Molecular markers, marker combinations and applications of SNP genes associated with chest girth in Suffolk sheep

Abstract

This invention belongs to the fields of molecular biology, molecular marker technology, and animal genetics and breeding. It discloses SNP molecular markers, marker combinations, and their applications related to the chest girth trait in Suffolk sheep. Based on genome-wide association analysis, this invention screens molecular markers associated with the chest girth trait in Suffolk sheep. Using these molecular markers, through marker-assisted breeding, individuals with favorable genotypes are retained while those with unfavorable genotypes are eliminated. This significantly improves the chest girth trait in Suffolk sheep, thereby improving their growth performance and ultimately increasing their breeding efficiency and economic value.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology, molecular marker technology and animal genetics and breeding. Specifically, it relates to SNP molecular markers, marker combinations and their applications for genes related to chest girth in Suffolk sheep. Background Technology

[0002] Suffolk sheep are a superior meat sheep breed, renowned worldwide for their large size, rapid growth, and excellent meat quality. With the development of molecular biology techniques, screening for molecular markers associated with productive traits through genome-wide association studies (GWAS) has become an important direction in animal genetics and breeding. These molecular markers can be used for early selection and genetic improvement, thereby increasing the efficiency of genetic progress.

[0003] In sheep production, chest circumference is an important indicator of growth and development, and is closely related to meat value. However, traditional phenotypic selection methods have limited effectiveness in improving traits with low heritability. Furthermore, obtaining phenotypic data on chest circumference is relatively difficult, requiring professional measurement and evaluation, which limits its application in breeding practice.

[0004] To overcome these limitations, genome-wide association studies (GWAS) can be used to screen for molecular markers associated with the chest girth trait in Suffolk sheep. By detecting a large number of single nucleotide polymorphism (SNP) sites at the whole genome level, genetic variations significantly associated with the chest girth trait can be identified. These genetic variations can serve as molecular markers for selection-aided improvement and genetic enhancement.

[0005] Previous studies on molecular markers of sheep production traits have reported molecular markers associated with growth traits. For example, Zhang et al. used a 50K SNP chip to perform GWAS analysis on 329 purebred sheep and identified candidate genes associated with growth and meat quality traits. Furthermore, Tao et al. discovered the RXFP2 gene associated with body size traits in Qira black sheep. These studies provide valuable information for understanding the genetic basis of sheep growth traits.

[0006] However, current research on molecular markers for the chest girth trait in Suffolk sheep is relatively limited. Therefore, the aim of this study is to screen for molecular markers associated with the chest girth trait in Suffolk sheep using GWAS analysis and to explore their potential application in marker-assisted breeding. This will provide new strategies and tools for the genetic improvement of Suffolk sheep.

[0007] These studies aim to provide new molecular tools for the genetic improvement of Suffolk sheep, thereby enhancing their production performance and economic value. This has significant theoretical and practical implications for promoting the development of the meat sheep industry. Summary of the Invention

[0008] The purpose of this invention is to provide molecular markers for SNP genes related to chest girth traits in Suffolk sheep, marker combinations, and their applications.

[0009] To achieve the objectives of this invention, in a first aspect, this invention provides SNP molecular markers for genes related to chest girth traits in Suffolk sheep, wherein the markers are selected from any one of ① to ⑥:

[0010] ①The marker contains a nucleotide sequence with a polymorphism of G / A at position 101919591 bp on chromosome 5 of Suffolk sheep;

[0011] ②The marker contains a nucleotide sequence with a polymorphism of A / G at position 101927152 bp on chromosome 5 of Suffolk sheep;

[0012] ③The marker contains a nucleotide sequence with a polymorphism of G / A at position 101935914 bp on chromosome 5 of Suffolk sheep;

[0013] ④ The marker contains a nucleotide sequence with a T / C polymorphism at position 101936632 bp on chromosome 5 of Suffolk sheep;

[0014] ⑤ The marker contains a nucleotide sequence with a polymorphism of A / G at position 101972459 bp on chromosome 5 of Suffolk sheep;

[0015] ⑥ The marker contains a nucleotide sequence with a polymorphism of A / G at position 101975776 bp on chromosome 5 of Suffolk sheep;

[0016] The physical location mentioned above corresponds to the sheep (Ovis aries) reference genome version number assembly ARS-UI_Ramb_v2.0.

