A SNP molecular marker affecting the number of ribs in Hu sheep and its application

Abstract

This invention provides a SNP molecular marker affecting the number of ribs in Hu sheep and its application. The SNP molecular marker is located at position 81850060 on chromosome 6 of Hu sheep, and its polymorphism is G / A. This invention also provides a primer set for amplifying the above-mentioned SNP molecular marker to detect the genotype of the rib trait in Hu sheep. By detecting this genotype, the rib number trait in Hu sheep can be identified efficiently and rapidly, allowing individuals with genotypes exhibiting a higher rib number to be selected, thereby improving the meat production capacity and economic value of Hu sheep, and providing technical support for the early breeding of high-quality Hu sheep.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to an SNP molecular marker that affects the number of ribs in Hu sheep and its application. Background Technology

[0002] The Hu sheep is a unique and high-quality meat sheep breed in China, renowned for its tender meat and distinctive flavor. The number of ribs is a key economic trait in Hu sheep, directly impacting meat yield and market value. Rib number is closely related to sheep growth and development, making rib optimization crucial for improving the economic benefits of Hu sheep farming. Traditional breeding methods largely rely on phenotypic selection; however, due to the complexity of the interaction between environmental and genetic factors, the results of phenotypic selection are not ideal. In recent years, the continuous development of molecular marker technology has opened up new pathways for animal genetic improvement.

[0003] The EPHA5 gene is located on chromosome 6 of the Hu sheep, containing 18 exons with a total coding region of 3975 bp. The EPHA5 gene is involved in regulating angiogenesis and development, and the Eph receptor family and its ligands are involved in regulating osteoblast differentiation, influencing osteoblast proliferation, and guiding the spatial distribution of bone tissue. However, no molecular markers for economically relevant traits in Hu sheep associated with this gene have yet been discovered using current technology. Summary of the Invention

[0004] This invention provides an SNP molecular marker that affects the number of ribs in Hu sheep and its application. The SNP molecular marker is located on the EPHA5 gene on chromosome 6 of Hu sheep and is related to the number of ribs in Hu sheep. It can be used to detect the phenotype of Hu sheep with a high number of ribs to assist in breeding and improve economic benefits.

[0005] In a first aspect, the present invention provides a SNP molecular marker related to the number of ribs in Hu sheep, wherein the polymorphic site of the SNP molecular marker is located at position 81850060 on chromosome 6 of Hu sheep, with the reference genome NCBI accession number ARS-UI_Ramb_v2.0 (GCF_016772045.1), and the polymorphism of the SNP molecular marker is G / A.

[0006] The present invention further provides an SNP molecular marker related to the number of ribs in Hu sheep, the SNP molecular marker comprising a nucleotide sequence as shown in SEQ ID NO 3, wherein the 51-position polymorphism of the nucleotide sequence is G / A.

[0007] Secondly, the present invention provides a set of primer combinations for amplifying any of the above-mentioned SNP molecular markers, the primer combination comprising an upstream primer F and a downstream primer R, wherein the nucleotide sequence of the upstream primer F is shown in SEQ ID NO.1 and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.2.

[0008] The present invention further provides a kit comprising the primer combination described above.

[0009] Thirdly, the present invention provides the application of the above-mentioned SNP molecular markers, primer combinations or kits in the detection of the number of ribs in Hu sheep.

[0010] This invention also provides the application of the above-mentioned SNP molecular markers, primer combinations or kits in the breeding of multi-ribbed Hu sheep.

[0011] Fourthly, the present invention provides a method for determining the number of ribs in Hu sheep. Specifically, the genomic DNA of the Hu sheep to be tested is amplified by PCR using the above primer combination or kit, and the amplification product is detected to contain the nucleotide sequence shown in SEQ ID NO 3. When the SNP at 51 bp is A / A or G / A, it is a multiple rib number phenotype.

[0012] Fifthly, the present invention provides a method for determining the rib number phenotype of Hu sheep, characterized in that the genomic DNA of the Hu sheep to be tested is amplified by PCR using the primer combination described in claim 3, and the amplification product is detected to contain the nucleotide sequence shown in SEQ ID NO 3. When the SNP at 51 bp is G / G genotype, it is a low rib number phenotype.

[0013] Most Hu sheep have 26 ribs, but when the multi-rib trait is present, it has 28 ribs. This trait accounts for about 15% of the entire Hu sheep population. When the genotype of the above-mentioned SNP locus is A / A, the proportion of individuals with 28 ribs is the highest, at 66.67%; when the genotype is A / G, the proportion is 45.23%; and when the genotype is G / G, the proportion is 10.44%.

