep in question are Suffolk sheep.

2. The application as described in claim 1, characterized in that, The method for identifying the backfat thickness trait in sheep includes the following steps: Extracting genomic DNA from sheep; Using sheep genomic DNA as a template, the genotype at the 101 bp site of the molecular marker was detected; If the genotype is CC, the sheep is determined to have low backfat.

3. The application as described in claim 2, characterized in that, The sheep's genomic DNA was derived from the sheep's blood.

4. The application as described in claim 3, characterized in that, The sheep's genomic DNA was extracted using the phenol-chloroform method.

5. The application as described in claim 1, characterized in that, The method to reduce the backfat thickness of sheep offspring is as follows: select sheep individuals with the genotype CC at the 101bp locus of the molecular marker as parents to reduce the backfat thickness of sheep offspring.

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