[0017] Furthermore, for the marker ①, the genotype AA containing the polymorphic site has a larger average chest circumference compared to Suffolk sheep with genotypes AG and GG;

[0018] For marker ②, the genotype of the polymorphic site containing GG has a larger average chest circumference compared to Suffolk sheep with genotypes GA and AA;

[0019] For marker ③, the genotype AA containing the polymorphic site has a larger average chest circumference compared to Suffolk sheep with genotypes AG and GG;

[0020] For marker ④, the genotype CC containing the polymorphic site has a larger average chest circumference compared to Suffolk sheep with genotypes CT and TT;

[0021] For marker ⑤, the genotype of the polymorphic site containing GG has a larger average chest circumference compared to Suffolk sheep with genotypes GA and AA;

[0022] For the marker ⑥, the genotype of the polymorphic site containing GG has a larger average chest circumference compared to Suffolk sheep with genotypes GA and AA.

[0023] Preferred markings ③ and ④.

[0024] Secondly, the present invention provides a combination of SNP molecular markers related to the chest girth trait of Suffolk sheep, wherein the combination of markers includes two or more of the markers ① to ⑥.

[0025] Thirdly, the present invention provides primers for amplifying the markers or primer sets for amplifying the marker combinations.

[0026] Fourthly, the present invention provides detection reagents or kits containing the said primers or primer sets.

[0027] Fifthly, the present invention provides any of the following applications of the marker, the marker combination, the primer or primer set, or the detection reagent or kit:

[0028] (1) Used for the identification, selection and improvement of breast girth traits in Suffolk sheep;

[0029] (2) Used for early prediction of breast girth traits in Suffolk sheep;

[0030] (3) Used for molecular marker-assisted breeding of breast girth in Suffolk sheep.

[0031] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] This invention screens molecular markers associated with the chest girth trait of Suffolk sheep based on genome-wide association analysis. By utilizing these molecular markers and employing marker-assisted breeding, individuals with favorable genotypes can be retained while those with unfavorable genotypes are eliminated, which can significantly improve the chest girth trait of Suffolk sheep and thus improve their growth performance.

[0033] This invention provides a combination of SNP markers related to the chest circumference trait of Suffolk sheep based on genome-wide association analysis and a breeding method. By detecting SNP molecular markers, the chest circumference of Suffolk sheep can be predicted quickly, cost-effectively and efficiently, thereby improving its breeding efficiency and economic value. Attached Figure Description

[0034] Figure 1 This is a technical roadmap for the present invention.

[0035] Figure 2 This is a chest circumference phenotype distribution diagram in a preferred embodiment of the present invention.

[0036] Figure 3 This is a SNP distribution diagram in a preferred embodiment of the present invention.

[0037] Figure 4 This is an SNP density map in a preferred embodiment of the present invention.

[0038] Figure 5 This is a Manhattan plot showing the genome-wide association analysis results of chest circumference in Tan sheep in a preferred embodiment of the present invention; the vertical axis represents -lg of the association value, and each point represents one SNP locus.

[0039] Figure 6 This is a QQ diagram representing the genome-wide association analysis of SNPs related to chest girth in Tan sheep in a preferred embodiment of the present invention.

[0040] Figure 7 This is a GO and KEGG functional annotation of candidate genes related to significant SNPs in chest circumference of Tan sheep in a preferred embodiment of the present invention.

[0041] Figure 8 This invention provides a preferred embodiment of the analysis of genotypic differences in SNPs related to chest girth in Tan sheep. Detailed Implementation

[0042] This invention provides a method for screening candidate markers related to the chest girth trait in Suffolk sheep based on genome-wide association analysis, the method comprising the following steps ( Figure 1 The method involves collecting blood samples from individual Suffolk sheep and performing 10× resequencing on each sample, with a data volume of no less than 30G per sample. Simultaneously, liquid-phase capture sequencing was performed on some samples to improve the sequencing depth and coverage of specific genomic regions. Quality control and filtering were applied to the resequencing and liquid-phase capture sequencing data. High-quality sequencing data were compared with a reference genome for variant detection to obtain SNP markers. Chest girth data corresponding to the sequencing samples were collected. Genome-wide association analysis (GWAS) was performed using GMAT software, integrating the analysis results of resequencing and liquid-phase capture data to screen for SNP loci significantly associated with chest girth. Functional annotation was performed on the significantly associated SNP loci, integrating gene function information to screen for candidate genes potentially associated with chest girth. Candidate SNP markers were determined based on statistical significance, functional relevance, and consistency across different datasets. This method, combining high-throughput sequencing technology and genome-wide association analysis, can effectively identify genetic markers associated with chest girth in Suffolk sheep, providing a scientific basis for subsequent marker-assisted selection breeding.