[0014] Preferably, the PCR amplification system in the above method is as follows: 1 µL of 20-50 ng / μL genomic DNA, 0.5 µL of 10× PCR reaction buffer, 0.4 µL of 25 mmol / L MgCl2, 0.1 µL of 25 μmol / L dNTPs, 1 µL of PCR Primer mix, 0.2 µL of 5 U / μL Taq DNA polymerase, and deionized water to a final volume of 5 µL.

[0015] And / or the PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 56℃ annealing for 30 s, 72℃ extension for 60 s, 45 cycles; 72℃ further extension for 5 min; 25℃ incubation.

[0016] In a sixth aspect, the present invention provides a breeding method for multi-rib number Hu sheep, specifically, using the above method to detect the SNP phenotype of Hu sheep, and selecting G / A or A / A individuals with the multi-rib number trait.

[0017] The SNP molecular markers and related detection primer combinations and kits provided by this invention can rapidly and efficiently screen Hu sheep with multiple ribs, which is beneficial for the breeding of Hu sheep with multiple ribs, improves economic benefits, and provides a theoretical basis for the molecular breeding of Hu sheep. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The proportion of 28-rib individuals in Hu sheep corresponding to different genotypes at SNP loci in Example 2 of this invention.

[0020] Figure 2 This represents the proportion of Hu sheep individuals with 28 ribs corresponding to different genotypes at different SNP loci in the experimental examples of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0022] Example 1: Genomic DNA Extraction, PCR Amplification, and Sequencing

[0023] Genomic DNA extraction: 245 Hu sheep raised by Jiangsu Qianbao Animal Husbandry Co., Ltd. were selected as the detection objects. 1 mL of jugular vein blood was collected from the Hu sheep and anticoagulated with EDTA. First, red blood cell lysate (TIANGEN RT122-02) was used to lyse and remove red blood cells without DNA, and nuclear lysate (TIANGEN DP304-03) was used to lyse cells to release genomic DNA. Then, protein precipitation solution was used to selectively precipitate and remove proteins. Finally, pure genomic DNA was precipitated with isopropanol and redissolved in DNA dissolution solution.

[0024] PCR amplification: Using the genomic DNA extracted above as a template, PCR amplification was performed on the fragment containing the SNP molecular marker at position 81850060 on chromosome 6 of Hu sheep. The nucleotide sequence of the upstream primer F used for amplification is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.2.

[0025] Upstream primer F: 5' -GTTAGGTCTAAAGTGCAGCCTG -3' (SEQ ID NO.1)

[0026] Downstream primer R: 5' -GTTGACATCTCCTCTCCATTGC-3' (SEQ ID NO.2)

[0027] The PCR reaction system was as follows: 1 µL of genomic DNA at 20 - 50 ng / µL, 0.5 µL of 10×PCR reaction buffer, 0.4 µL of 25 mmol / L MgCl2, 0.1 µL of 25 µmol / L dNTPs, 1 µL of PCR Primer mix, 0.2 µL of 5 U / µL Taq DNA polymerase, and made up to 10 µL with deionized water; The upstream primer F and the downstream primer R constituted the PCR Primer mix, and the final concentrations of both the upstream primer F and the downstream primer R were 100 μmol / L.

[0028] The PCR program was: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 60 s, for 45 cycles; 72℃ for 5 min, and left standing at 25℃.

[0029] The PCR products of each sample were subjected to Sanger sequencing.

[0030] Analysis of sequencing results in Example 2

[0031] The number of ribs of 245 Hu sheep was counted. Among them, 203 individuals had 26 ribs and 42 individuals had 28 ribs.

[0032] A Lasso regression model was built using the glmnet R package. The model after logit transformation is as follows:

[0033]

[0034] Where G∈{0,1} is a binary variable, G=0 represents a rib count of 26, and G=1 represents a rib count of 28. p represents the probability of G=1. The loss function is calculated using Lasso regression with L1 regularization to constrain the model.

[0035] The Lasso fitting results yielded a minimum misclassification rate of 16.85% and an interpretability of 88.12%, retaining a total of 52 sites. Based on these 52 sites, a random forest model was further constructed using the R package randomForest, resulting in a machine learning model with an error rate reduced to 7.49%. The 12 most important sites were selected as candidates based on the mean decrease accuracy (Table 1).

[0036] Table 1. Features of the 12 candidate sites retained by the Lasso and Random Forest models.

