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 3605393 on chromosome 18 of Hu sheep, and its polymorphism is T / C. This invention also provides a primer set capable of amplifying the above-mentioned SNP molecular marker for detecting 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 GABRA5 gene, located on chromosome 18 of the Hu sheep, contains 12 exons. GABRA5 is a major inhibitory neurotransmitter in the mammalian brain, acting on GABA-A receptors, ligand-gated chloride channels. From a developmental biology perspective, skeletal development, including rib development, is regulated by multiple signaling pathways. Complex interactions exist between neural and developmental signaling pathways. Although GABRA5 primarily functions in the nervous system, the nervous system can influence the development of surrounding tissues by secreting neurotransmitters or neuropeptides. However, no molecular markers for economically relevant traits in Hu sheep associated with this gene have yet been identified 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 in the upstream regulatory region of the GABRA5 gene on chromosome 18 of Hu sheep and is related to the number of ribs in Hu sheep. It can be used to breed Hu sheep with a high number of ribs and improve economic benefits.

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

[0006] The present invention also provides an SNP molecular marker related to the number of ribs in Hu sheep, wherein the SNP molecular marker is located at position 51 of the nucleotide sequence as 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 including 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)

[0009] Downstream primer R: 5'-GACCCATAACCATGTGGGATTTG-3' (SEQ ID NO.2)

[0010] The present invention also provides a kit containing the above-described primer combination.

[0011] Secondly, the present invention provides the application of the above-mentioned SNP molecular markers, primer combinations, and kits in at least one of the following:

[0012] (1) Phenotypic detection of the number of ribs in Hu sheep;

[0013] (2) Breeding of multi-ribbed Hu sheep;

[0014] (3) Analysis of genetic diversity of Hu sheep.

[0015] Thirdly, the present invention provides a method for determining the number of ribs in Hu sheep, which involves PCR amplifying the genomic DNA of the Hu sheep to be tested using the primer combination or kit described above, and detecting whether the amplification product contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at 51 bp is T / C or C / C, it is a multiple rib number phenotype.

[0016] This invention also provides a method for determining the rib count phenotype of Hu sheep, which involves PCR amplifying the genomic DNA of the Hu sheep to be tested using the primer combination or kit described above, and detecting whether the amplification product contains the nucleotide sequence shown in SEQ ID NO 3. When the SNP at 51 bp is T / T, it is a low rib count phenotype.

[0017] Most Hu sheep have 26 ribs, with 28 ribs observed when the trait of multiple ribs is present. This trait occurs in approximately 15% of the entire Hu sheep population. The highest proportion of individuals with 28 ribs (81.82%) is observed when the genotype at the aforementioned SNP locus is C / T; the proportion is 50.00% when the genotype is C / C; and 13.73% when the genotype is T / T. However, individuals with the C / T genotype at the aforementioned SNP locus constitute a very small percentage of the Hu sheep population. Therefore, using the C / C genotype to determine the multiple rib trait in Hu sheep is more meaningful.

[0018] Preferably, the PCR amplification system described above 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.

[0019] Preferably, the PCR amplification program described above 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; and incubation at 25℃.

[0020] Preferably, the detection of the nucleotide sequence of the amplification product in the above method is performed using Sanger sequencing.

[0021] Fourthly, the present invention provides a breeding method for multi-rib number Hu sheep, characterized in that the SNP phenotype of Hu sheep is detected by the method described in any one of claims 6-8, and C / T heterozygous individuals with the multi-rib number trait are selected.

[0022] 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

[0023] 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.

[0024] Figure 1 The proportion of 28-rib individuals in Hu sheep corresponding to different genotypes at SNP loci in Example 2 of this invention.

