SNP (Single Nucleotide Polymorphism) molecular marker capable of influencing hu sheep rib number and application of SNP molecular marker

Abstract

The invention provides an SNP molecular marker influencing the rib number of Hu sheep and application of the SNP molecular marker, the SNP molecular marker is located at the 81850060th site of a Hu sheep chromosome 6, and the polymorphism of the SNP molecular marker is G / A. The invention further provides a primer group for amplifying the SNP molecular marker and a genotype for detecting the rib character of the Hu sheep, the rib number character of the Hu sheep can be efficiently and rapidly identified by detecting the genotype, and genotype individuals with the multi-rib number character are selected and reserved, so that the meat production capacity of the Hu sheep is improved, the economic value is increased, and the economic benefit of the Hu sheep is increased. And a technical support is provided for early breeding of the high-quality Hu sheep.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a SNP molecular marker affecting the number of ribs of Hu sheep and an application thereof. Background Art

[0002] Hu sheep is a high-quality meat sheep breed unique to China. Its meat is tender and has a unique flavor, and it is quite famous in the market. The number of ribs of Hu sheep is a key economic trait, which has a direct impact on meat production and market value. The number of ribs is closely related to the growth and development of sheep, so optimizing the number of ribs is crucial to improving the efficiency of Hu sheep farming. Traditional breeding methods mostly 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 paths for animal genetic improvement.

[0003] The EPHA5 gene is located on chromosome 6 of Hu sheep, contains 18 exons, and the total length of the coding region is 3975bp. The EPHA5 gene is involved in regulating the growth and development of blood vessels, and the Eph receptor family and its ligands are involved in regulating the differentiation process of osteoblasts, affecting the proliferation of osteoblasts and guiding the spatial distribution of bone tissue. However, no molecular markers for economic traits of Hu sheep related to this gene have been found in the prior art. Summary of the invention

[0004] The present invention provides a SNP molecular marker affecting the number of ribs of Hu sheep and an application thereof. The SNP molecular marker is located on the EPHA5 gene on chromosome 6 of Hu sheep, is related to the number of ribs of Hu sheep, and can be used to detect the phenotype of Hu sheep with a large number of ribs to assist breeding and improve economic benefits.

[0005] In the first aspect, the present invention provides a SNP molecular marker associated with the number of ribs of Hu sheep, the polymorphic site of the SNP molecular marker is located at position 81850060 on chromosome 6 of Hu sheep, the reference genome NCBI accession number is 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 a SNP molecular marker associated with the number of ribs of Hu sheep, wherein the SNP molecular marker comprises a nucleotide sequence as shown in SEQ ID NO 3, and the 51-position polymorphism of the nucleotide sequence is G / A.

[0007] In a second aspect, 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, 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 above primer combination.

[0009] In a third aspect, the present invention provides the use of the above-mentioned SNP molecular marker, primer combination or kit in the detection of the rib number phenotype of Hu sheep.

[0010] The present invention also provides the use of the above-mentioned SNP molecular marker, primer combination or kit in the breeding of Hu sheep with many ribs.

[0011] In a fourth aspect, the present invention provides a method for determining the rib number phenotype of Hu sheep, specifically, using the above-mentioned primer combination or kit to PCR amplify the genomic DNA of the Hu sheep to be tested, and detecting whether the amplified product contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at the 51bp position is A / A or G / A, it is a multiple rib number phenotype.

[0012] In a fifth aspect, 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 amplified product is detected to see whether it contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at the 51bp position is the G / G genotype, it is a low rib number phenotype.

[0013] Most Hu sheep have 26 ribs, and when the multi-rib trait appears, the number of ribs is 28. The proportion of this trait in the entire Hu sheep population is about 15%. When the genotype of the above SNP site 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, in the above method, the PCR amplification system is: 20-50 ng / μL genomic DNA 1μL, 10× PCR reaction buffer 0.5μL, 25mmol / L MgCl2 0.4μL, 25μmol / L dNTPs 0.1μL, PCR Primer mix 1μL, 5U / μL Taq DNA polymerase 0.2μL, and deionized water is added to 5μL; And / or the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 60 s, 45 cycles; extension at 72°C for another 5 min; and standing at 25°C.

[0015] In a sixth aspect, the present invention provides a breeding method for Hu sheep with a large number of ribs, specifically, using the above method to detect the SNP phenotype of Hu sheep, and selecting G / A or A / A individuals with the trait of large number of ribs.

[0016] The SNP molecular markers and related detection primer combinations and kits provided by the present invention can quickly and efficiently screen Hu sheep with a large number of ribs, which is beneficial to the breeding of Hu sheep with a large number of ribs, improves economic benefits, and provides a theoretical basis for the molecular breeding of Hu sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 It is the proportion of 28 ribs of Hu sheep individuals corresponding to different genotypes of SNP sites in Example 2 of the present invention.

[0019] Figure 2 It is the proportion of 28 ribs of Hu sheep individuals corresponding to different genotypes at different SNP sites in the test example of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 Genomic DNA extraction, PCR amplification and sequencing Genomic DNA extraction: 245 Hu sheep raised by Jiangsu Qianbao Animal Husbandry Co., Ltd. were selected as the test objects. 1 mL of blood was collected from the jugular vein of the Hu sheep and anticoagulated with EDTA. First, red blood cell lysis buffer (TIANGEN / Tiangen Biochemical RT122-02) was used to lyse and remove red blood cells without DNA, and cell nucleus lysis buffer (TIANGEN / Tiangen Biochemical DP304-03) was used to lyse cells to release genomic DNA, and then protein precipitation buffer was used to selectively precipitate and remove protein. Finally, the pure genomic DNA was precipitated by isopropanol and redissolved in DNA dissolving buffer.

