Method for rapidly detecting FecB mutation site of sheep BMPR1B gene by using KASP

Abstract

The invention discloses a method for rapidly detecting FecB mutation of a sheep BMPR1B gene by using KASP. According to the present invention, sheep genome DNA is adopted as a template, the sheep BMPR1B gene FecB mutation 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 lambing number trait, such that the important effect is provided in the early selection of the sheep lambing number trait. According to the method, the FecB mutation site genotype related to the sheep lambing number character is rapidly and accurately detected, so that a high-fecundity sheep genetic resource group can be established in an auxiliary manner, and the genetic progress of the sheep lambing number character is improved.

Description

Technical Field

[0001] The invention relates to a method for rapidly detecting FecB mutation sites of sheep BMPR1B gene by using KASP, and belongs to the technical field of sheep SNP molecular markers. Background Art

[0002] The trait of lambing number is an important economic trait in the actual production of mutton sheep. Ewes with multiple lambs can provide more lambs for mutton sheep farms, thereby increasing the number of mutton sheep on hand and the number of lambs slaughtered, and improving the overall production capacity of mutton. This is of great significance for meeting the market demand for mutton products and improving the economic benefits of farms. There are differences in the reproductive capacity of different breeds of mutton sheep. Increasing the number of lambs born by ewes through genetic improvement is one of the focuses of breeding work. By selecting ewes with high lambing performance for breeding, mutton sheep breeds with high lamb production can be gradually cultivated, providing strong support for the sustainable development of the mutton sheep industry.

[0003] It is reported that the heritability of lambing number is only 0.1, but the heritability of ovulation number, which affects lambing number, can reach more than 0.45, which is a medium heritability trait. Therefore, there are often major regulatory genes for lambing number and ovulation number traits in ewes. BMPR1B Since the discovery of the FecB mutation site, it has been regarded as the most effective mutation site for molecular marker-assisted selection of sheep lambing number and ovulation number. This site shows an additive effect in the selection of sheep ovulation number and a partial dominant effect in the selection of lambing number.

[0004] Currently, it has been reported BMPR1B The PCR-SSCP, PCR-RFLP, SNaPshot and TaqMan probe methods for typing detection of FecB mutation sites in the gene have problems such as long detection time, high detection cost or low detection throughput. BMPR1B The method of gene FecB mutation can efficiently detect the mutation of the molecular marker site of the sheep lamb number trait; and in the future, it can be simultaneously detected with other important trait effect sites to achieve joint detection of multiple sites and breeding screening of multiple excellent traits. Summary of the invention

[0005] The present invention discloses a method for rapidly detecting FecB mutation sites of sheep BMPR1B gene by using KASP. Through the specific KASP primers disclosed in the present invention, the genotype of FecB mutation sites related to the sheep lamb number trait can be rapidly and accurately detected, thereby assisting in establishing a high-fertility sheep genetic resource population, thereby improving the genetic progress of the sheep lamb number trait.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A method for rapidly detecting FecB mutation sites of sheep BMPR1B gene using KASP, characterized in that sheep genomic DNA is used as a template, a BMPR1B gene partial fragment containing FecB mutation at Chr6: g. 30050621 site is amplified by specific KASP primers, and then the FecB mutation genotype at Chr6: g. 30050621 site is identified, wherein Chr6: g. 30050621 site is located in the sheep reference sequence ARS-UI_Ramb_v3.0, Chr6: g. 30050621 A>G variation site. The specific KASP primers, site A typing primer FAM, site G typing primer VIC sequence and universal primer sequence are as follows: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctTGGTTCCGAGAGACAGAAATATATCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattTGGTTCCGAGAGACAGAAATATATCG-3'; Universal Primer: 5'-CCAAGATGTTTTCATGCCTCATCA-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 genotype determination feature of the method for rapidly detecting FecB mutation of sheep BMPR1B gene using KASP of the present invention is that the three genotypes of AA, AG and GG of FecB mutation at Chr6: g.30050621 site are typed 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 FecB mutation of sheep BMPR1B gene using KASP in sheep molecular marker assisted selection breeding, and the application refers to: efficiently detecting the mutation of the molecular marker site of sheep lamb number trait. The FecB mutation at the g. 30050621 site of the BMPR1B gene is an A>G variation site located in the sheep reference sequence ARS-UI_Ramb_v3.0, Chr6: g. 30050621. The site is used in molecular marker assisted selection breeding for early selection of sheep lamb number trait.

[0011] The method of the present invention for rapidly detecting FecB mutation of sheep BMPR1B gene by using KASP is that the AG and GG genotypes of the A>G mutation site located in the sheep reference sequence ARS-UI_Ramb_v3.0, Chr6: g. 30050621 can be used as molecular markers for improving the lamb number trait of sheep.

[0012] Compared with the prior art, the method disclosed in the present invention for rapidly detecting FecB mutation sites of sheep BMPR1B gene using KASP has the following positive effects: (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.

[0013] (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

[0014] Figure 1 This is the Sanger sequencing map of sheep Chr6: g. 30050621 site; Figure 2 This is the KASP typing map of sheep Chr6: g. 30050621 site. DETAILED DESCRIPTION

[0015] 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

[0016] 1. A total of 247 multiparous Aodu Lake sheep (all second-born) were selected as the experimental subjects (all from Tianjin Aoqun Animal Husbandry Co., Ltd.), and the number of lambs born was recorded under the same breeding and feeding management conditions.

