Method for rapidly detecting single nucleotide polymorphism of sheep Chr11: g.42139093 site by using KASP

Abstract

The invention discloses a method for rapidly detecting single nucleotide polymorphism (SNP) of a sheep Chr11: g.42139093 site by using KASP (Kinase Assay Specific Protein). According to the present invention, the sheep genome DNA is adopted as the template, the single nucleotide polymorphism of the sheep Chr11: g.42139093 site is subjected to rapid and accurate typing through the published specific KASP primer sequence by using the KASP detection method, and the different genotypes of the SNP site are significantly related to the sheep lactation performance related traits, such that the important effect is provided in the early selection of the sheep lactation traits. According to the present invention, the mutation genotype of the Chr11: g.42139093 site related to the sheep lactation character can be rapidly and accurately detected, such that the establishment of the high lactation ability sheep genetic resource group can be assisted so as to improve the genetic progress of the sheep lactation character.

Description

Technical Field

[0001] The present invention relates to a method for rapidly detecting single nucleotide polymorphism at Chr11: g. 42139093 site of sheep by utilizing KASP, and belongs to the field of sheep SNP molecular marker detection. Background Art

[0002] The lactation traits of sheep are also important economic traits that need to be considered in actual production. These traits are directly related to the output and quality of milk, specifically involving traits such as total milk production, milk protein rate, lactose rate, milk fat rate, and somatic cell count. The milk production of lactating ewes directly affects whether the lambs can obtain sufficient colostrum and subsequent nutritional support. Ewes with high milk production can provide lambs with more sufficient milk, which helps the growth and development of lambs and improves their immunity. Insufficient milk production may lead to malnutrition in lambs, which in turn affects their survival rate and health.

[0003] Genetic factors have a huge impact on lactation traits. For example, as a dairy sheep breed, the East Friesian sheep can produce 2kg of milk per day, which is about 4 times the daily milk production of Hu sheep. There are many genes related to sheep lactation that have been reported, and these genes play an important role in the lactation process of sheep. Among them, prolactin is an important lactogenic hormone that can promote mammary gland development and milk secretion. Therefore, previous studies have reported that some related mutation sites of prolactin genes have a certain correlation with their lactation traits.

[0004] Earlier, the STAT5a gene at the Chr11: g. 42139093 site was also reported to have a regulatory relationship with lactation traits in dairy cows. A Sanger sequencing detection method has been reported for the A>G variant site at the Chr11: g. 42139093 site related to the lactation traits of sheep. This patent discloses a specific primer sequence for a KASP rapid detection method for this site, which can efficiently detect the mutation of the A>G variant site at the Chr11: g. 42139093 site in sheep; and in the future, it can be detected simultaneously with other important trait effect sites to achieve joint detection of multiple sites and breeding screening for multiple excellent traits. Summary of the invention

[0005] The present invention discloses a method for rapidly detecting single nucleotide polymorphism at Chr11: g. 42139093 site of sheep using KASP. By using the specific KASP primers disclosed in the present invention, the mutation of A>G variant site at Chr11: g. 42139093 site related to the lactation trait of sheep can be rapidly and accurately detected, thereby assisting in establishing a genetic resource group of sheep with high lactation ability, thereby improving the genetic progress of the lactation trait of sheep.

[0006] To achieve the above object, the present invention adopts the following technical solution: The present invention discloses a method for rapidly detecting single nucleotide polymorphism of Chr11: g. 42139093 site of sheep by using KASP, which is characterized by taking sheep genomic DNA as template, amplifying a partial fragment containing the A>G variation site of Chr11: g. 42139093 by using specific KASP primers, and then identifying the genotype of the A>G variation site of Chr11: g. 42139093, wherein the sheep reference genome version of the Chr11: g. 42139093 site is ARS-UI_Ramb_v3.0.

[0007] The specific KASP primer, site A typing primer FAM, site G typing primer VIC sequence and universal primer sequence are as follows: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctCCTCCCACTCTTTGTCTTGCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattCCTCCCACTCTTTGTCTTGCG-3'; Universal Primer: 5'- GGGACACCCTAATAGTGAAATCCT-3'.

[0008] The reaction program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61°C for 1 min for a total of 10 cycles, with the annealing temperature decreasing by 0.6°C after each cycle; denaturation at 94°C for 20 s, annealing at 55°C for 1 min for a total of 26 cycles.

[0009] The present invention types the three genotypes of AA, AG and GG at the Chr11:g. 42139093 site according to the intensity of different types of fluorescence matching the linker sequence carried by the KASP primer.

[0010] The present invention further discloses the application of a method for rapidly detecting single nucleotide polymorphism of Chr11: g. 42139093 site of sheep using KASP in sheep molecular marker assisted selection breeding, characterized in that: the reference genome version of the Chr11: g.42139093 site is ARS-UI_Ramb_v3.0. According to the detection method, the AG and GG genotypes of the A>G variant site of the Chr11: g.42139093 site are used as molecular markers for improving the lactation traits of sheep.

[0011] The method disclosed in the present invention for rapid detection of single nucleotide polymorphism at Chr11: g.42139093 site in sheep using KASP has the following positive effects compared with the prior art: (1) More efficient detection accuracy, site detection accuracy> 99.8%; (2) It has lower costs, requiring only primer synthesis costs, without the high cost of site-labeling probes, and has lower requirements for DNA sample volume.