[0022] PCR amplification: Using the genomic DNA extracted as above as a template, PCR amplification was performed on the fragment containing the 81850060th SNP molecular marker of 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.

[0023] Upstream primer F: 5'-GTTAGGTCTAAAGTGCAGCCTG -3' (SEQ ID NO.1) Downstream primer R: 5'-GTTGACATCTCCTCTCCATTGC-3' (SEQ ID NO.2) The PCR reaction system was as follows: 1µL of 20-50ng / µL genomic DNA, 0.5µL of 10× PCR reaction buffer, 0.4µL of 25mmol / LMgCl2, 0.1µL of 25µmol / L dNTPs, 1µL of PCR Primer mix, 0.2µL of 5U / µL Taq DNA polymerase, and deionized water to make up to 10µL; the upstream primer F and the downstream primer R constituted the PCR Primer mix, and the final concentrations of the upstream primer F and the downstream primer R were both 100μmol / L.

[0024] The PCR program was: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 60 s, 45 cycles; 72°C for 5 min, and rest at 25°C.

[0025] The PCR products of each sample were subjected to Sanger sequencing.

[0026] Example 2 Sequencing Results Analysis The rib numbers of 245 Hu sheep were counted, of which 203 individuals had 26 ribs and 42 individuals had 28 ribs.

[0027] The Lasso regression model was constructed using the glmnet R package. The model after logit transformation is as follows: Where G∈{0,1} is a binary variable, G=0 means the number of ribs is 26, G=1 means the number of ribs is 28. p represents the probability of G=1. The calculation of the loss function introduces Lasso regression and adds L1 regularization to constrain the model.

[0028] The Lasso fitting results showed that the minimum error rate was 16.85%, the explanation was 88.12%, and a total of 52 loci were retained. Based on these 52 loci, the random forest model was further constructed using the R package randomForest, and the error rate of the obtained machine learning model was reduced to 7.49%. The 12 most important loci were selected as candidates based on the mean decrease accuracy (Table 1).

[0029] Table 1 Characteristics of the 12 candidate sites retained by the Lasso and random forest models The overlapping sites in the table above and the GWAS results are marked in red. The columns in the table are chromosome, site name, average dropout accuracy, 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 frequency between the two populations, and Fst value of the two populations at the target site. As can be seen from the table above, among the above loci, 6_81850060 has a strong signal (P value < 10 -4 ), and the Fst values ​​of the groups with different rib numbers were larger (Table 1). This locus is located within the gene EPHA5. When the genotype of the 6_81850060 locus 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 show that the EPHA5 gene molecular marker is significantly correlated with the rib number trait in Hu sheep. According to the actual breeding goals, G / A or A / A genotype individuals can be selected to increase the rib number and increase the economic value.

[0030] Test example: Hu sheep rib number verification test A total of 76 Hu sheep raised by Jiangsu Qianbao Animal Husbandry Co., Ltd. were selected as test cases, of which 14 had 28 ribs and the rest had 26 ribs. We sequenced these samples and calculated the phenotypic ratios corresponding to different genotypes of 6_81850060. The results are as follows: Figure 2 The proportion of individuals with 28 ribs corresponding to the GG genotype was 16.92%, the proportion of individuals with 28 ribs corresponding to the AG genotype was 28.57%, and the proportion of individuals with 28 ribs corresponding to the AA genotype was 50%, which was consistent with the previous results.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SNP molecular marker associated with the number of ribs of Hu sheep, characterized in that: The polymorphic site of the SNP molecular marker is located at position 81850060 on chromosome 6 of Hu sheep, the reference genome NCBI accession number is ARS-UI_Ramb_v2.0 (GCF_016772045.1), and the polymorphism of the SNP molecular marker is G / A.

2. A SNP molecular marker associated with the number of ribs of Hu sheep, characterized in that: The SNP molecular marker includes a nucleotide sequence as shown in SEQ ID NO 3, and the polymorphism at position 51 of the nucleotide sequence is G / A.

3. A primer combination for amplifying the SNP molecular marker according to any one of claims 1 or 2, characterized in that: The primer combination includes an upstream primer F and a downstream primer R, the nucleotide sequence of the upstream primer F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer R is shown as SEQ ID NO.

2.

4. A kit, characterized in that The method comprises the primer combination as claimed in claim 3.

5. Use of the SNP molecular marker in any one of claims 1 or 2, the primer combination in claim 3 or the kit in claim 4 in the detection of the rib number phenotype of Hu sheep.

6. Use of the SNP molecular marker in any one of claims 1 or 2, the primer combination in claim 3 or the kit in claim 4 in the breeding of Hu sheep with many ribs.

7. A method for determining the phenotype of the number of ribs 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 4 or the kit described in claim 5, and the amplified product is detected to see whether it contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at the 51 bp position is A / A or G / A, it is a multiple rib number phenotype.

8. A method for determining the phenotype of the number of ribs 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 amplified product is detected to see whether it contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at the 51 bp position is a G / G genotype, it is a low rib number phenotype.

9. The method according to claim 6, characterized in that The PCR amplification system is: 20-50 ng / μL genomic DNA 1μL, 10× PCR reaction buffer 0.5μL, 25mmol / L MgCl2 0.4μL, 25μmol / L dNTPs 0.1μL, PCR Primer mix 1μL, 5U / μL Taq DNA polymerase 0.2μL, and deionized water to 5μL; And / or the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 60 s, 45 cycles; extension at 72°C for another 5 min; and standing at 25°C.

10. A breeding method for Hu sheep with many ribs, characterized in that: The SNP phenotype of Hu sheep is detected by the method described in any one of claims 6 to 8, and G / A or A / A individuals with multiple rib number traits are selected.

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