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 3605393 site of the 18th chromosome of the Hu sheep, and the polymorphism of the SNP molecular marker is T / C. The invention further provides a primer group capable of 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, and the economic value 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 GABRA5 gene is located on chromosome 18 of Hu sheep and contains 12 exons. The GABRA5 gene is the main inhibitory neurotransmitter in the mammalian brain. It acts on the GABA-A receptor, which is a ligand-gated chloride ion channel. From the perspective of developmental biology, skeletal development, including rib development, is regulated by multiple signaling pathways. There are complex interactions between neural signaling pathways and developmental signaling pathways. Although GABRA5 mainly plays a role in the nervous system, the nervous system can affect the development of peripheral tissues by secreting neurotransmitters or neuropeptides. However, no molecular markers for economically relevant 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 in the upstream regulatory region of the GABRA5 gene on chromosome 18 of Hu sheep, is related to the number of ribs of Hu sheep, and can be used to breed Hu sheep with more ribs to improve economic benefits.

[0005] In the first aspect, the present invention provides a SNP molecular marker that affects the number of ribs of Hu sheep. The SNP molecular marker is located at position 3605393 on chromosome 18 of Hu sheep, the reference genome NCBI accession number is ARS-UI_Ramb_v2.0 (GCF_016772045.1), and its polymorphism is T / C.

[0006] The present invention also provides a SNP molecular marker related to the number of ribs of Hu sheep. The SNP molecular marker is located at position 51 of the base sequence shown in SEQ ID NO 3, and its polymorphism is T / C.

[0007] The present invention also provides a set of primer combinations for amplifying 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] Upstream primer F: 5'-GAGAAGCTGCTGTATAACACAGG -3' (SEQ ID NO.1) Downstream primer R: 5'-GACCCATAACCATGTGGGATTTG-3' (SEQ ID NO.2) The present invention also provides a kit comprising the above primer combination.

[0009] In a second aspect, the present invention provides the use of the above-mentioned SNP molecular marker, primer combination, and kit in at least one of the following: (1) Phenotypic detection of rib number in Hu sheep; (2) Breeding of Hu sheep with a high number of ribs; (3) Analysis of genetic diversity of Hu sheep.

[0010] In the third aspect, the present invention provides a method for determining the rib number phenotype of Hu sheep, wherein the genomic DNA of the Hu sheep to be tested is amplified by PCR using the above-mentioned primer combination or kit, and the amplified product is detected to determine whether it contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at the 51bp position is T / C or C / C, it is a multiple rib number phenotype.

[0011] The present invention also provides a method for determining the rib number phenotype of Hu sheep, wherein the genomic DNA of the Hu sheep to be tested is amplified by PCR using the above-mentioned primer combination or kit, and the amplified product is detected to determine whether it contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at the 51 bp position is T / T, it is a low rib number phenotype.

[0012] Most Hu sheep have 26 ribs, and when the multi-rib trait appears, it has 28 ribs. The proportion of this trait in the entire Hu sheep population is about 15%. When the genotype of the above SNP site is C / T, the proportion of individuals with 28 ribs is the highest, at 81.82%; when the genotype of the above SNP site is C / C, the proportion is 50.00%, and when the genotype of the above SNP site is T / T, the proportion is 13.73%. However, the proportion of individuals with the genotype of the above SNP site C / T in the Hu sheep population is extremely low, so it is more meaningful to judge the multi-rib trait of Hu sheep by the C / C genotype.

[0013] Preferably, the PCR amplification system described in the above method is: 20~50ng / 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.

[0014] Preferably, the PCR amplification program described in the above method 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] Preferably, the nucleotide sequence of the amplified product detected in the above method is completed by Sanger sequencing.

[0016] In a fourth aspect, the present invention provides a breeding method for Hu sheep with a large number of 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 C / T heterozygous individuals with the trait of large number of ribs are selected.

[0017] 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

[0018] 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.

[0019] 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.

[0020] Figure 2 It is the proportion of 28 ribs of Hu sheep individuals corresponding to different genotypes of SNP sites in the test example of the present invention. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] PCR amplification: Using the genomic DNA extracted as above as a template, PCR amplification was performed on the fragment containing the 3605393 SNP molecular marker of chromosome 18 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.

[0024] The PCR reaction system was: 20-50ng / μL genomic DNA 1µL, 10× PCR reaction buffer 0.5µL, 25mmol / LMgCl2 0.4µL, 25μmol / L dNTPs 0.1µL, PCR Primer mix 1µL, 5U / μL Taq DNA polymerase 0.2µL, and deionized water to 10µL; 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, 18_3605393 has a strong signal (P value < 10 -4 ), and the Fst values ​​in the populations with different rib numbers were larger (Table 1). This locus is located within the gene GABRA5. When the genotype of the 18_3605393 locus was C / T, the proportion of individuals with 28 ribs was the highest, at 81.82%; when the genotype was C / C, the proportion was 50.00%, and when the genotype was T / T, the proportion was 13.73% ( Figure 1 The above results show that the molecular marker of GABRA5 gene in Hu sheep is significantly correlated with the rib number trait. According to the actual breeding goals, C / T or C / C 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 proportions corresponding to different genotypes of 18_3605393. The results are shown in the figure. The proportion of individuals with 28 ribs corresponding to the TT genotype is 18.06%, and the proportion of individuals with 28 ribs corresponding to the CC genotype is 50%, which is consistent with the trend of previous results. However, the abundance of the CT genotype in Hu sheep is extremely low, so no individuals with the CT genotype at this site were found in the test set.

[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 SNP molecular marker is located at position 3605393 on chromosome 18 of Hu sheep, the reference genome NCBI accession number is ARS-UI_Ramb_v2.0 (GCF_016772045.1), and its polymorphism is T / C.

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

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 2.

5. Use of the SNP molecular marker of claim 1, the primer combination of claim 2 or the kit of claim 3 in at least one of the following: (1) Phenotypic detection of rib number in Hu sheep; (2) Breeding of Hu sheep with a high number of ribs; (3) Analysis of genetic diversity of Hu sheep.

6. 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 or the kit described in claim 4, 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 T / C or C / C, it is a multiple rib number phenotype.

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 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 T / T genotype, it is a low rib number phenotype.

8. The method according to claim 6, characterized in that The PCR amplification system is: 1µL of 20-50ng / µL genomic DNA, 0.5µL of 10×PCR reaction buffer, 0.4µL of 25mmol / L MgCl2, 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 5µL.

9. The method according to any one of claims 6 or 7, characterized in that: 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 C / T or C / C individuals with multiple rib number traits are selected.

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