[0012] (3) It has strong detection flexibility and can be detected together with multiple other mutation sites to meet the needs of low, medium, and high throughput individual detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the Sanger sequencing map of sheep Chr11: g. 42139093 site; Figure 2 This is the KASP genotyping map of sheep Chr11: g. 42139093 locus. DETAILED DESCRIPTION

[0014] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available. Example

[0015] 1. A total of 136 Huyang reserve ewes were used as test subjects (all from Tianjin Tengyuan Animal Husbandry Co., Ltd.). Their lactation-related traits were measured and recorded under uniform breeding and postpartum feeding management conditions.

[0016] 2. Collect 2 mL of anticoagulated blood from the jugular vein of all test sheep and extract the target individual DNA using the phenol imitation method; 3. Use specific KASP primers and universal primers. The specific sequence information is as follows: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctCCTCCCACTCTTTGTCTTGCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattCCTCCCACTCTTTGTCTTGCG-3'; Universal Primer: 5'- GGGACACCCTAATAGTGAAATCCT-3'.

[0017] 4. Using the extracted DNA as a template, the specific KASP primers and universal primers in step 3 were used for typing amplification detection. The amplification program was: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃ annealing for 1 min for a total of 10 cycles, and the annealing temperature was reduced by 0.6℃ after each cycle; 94℃ denaturation for 20 s, 55℃ annealing for 1 min, for a total of 26 cycles. The final sheep Chr11: g. 42139093 site KASP typing results were obtained; 5. Perform Sanger sequencing on the amplified product in step 4 and analyze the sequencing results using ChromasPro software; based on the sequencing peak graph, the genotype of the sheep Chr11: g. 42139093 site can be determined as AA, AG and GG types, such as Figure 1 As shown; 6. The results of the measurement of various lactation traits of primiparous Hu sheep ewes (mean ± standard error) showed that: when the Chr11: g. 42139093 locus genotype was GG or AG, the total milk production from 7 to 56 days after delivery was significantly higher than that of the AA genotype, and the average somatic cell count was significantly lower than that of the AA genotype (P<0.05); when the Chr11: g. 42139093 locus genotype was GG, the lactose rate index was significantly higher than that of the AA genotype (P<0.05). (Table 1).

[0018]

[0019] According to the analysis results of lactation-related traits of primiparous Hu sheep, the lactation performance of newly born Hu sheep can be determined by blood sampling and DNA extraction, and then the patented KASP rapid genotyping technology can be used to determine the lactation performance of adult breeding and production. In order to breed Hu sheep with good lactation performance, the genotype offspring of the G mutant site at the Chr11: g. 42139093 site in the flock should be continuously selected, and the genotype offspring of the A wild-type site at the Chr11: g. 42139093 site in the flock should be continuously eliminated. Example

[0020] 1. Take Donghu hybrid sheep, Dorper sheep, Aoduhu hybrid sheep, Small-tailed Han sheep and Hu sheep as test subjects, collect 2mL of anticoagulated blood from their jugular vein, and use phenol imitation method to extract DNA of target individuals; 2. Use specific KASP primers and universal primers. The specific sequence information is as follows: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctCCTCCCACTCTTTGTCTTGCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattCCTCCCACTCTTTGTCTTGCG-3'; Universal Primer: 5'- GGGACACCCTAATAGTGAAATCCT-3'.

[0021] 3. Using the extracted DNA as a template, the specific KASP primers and universal primers in step 3 were used for typing amplification detection. The amplification program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃ annealing for 1 min for a total of 10 cycles, with the annealing temperature reduced by 0.6℃ after each cycle; 94℃ denaturation for 20 s, 55℃ annealing for 1 min for a total of 26 cycles. The final sheep Chr11: g. 42139093 site KASP typing diagram is shown below. Figure 1 The statistical genotyping results are shown in Table 2;

[0022] The results of KASP typing showed that the three genotypes of Chr11: g. 42139093, AA, AG and GG, were distributed in Donghu hybrid sheep, Dorper sheep, Aoduhu hybrid sheep, Small-tailed Han sheep and Hu sheep.

[0023] Therefore, the above-mentioned sheep breeds can further improve the lactation performance of ewes by selecting the G mutation site at Chr11: g. 42139093 within the group.

Claims

1. A specific primer for rapid detection of single nucleotide at position 42139093 of Chr11 in sheep Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctCCTCCCACTCTTTGTCTTGCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattCCTCCCACTCTTTGTCTTGCG-3'; Universal Primer: 5'- GGGACACCCTAATAGTGAAATCCT-3'.

2. A method for rapidly detecting single nucleotide polymorphism at Chr11: g.42139093 site in sheep using the specific primers described in claim 1, characterized in that: Using sheep genomic DNA as a template, a partial fragment containing the A>G variant site of Chr11: g. 42139093 was amplified by specific KASP primers, and then the genotype of the A>G variant site of Chr11: g. 42139093 was identified, wherein the sheep reference genome version of Chr11: g. 42139093 site was ARS-UI_Ramb_v3.0; the KASP primers were: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctCCTCCCACTCTTTGTCTTGCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattCCTCCCACTCTTTGTCTTGCG-3'; Universal Primer: 5'- GGGACACCCTAATAGTGAAATCCT-3'; The identification of the genotype includes: typing the three genotypes of AA, AG, and GG at the Chr11: g. 42139093 site according to the intensity of different types of fluorescence that match the linker sequence carried by the KASP primer.

3. Application of the method for rapid detection of single nucleotide polymorphism at Chr11: g.42139093 site in sheep using KASP as described in claim 2 in sheep molecular marker-assisted selection breeding.

4. The use described in claim 3, characterized in that the AG and GG genotypes of the A>G mutation site at Chr11: g. 42139093 can be used as molecular markers for improving sheep lactation traits.

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