[0025] Figure 2 This represents the proportion of Hu sheep individuals with 28 ribs corresponding to different genotypes at SNP loci in the experimental examples of this invention. Detailed Implementation

[0026] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0027] Example 1: Genomic DNA extraction, PCR amplification and sequencing

[0028] 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, the red blood cell lysate (TIANGEN Biochemical Technology RT122-02) was used to lyse and remove the red blood cells without DNA, and the nuclear lysate (TIANGEN Biochemical Technology DP304-03) was used to lyse the cells to release genomic DNA. Then, the protein precipitation solution was used to selectively precipitate and remove proteins. Finally, the pure genomic DNA was precipitated with isopropanol and redissolved in the DNA dissolution solution.

[0029] PCR amplification: Using the genomic DNA extracted above as a template, the fragment containing the SNP molecular marker at the 3,605,393rd position on chromosome 18 of Hu sheep was amplified by PCR. The nucleotide sequence of the upstream primer F used for amplification was as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer R was as shown in SEQ ID NO.2.

[0030] The PCR reaction system was: 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;

[0031] 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 left standing at 25°C.

[0032] The PCR products of each sample were subjected to Sanger sequencing.

[0033] Example 2: Analysis of sequencing results

[0034] The number of ribs of 245 Hu sheep was counted. Among them, 203 individuals had 26 ribs and 42 individuals had 28 ribs.

[0035] A Lasso regression model was built using the glmnet R package. The model after logit transformation is as follows:

[0036]

[0037] 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.

[0038] 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).

[0039] Table 1. Features of the 12 candidate sites retained by the Lasso and Random Forest models.

[0040]

[0041] 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.

[0042] As shown in the table above, locus 18_3605393 exhibited a strong signal (P-value < 10) in GWAS analysis (calculated using GCTA software). -4 Furthermore, the Fst value was larger in populations with different numbers of ribs (Table 1). This locus is located within the GABRA5 gene range. When the genotype at locus 18_3605393 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 indicate that the GABRA5 gene molecular marker in Hu sheep is significantly correlated with the rib number trait. Individuals with C / T or C / C genotypes can be selected to increase the rib number and economic value, depending on actual breeding goals.

[0043] Experimental Example: Verification Experiment on the Number of Ribs in Hu Sheep

[0044] 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 proportions corresponding to different genotypes of 18_3605393 were calculated. The results are shown in the figure. The proportion of individuals with 28 ribs corresponding to the TT genotype was 18.06%, and the proportion of individuals with 28 ribs corresponding to the CC genotype was 50%, consistent with previous results. However, the CT genotype is extremely rare in Hu sheep, therefore no individuals with the CT genotype at this locus were found in the test set.

[0045] 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. The application of SNP molecular markers, primer combinations, or kits in at least one of the following: (1) Phenotypic detection of the number of ribs in Hu sheep; (2) Breeding of multi-ribbed Hu sheep; The SNP molecular marker is located at position 3605393 on chromosome 18 of the Hu sheep genome, with the NCBI accession number ARS-UI_Ramb_v2.0 (GCF_016772045.1) and a polymorphism of T / C; or The SNP molecular marker is located at position 51 of the nucleotide sequence as shown in SEQ ID NO 3, and its polymorphism is T / C. 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.

2. A method for determining the rib count phenotype of Hu sheep, characterized in that, The genomic DNA of the Hu sheep to be tested was amplified by PCR using primer combination. The amplification product was tested to see if it contained the nucleotide sequence shown in SEQ ID NO 3. When the SNP at 51 bp of SEQ ID NO 3 is T / C or C / C, it is a multi-rib number phenotype. 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.

3. A method for determining the rib count phenotype of Hu sheep, characterized in that, The genomic DNA of the Hu sheep to be tested was amplified by PCR using primer combination, and the amplification product was detected to contain the nucleotide sequence shown in SEQ ID NO 3. When the SNP at 51 bp of SEQ ID NO 3 is T / T genotype, it is a low rib number phenotype. 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.

5. The method according to any one of claims 3 or 4, characterized in that, The PCR amplification program was 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; and incubation at 25℃.

6. A breeding method for multi-ribbed Hu sheep, characterized in that, Using the method described in any one of claims 3-5, the SNP phenotype of the Hu sheep is detected, and individuals with the multiple rib number trait (C / T or C / C) are selected.

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