[0043] The SNP marker sites correspond to the following chromosomes in the sheep reference genome Ovis aries (version: assembly ARS-UI_Ramb_v2.0): chr5:101919591, chr5:101927152, chr5:101935914, chr5:101936632, chr5:101972459, and chr5:101975776. Using these SNP markers, through marker-assisted breeding, individuals with favorable genotypes can be retained while those with unfavorable genotypes are eliminated, significantly improving the chest girth trait in Suffolk sheep, thereby achieving the goal of improving their growth performance.

[0044] The present invention adopts the following technical solution:

[0045] The SNP molecular marker combination related to the chest girth trait of Suffolk sheep provided by this invention includes at least one or more combinations of the following SNP markers, wherein the SNP sites of the molecular markers are mutations located at positions 101919591 (chr5:101919591), 101927152 (chr5:101927152), 101935914 (chr5:101935914), 101936632 (chr5:101936632), 101972459 (chr5:101972459), and 101975776 (chr5:101975776) on chromosome 5. These correspond to the 51st base of the sequence shown in SEQ ID NO:1-6, respectively, with n being g or a, a or g, g or a, t or c, a or g, and a or g.

[0046] The polymorphisms of the SNP loci are as follows: alleles of locus chr5:101919591 are G and A, alleles of locus chr5:101927152 are A and G, alleles of locus chr5:101935914 are G and A, alleles of locus chr5:101936632 are T and C, alleles of locus chr5:101972459 are A and G, and alleles of locus chr5:101975776 are A and G.

[0047] This invention also provides a method for screening candidate markers for the chest girth trait in Suffolk sheep based on genome-wide association analysis.

[0048] Furthermore, after quality control, the genome-wide association sequencing data were analyzed using GMAT software based on the LMM model to identify SNPs that were significantly associated with the chest girth trait of Suffolk sheep.

[0049] The present invention also provides the application of the aforementioned SNP marker combination in marker-assisted selection of breast girth traits in Suffolk sheep.

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] Example 1

[0052] 1. Materials and Methods

[0053] 1.1 Collection of experimental animals and samples

[0054] The experiment selected 427 Suffolk sheep as research subjects. The sheep came from various sources, including 48 from a sheep farm in Hohhot, 18 from a sheep farm in Ningxia, 49 from a sheep farm in Huade County, Ulanqab, 30 from a sheep farm in Shangdu County, Ulanqab, and 282 from a sheep farm in Xinjiang. Ear tissue or blood samples were collected from each sheep, and genomic DNA was extracted.

[0055] 1.2 Whole genome resequencing

[0056] Genomic DNA was extracted using a magnetic bead method, following standard extraction procedures. Integrity and purity were tested. For samples that passed the tests, appropriate fragment sizes were selected by gel electrophoresis, followed by PCR enrichment to construct libraries. After library construction, Quantitative quality control was performed using Qubit, and qualified libraries were sequenced. Following sequencing, base sequencing quality distribution analysis, base content distribution analysis, and filtering of the raw image data (Raw reads) obtained from high-throughput sequencing were performed.

[0057] The final sequence obtained from sequencing was re-aligned onto the reference genome for further analysis. The reference genome species was sheep, and the link to the reference genome was: Ovis aries genome assembly ARS-UI_Ramb_v2.0 -NCBI - NLM (nih.gov).

[0058] 1.3 Genome-wide association analysis

[0059] After quality control, the sequencing data were analyzed using GMAT software based on the LMM model to determine their association with chest circumference and to identify SNPs with significant effects. The LMM model is as follows:

[0060]

[0061] In the formula, y is the phenotypic vector. For fixed effects or covariates, for The correlation matrix, The labeling effect to be tested. for The correlation matrix, 'e' represents a polygenic effect, and 'e' represents a residual effect.

[0062] 1.4 Group Stratification

[0063] Population stratification refers to the difference in allele frequencies due to different ancestors. It has been proven to be a confounding factor that may lead to false positive results. Therefore, a QQ plot was drawn on the chest circumference of Suffolk sheep to determine whether there were biased samples and population stratification in the association analysis.