[0017] 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'-gaaggtgaccaagttcatgctTGGTTCCGAGAGACAGAAATATATCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattTGGTTCCGAGAGACAGAAATATATCG-3'; Universal Primer: 5'-CCAAGATGTTTTCATGCCTCATCA-3'.

[0018] 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 Chr6: g. 30050621 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 Chr6: g. 30050621 site can be determined as AA, AG and GG types, such as Figure 1 As shown; 6. The results of statistics on the number of lambs born by Aodu Lake sheep ewes (mean ± standard error) show that: when the genotype of Chr6: g.30050621 locus is GG, the average number of lambs born is 2.43±0.79; when the genotype of Chr6: g. 30050621 locus is AG, the average number of lambs born is 1.97±0.71; when the genotype of Chr6: g. 30050621 locus is AA, the average number of lambs born is 1.33±0.52.

[0019] The chi-square fitness test method was used to analyze the significance level of differences among genotype groups. It was found that the average number of lambs born by multiparous ewes of GG type Aodu Lake sheep was significantly higher than that of AG and AA type ewes (P<0.05), while the average number of lambs born by multiparous ewes of AG type Aodu Lake sheep was significantly higher than that of AA type ewes (P<0.05) (Table 1).

[0020] Table 1 Statistical comparison of the number of lambs born by each genotype of Aodu Lake multiparous ewes Note: Different lowercase letters in the same column represent significant differences (P<0.05).

[0021] According to the results of the correlation analysis between the number of lambs born of Audu Lake sheep ewes and the genotype, the number of lambs born after adult breeding and production can be determined by blood sampling and DNA extraction from newly born Audu Lake sheep using the patented KASP rapid genotyping technology. In order to breed Audu Lake sheep with a high number of lambs, the genotype offspring of the G mutant site at the Chr6: g.30050621 site in the flock should be continuously selected and retained, and the genotype offspring of the A wild-type site at the Chr6: g. 30050621 site in the flock should be continuously eliminated. Example

[0022] 1. For Aodu Lake sheep, Donghu sheep, Australian white sheep and small-tailed Han sheep, collect 2 mL of anticoagulated blood from the jugular vein, and use the phenol imitation method to extract the target individual DNA; 2. Use specific KASP primers and universal primers. The specific sequence information is as follows: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctTGGTTCCGAGAGACAGAAATATATCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattTGGTTCCGAGAGACAGAAATATATCG-3'; Universal Primer: 5'-CCAAGATGTTTTCATGCCTCATCA-3'.

[0023] 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 Chr6: g. 30050621 site KASP typing results are as follows: Figure 2As shown, the statistical genotyping results are shown in Table 2;

[0024] 4. The KASP typing results show that the frequency of the mutant G allele at Chr6: g. 30050621 in Aodu Lake sheep, Donghu sheep and Small Tail Han sheep is higher than 50%, while the frequency of the mutant G allele at Chr6: g. 30050621 in Australian White sheep is only 0.8%.

[0025] Therefore, the lambing rate of sheep breeds such as Australian Lake Sheep, Donghu Sheep and Small Tail Han Sheep can be further improved by selecting the G mutation site at the Chr6: g. 30050621 site within the group; and Australian White Sheep can introduce the G mutation site at the Chr6: g. 30050621 site through hybridization and other means to increase its lambing level.

Claims

1. A specific primer for rapid detection of FecB mutation site of sheep BMPR1B gene using KASP, characterized in that: The primer KASP primer is: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctTGGTTCCGAGAGACAGAAATATATCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattTGGTTCCGAGAGACAGAAATATATCG-3'; Universal Primer: 5'-CCAAGATGTTTTCATGCCTCATCA-3'.

2. A method for rapidly detecting FecB mutation sites in sheep BMPR1B gene using the KASP primers described in claim 1, characterized in that: Using sheep genomic DNA as a template, a partial fragment of the BMPR1B gene containing the FecB mutation at the Chr6: g. 30050621 site was amplified by specific KASP primers, and then the FecB mutant genotype at the Chr6: g. 30050621 site was identified, wherein the Chr6: g. 30050621 site was located at the A>G variation site of the sheep reference sequence ARS-UI_Ramb_v3.0, Chr6: g. 30050621; the primers KASP primers were: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctTGGTTCCGAGAGACAGAAATATATCA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattTGGTTCCGAGAGACAGAAATATATCG-3'; Universal Primer: 5'-CCAAGATGTTTTCATGCCTCATCA-3'; The three genotypes of FecB mutation at Chr6: g. 30050621 site, AA, AG, and GG, were typed according to the intensity of different types of fluorescence that matched the linker sequence carried by the KASP primer.

3. Use of the method for rapidly detecting FecB mutation sites of sheep BMPR1B gene using KASP as described in claim 2 in sheep molecular marker-assisted selection breeding.

4. The use according to claim 3, characterized in that: The FecB mutation at the g. 30050621 site of the BMPR1B gene is located at the A>G variation site of the sheep reference sequence ARS-UI_Ramb_v3.0, Chr6: g. 30050621; specifically, the AG and GG genotypes of the A>G variation site of Chr6: g. 30050621 are used as molecular markers for improving the lamb number trait of sheep.

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