[0037]

[0038] The sites overlapping with the GWAS results in the table above are marked in red. The columns in the table are, in order: chromosome, site name, average descent precision, reference base, mutant base, mutant base frequency in the 26-rib population, number of individuals in the 26-rib population, mutant base frequency in the 28-rib population, number of individuals in the 28-rib population, difference in mutant base frequencies between the two populations, and Fst value at the target site in both populations.

[0039] As shown in the table above, locus 6_81850060 exhibited a strong signal (P-value < 10) in GWAS analysis (calculated using GCTA software). -4 The Fst value was higher in populations with different numbers of ribs (Table 1). This locus is located within the EPHA5 gene range. When the genotype at locus 6_81850060 was A / A, the proportion of individuals with 28 ribs was the highest, at 66.67%; when the genotype was A / G, the proportion was 45.23%; and when the genotype was G / G, the proportion was 10.44%. Figure 1 The above results indicate that the EPHA5 gene molecular marker in Hu sheep is significantly correlated with the number of ribs. Individuals with the G / A or A / A genotypes can be selected to increase the number of ribs and thus improve economic value, depending on the actual breeding objectives.

[0040] Experimental Example: Verification Experiment on the Number of Ribs in Hu Sheep

[0041] A total of 76 Hu sheep raised by Jiangsu Qianbao Animal Husbandry Co., Ltd. were selected as experimental cases, including 14 individuals with 28 ribs and the rest with 26 ribs. These samples were sequenced, and the phenotypic ratios corresponding to different genotypes of 6_81850060 were calculated. The results are as follows: Figure 2 The proportion of individuals with 28 ribs corresponding to the GG genotype was 16.92%, the proportion corresponding to the AG genotype was 28.57%, and the proportion corresponding to the AA genotype was 50%, which is consistent with the previous trend.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of SNP molecular markers related to the number of ribs in Hu sheep, primer combinations or kits for amplifying these SNP molecular markers, in the detection of the rib number phenotype in Hu sheep; The polymorphic site of the SNP molecular marker is located at position 81850060 on chromosome 6 of the Hu sheep genome, with the NCBI accession number GCF_016772045.

1. The polymorphism of the SNP molecular marker is G / A; or The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO 3, and the 51-position polymorphism of the nucleotide sequence is G / A; The primer combination includes an upstream primer F and a downstream primer R, the nucleotide sequence of the upstream primer F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.2; The kit is a kit containing the primer combination; When the genotype of the SNP molecular marker locus is A / A or G / A, it indicates a high rib number phenotype; when the genotype is G / G, it indicates a low rib number phenotype.

2. Application of SNP molecular markers related to the number of ribs in Hu sheep, primer combinations or kits for amplifying these SNP molecular markers in the breeding of Hu sheep with multiple ribs; The polymorphic site of the SNP molecular marker is located at position 81850060 on chromosome 6 of the Hu sheep genome, with the NCBI accession number GCF_016772045.

1. The polymorphism of the SNP molecular marker is G / A; or The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO 3, and the 51-position polymorphism of the nucleotide sequence is G / A; The primer combination includes an upstream primer F and a downstream primer R, the nucleotide sequence of the upstream primer F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.2; The kit is a kit containing the primer combination; When the genotype of the SNP molecular marker locus is A / A or G / A, it indicates a high rib number phenotype; when the genotype is G / G, it indicates a low rib number phenotype.

3. A method for determining the rib count phenotype of Hu sheep, characterized in that, The genomic DNA of the target sheep was amplified by PCR using the primer combination or a kit containing the primer combination. The amplification product was tested to see if it contained the nucleotide sequence shown in SEQ ID NO 3. When the genotype of the SNP site at 51 bp was A / A or G / A, it was a phenotype with a high number of ribs. When the genotype of the SNP site at 51 bp was G / G, it was a phenotype with a low number of ribs. The primer combination includes an upstream primer F and a downstream primer R, the nucleotide sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO.

2.

4. The method according to claim 3, characterized in that, The PCR amplification system consisted of: 1 µL of 20–50 ng / μL genomic DNA, 0.5 µL of 10× PCR reaction buffer, 0.4 µL of 25 mmol / L MgCl2, 0.1 µL of 25 μmol / L dNTPs, 1 µL of PCR Primer mix, 0.2 µL of 5 U / μL Taq DNA polymerase, and deionized water to a final volume of 5 µL. And / or the PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 56℃ annealing for 30 s, 72℃ extension for 60 s, 45 cycles; 72℃ further extension for 5 min; 25℃ incubation.

5. A breeding method for multi-ribbed Hu sheep, characterized in that, Using the method described in any one of claims 2-3, the SNP phenotype of the Hu sheep is detected, and individuals with the G / A or A / A genotype who have the multiple rib number trait are selected.

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