[0064] 1.5 Gene annotation of significant SNPs

[0065] After obtaining significant SNP markers from genome-wide association analysis, the reference genome information of the corresponding species was downloaded from the ENSEMBL website, and genes near the significant SNPs were annotated using the ANNOVAR software. The clusterProfiler package was then used to perform gene function enrichment analysis on the annotated candidate genes based on the GO database.

[0066] 2. Results and Analysis

[0067] 2.1 Sequencing data quality control and alignment results with the reference genome ( Figure 3 )

[0068] The quality control data for sample sequencing are shown in Table 1. Base type distribution detection was mainly used to check for AT and CG segregation. As shown in Table 1, the average percentage of G and C in the total bases was 42.89%, bases with a mass value greater than or equal to 20 accounted for 98.79% of the total bases, and bases with a mass value greater than 30 accounted for 95.7%. The average alignment efficiency between sample DNA and genomic DNA was 99.7%. This indicates that the library construction and sequencing of this population of samples were normal.

[0069] Table 1. Quality control statistics of sample sequencing data

[0070]

[0071] 2.2 Statistical analysis of different chest circumference traits in Suffolk sheep

[0072] Table 2 shows that a detailed statistical analysis was conducted on the chest circumference traits of 427 Suffolk sheep. In this statistical analysis, we observed that the maximum, minimum, and mean chest circumference were 120 cm, 76 cm, and 98.123 cm, respectively. Figure 4 (See chest circumference phenotype distribution chart) Figure 2 .

[0073] Table 2. Statistics on chest circumference of Suffolk sheep

[0074]

[0075] 2.3 Genome-wide association analysis of chest girth trait in Suffolk sheep

[0076] Based on resequencing, 25,510,198 SNPs were selected for further analysis, and an SNP density map was plotted. Figure 3 ) and distribution diagrams before and after quality control at each chromosome location ( Figure 4 Using the commonly used GWAS model LMM statistical analysis, 373 SNPs were found to be significantly correlated with chest circumference in Suffolk sheep (see Manhattan plot). Figure 5 See QQ image Figure 6 Sequence alignment using NCBI and Ensembl was performed, and the sequences were annotated onto 57 genes. Through literature review and multiple comparisons, SNP sites 5_101919591, 5_101927152, 5_101935914, 5_101936632, 5_101972459, and 5_101975776 were anchored and annotated onto the NUDT12 and EFNA5 genes. Detailed information for these six sites is shown in Table 3.

[0077] Table 3. Information on two significantly related SNPs in Suffolk sheep

[0078]

[0079] 2.4 Candidate genes related to chest circumference in Suffolk sheep and their GO functional annotations

[0080] To further identify genes related to the chest girth trait in Suffolk sheep, SNP loci 5_101919591, 5_101927152, 5_101935914, 5_101936632, 5_101972459, and 5_101975776 all annotated two important genes on chromosome 5 (GO annotations see [link]). Figure 7 See KEGG annotations. Figure 8 This gene is widely expressed in sheep tissues and may have a significant impact on sheep growth, development, and body size characteristics. It encodes a key enzyme involved in various biological processes, such as cell signal transduction, protein modification, and cell-cell interactions.

[0081] The product of this gene plays a crucial role in regulating cell growth, differentiation, and metabolism, and may influence the chest girth trait in Suffolk sheep by affecting bone and muscle development. Furthermore, variations in this gene may affect its expression levels or enzyme activity, leading to inter-individual differences in chest girth.

[0082] Example 2

[0083] 1. Materials and Methods

[0084] 1.1 Collection of experimental animals and samples

[0085] The 138 Suffolk sheep used for validation were collected from a sheep farm in Hohhot, including 2 rams and 136 ewes. Blood samples were collected from this group, and chest circumference data were recorded. Genotyping was performed using liquid chromatography-capture technology to validate the six SNP loci associated with chest circumference in Suffolk sheep.

[0086] 1.2 Association analysis of significant loci with traits and multiple comparisons

[0087] The association between marker genotypes and phenotypes was tested using R4.2 software. The model is as follows:

[0088]

[0089] y is the phenotypic vector. For fixed effects or covariates (gender, number of days of measurement). for The correlation matrix, The labeling effect to be tested. for The correlation matrix, This is to account for the residual effect. The LSD method was used to perform multiple comparisons between different genotypes.

