A method for detecting SNP of LPL gene of Hu sheep and its application in early screening of meat quality traits

Abstract

The invention discloses a kind of Hu sheep LPL The present invention is a method for detecting gene SNP and its application in early screening of meat quality traits. The present invention extracts DNA from Hu sheep tissues, performs typing using iMLDR technology, calculates allele frequency and genotype frequency, and compares the fatty acid percentage of Hu sheep longissimus dorsi with that of Hu sheep longissimus dorsi. LPL Association analysis of the gene mutation site g.23413T>C revealed the polymorphism of this site and the molecular markers that were significantly correlated with the percentage of beneficial fatty acids, providing a basis for breeding new sheep breeds with excellent meat quality through marker-assisted selection.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetics detection and breeding, and relates to a method for detecting Hu sheep by using DNA mixed pool sequencing. LPL The single nucleotide polymorphism (SNP) site of the gene was identified and the improved multiplex ligation detection reaction (iMLDR) technology was used to perform genotyping on the site and the association analysis was performed using the typing results and the fatty acid percentage. Background Art

[0002] Molecular markers are widely used in animal breeding, such as animal selection through marker-assisted selection. SNPs are widely present in the genome, with one SNP occurring approximately every 300 bp of sequence length. SNP detection technology is used to scan new polymorphic sites and determine the alleles of known polymorphisms in the target sequence. iMLDR technology is a new type of high-throughput SNP genotyping technology that has the advantages of high typing accuracy, low cost, fast speed, and the ability to detect multiple SNP sites simultaneously.

[0003] Meat is one of the basic foods people eat in their daily lives. It contains a large amount of easily digestible protein, vitamins, trace elements and fat, which are necessary for the normal functioning of many metabolic processes in the human body. Meat quality is a complex trait that can be defined by different attributes. Fatty acids are one of the important attributes that affect meat quality. Their content and composition determine the texture characteristics of fat and the oxidative stability of muscle, affecting the flavor, tenderness, shelf life, nutritional value, etc. of meat. At the same time, fatty acids are the main source of energy for the body. The content and composition of fatty acids are affected by breed, age, gender, feeding method, feed type, etc. Fatty acids are divided into saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) according to the degree of saturation of the carbon chain. C6, C8, and C10 in SFA can cause mutton to have a strong smell, and SFA can increase the risk of cardiovascular disease in the human body; MUFA is positively correlated with the aroma of meat and the overall acceptability, and MUFA also has the function of lowering blood sugar, blood lipids, and cholesterol levels; n-6 PUFA and n-3 PUFA in PUFA are essential fatty acids for the human body, and the human body cannot synthesize them by itself and can only obtain them from food. PUFA has a beneficial effect on inflammation, hypertension, ischemic stroke, peripheral arterial disease, and atherosclerosis.

[0004] Lipoprotein lipase LPL ) is a key enzyme in the decomposition of fatty acids. LPL Lipoprotein lipolysis mediated by lipoprotein releases fatty acids, which are transported to the heart through the blood and are the key source of fatty acids needed by the heart. Wei et al. found that LPL The gene expression level was significantly positively correlated with C16:1, C18:1n9c and MUFA in the longissimus dorsi muscle of Duroc × wild boar, and significantly negatively correlated with C16:0, C18:0 and SFA. LPL The correlation between SNPs in the gene and the percentage of fatty acids in the longissimus dorsi muscle tissue of Korean cattle was studied. LPL In gene c.322G>A, the percentages of C18:1n9c, C18:3n3, and MUFA in AA genotype individuals were significantly higher than those in GG genotype, and the percentages of C14:0, C16:0, and SFA in AA genotype individuals were significantly lower than those in GG genotype; in c.329A>T, the percentages of C18:1n9c, C18:3n3, and MUFA in AA genotype individuals were significantly higher than those in TT genotype, and the percentages of C16:0 and SFA were significantly lower than those in TT genotype. Both c.322G>A and c.329A>T had an additive effect on C18:1n9c, SFA, and MUFA. In addition to being an effective molecular marker that can be used to improve the quality of Duroc×Boar and Korean beef, Gao Hongmeng et al. found that LPL The A384T site in the gene has a significant effect on C16:0, C18:0, and C14:1 in the longissimus dorsi muscle of Ujumqin sheep, among which the C16:0, C18:0, and C14:1 of the TT genotype are significantly lower than those of the TA and AA genotypes.

[0005] although LPL The gene is a candidate gene for regulating fatty acid composition, but no relevant LPL The correlation between gene SNP and fatty acid phenotype of Hu sheep was studied, and there is a lack of effective molecular markers for beneficial fatty acid phenotypes of sheep. Summary of the invention

[0006] The object of the present invention is to provide a kind of Hu sheep LPL Detection methods of gene SNPs and their application in early screening of meat quality traits.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for detecting lake sheep LPL The method for detecting single nucleotide polymorphism of a gene comprises the following steps:

[0009] The extracted Hu sheep genomic DNA was used as a template for PCR amplification, and the single nucleotide polymorphism sites contained in the amplified products were genotyped. The single nucleotide polymorphism sites were located at LPL The mutation site on the gene is g.23413T>C (the reference sequence is NC_056055.1).

[0010] Preferably, the primers for PCR amplification are:

[0011] Upstream primer: 5'-TGTTCTGAACTTCTGGTTGACT -3'

[0012] Downstream primer: 5′-CCAATGGCAAGAGGAGAGTAAT -3′.