[0090] 2. Significant loci and trait association analysis and multiple comparison results

[0091] Association analysis and multiple comparisons were performed on multiple SNP loci and chest circumference traits in Suffolk sheep. The results showed that six SNP loci on chromosome 5 (chr5_101919591, chr5_101927152, chr5_101935914, chr5_101936632, chr5_101972459, chr5_101975776) were significantly associated with chest circumference. Among them, chr5_101935914 and chr5_101936632 showed the most significant additive effects, with significant differences among the three genotypes (P<0.01). Specifically, for chr5_101935914, the average chest circumference of individuals with the AA genotype was significantly greater than that of individuals with the AG genotype, and the AG genotype was significantly greater than that of individuals with the GG genotype. The chr5_101936632 locus showed the same pattern, CC>CT>TT. Notably, chr5_101936632 showed a significant association in both resequencing and resequencing plus liquid chromatography datasets, further confirming the stable relationship between this locus and chest circumference. Additionally, the chr5_101927152 and chr5_101975776 loci also showed clear allele dosage effects, with the GG genotype associated with significantly larger chest circumference (P<0.0001 and P<0.001, respectively). The chr5_101919591 locus showed a significant association between the GG genotype and smaller chest circumference (P<0.05). For chr5_101972459, despite the smaller sample size of the GG genotype, the GA genotype still showed a significantly larger chest circumference than the AA genotype (P<0.01). Detailed analysis results are shown in Table 4.

[0092] Table 4. Correlation test between SNP loci and chest circumference in Suffolk sheep

[0093]

[0094] Note: For the same locus, there is no significant difference between data with the same letter, but there is a significant difference between data with different lowercase letters.

[0095] This invention selected 427 Suffolk sheep as subjects for genome-wide association analysis (GWAS) based on pedigree. Chest circumference data were recorded, ear tissue was collected from samples of this population, genomic DNA was extracted, and the concentration, integrity, and purity of the DNA were tested. Qualified samples were resequencing, and then GWAS was performed on the chest circumference indicators of the Suffolk sheep to screen for candidate genes and SNP loci. Genotyping was performed on 138 Suffolk sheep using liquid-phase capture technology, and the six chest circumference-related loci screened by GWAS were validated, providing support for the future breeding of high-quality Suffolk sheep.

[0096] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of SNP molecular markers related to the breast girth trait in Suffolk sheep, or combinations of such markers: (1) Used for the identification, selection and improvement of breast girth traits in Suffolk sheep; (2) Used for early prediction of breast girth traits in Suffolk sheep; (3) Used for marker-assisted breeding of breast girth in Suffolk sheep; The SNP molecular markers associated with the breast girth trait in Suffolk sheep were selected from any one of ① to ⑥: ①The marker contains a nucleotide sequence with a polymorphism of G / A at position 101919591 bp on chromosome 5 of Suffolk sheep; ②The marker contains a nucleotide sequence with a polymorphism of A / G at position 101927152 bp on chromosome 5 of Suffolk sheep; ③The marker contains a nucleotide sequence with a polymorphism of G / A at position 101935914 bp on chromosome 5 of Suffolk sheep; ④ The marker contains a nucleotide sequence with a T / C polymorphism at position 101936632 bp on chromosome 5 of Suffolk sheep; ⑤ The marker contains a nucleotide sequence with a polymorphism of A / G at position 101972459 bp on chromosome 5 of Suffolk sheep; ⑥ The marker contains a nucleotide sequence with a polymorphism of A / G at position 101975776 bp on chromosome 5 of Suffolk sheep; The above physical location corresponds to the sheep reference genome version number assembly ARS-UI_Ramb_v2.0; Furthermore, For the marker ①, the genotype AA containing the polymorphic locus has a larger average chest circumference compared to Suffolk sheep with genotypes AG and GG; For marker ②, the genotype of the polymorphic site containing GG has a larger average chest circumference compared to Suffolk sheep with genotypes GA and AA; For marker ③, the genotype AA containing the polymorphic site has a larger average chest circumference compared to Suffolk sheep with genotypes AG and GG; For marker ④, the genotype CC containing the polymorphic site has a larger average chest circumference compared to Suffolk sheep with genotypes CT and TT; For the marker ⑤, the genotype of the polymorphic site containing GG has a larger average chest circumference compared to the Suffolk sheep with genotypes GA and AA; For the marker ⑥, the genotype of the polymorphic site containing GG has a larger average chest circumference compared to Suffolk sheep with genotypes GA and AA.

2. The application according to claim 1, characterized in that, The combination of markers includes two or more of the markers ① to ⑥ described in claim 1.

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