[0013] Preferably, the reaction system used for PCR amplification includes 1 µL of 30 ng / µL template and 0.5 µL of 10 pmol / µL upstream primer and 10 pmol / µL downstream primer respectively; the reaction program used for PCR amplification is: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 30 s, annealing at 63.8°C for 60 s, extension at 72°C for 90 s, for a total of 34 cycles; and extension at 72°C for another 9 min.

[0014] Preferably, the genotyping uses iMLDR technology.

[0015] Preferably, the genomic DNA is extracted from testicular tissue using the phenol-chloroform method.

[0016] Preferably, the mutation site is determined by DNA pool sequencing.

[0017] A method for detecting lake sheep LPL A kit for detecting single nucleotide polymorphism of a gene, the kit comprising a LPL Primers for a single nucleotide polymorphism site of a gene (such as the upstream primer and the downstream primer), wherein the single nucleotide polymorphism site is located at LPL The mutation site on the gene is g.23413T>C (the reference sequence is NC_056055.1).

[0018] The above detection of Huyang LPL Application of single nucleotide polymorphism method in sheep marker-assisted selection breeding.

[0019] sheep LPL Application of gene mutation site g.23413T>C (reference sequence NC_056055.1) and its detection reagent in sheep marker-assisted selection breeding.

[0020] Preferably, individuals with the TT genotype are superior in meat quality traits (for example, the C18:2n6c, n-6 PUFA, C18:3n6, percentage of PUFA, and P / S of individuals with the TT genotype are higher than those of individuals with the CT and CC genotypes).

[0021] The beneficial effects of the present invention are embodied in:

[0022] The present invention is to LPL A new SNP site on the gene (located at the gene mutation site g.23413T>C) was genotyped, and the significant correlation between this site and the percentage of fatty acids revealed by association analysis revealed that LPL This polymorphic site on the gene can serve as a molecular marker (SNP marker) that affects the composition and content of fatty acids, providing an important scientific basis for molecular breeding and meat quality improvement of sheep (such as Hu sheep) to improve the quality of meat (such as beneficial fatty acids related to nutritional value), thereby accelerating the selection and breeding of new sheep breeds with excellent meat quality.

[0023] Furthermore, the present invention uses high temperature (94°C) denaturation and low temperature (63.8°C) annealing of PCR to unwind the double-stranded genomic DNA extracted from the testicular tissue of Hu sheep, which is also conducive to extension; and 34 cycles can amplify a sufficient amount of DNA for use in DNA pool sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For Huyang LPL Electrophoresis of the PCR amplification products in the region near the gene mutation site g.23413T>C.

[0025] Figure 2 For Huyang LPL Sequencing peak diagram of gene mutation site g.23413T>C. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the protection scope of the present invention.

[0027] 1. Using DNA pool sequencing to obtain Hu sheep LPL Gene SNP site

[0028] S1 Sample selection

[0029] From August 2018 to August 2021, testicular tissue and longissimus dorsi muscle tissue were collected from 1029 (5 batches) 6-month-old healthy Hu sheep under the same breeding conditions from Minqin Defu Agricultural Technology Co., Ltd. (Minqin County, Wuwei City, Gansu Province), and the fatty acid composition and content of the longissimus dorsi muscle tissue were determined.

[0030] S2 DNA pool construction

[0031] a) The phenol-chloroform method was used to extract genomic DNA from the testicular tissue of 1029 Hu sheep individuals. The specific steps are as follows:

[0032] Ⅰ) Take 10 mg of ground testicular tissue powder and place it in a 2 mL centrifuge tube;

[0033] Ⅱ) Add 750 μL tissue lysis buffer and 10 μL 10 mg / mL proteinase K, mix by inversion, and incubate in a 55 ℃ water bath for 15 hours;

[0034] III) After incubation, cool to room temperature, add 800 μL of Tris-saturated phenol, shake on ice for 15 min, centrifuge at 12000 r / min for 10 min, and transfer 800 μL of supernatant to a new 2 mL centrifuge tube;

[0035] IV) Add 500 μL of Tris-saturated phenol and 500 μL of chloroform, shake on ice for 15 min, centrifuge at 12,000 r / min for 10 min, and transfer the supernatant to a new 2 mL centrifuge tube;

[0036] V) Add 1 mL of chloroform, shake on ice for 15 min, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube;

[0037] Ⅵ) Add 1 mL of anhydrous ethanol precooled at -20 °C, shake gently until precipitation occurs, place in a -20 °C refrigerator for 30 min, centrifuge at 12,000 r / min for 10 min, and discard the supernatant;

[0038] VII) Add 1 mL of 70% ethanol, shake gently for 10 min, centrifuge at 12000 r / min for 10 min, and discard the supernatant;

[0039] VIII) Repeat step VII, open the centrifuge tube, place it on a clean bench to dry until the ethanol evaporates, add 200 μL of sterile water, and dissolve the DNA in a 4 ℃ refrigerator overnight.

[0040] Ⅸ) The concentration and purity of DNA were tested by NanoPhotometer® spectrophotometer. An OD260 / 280 value between 1.8 and 2.0 indicated that the DNA was of good quality. DNA that passed the quality test was stored in a -80 ℃ refrigerator.

[0041] b) After 1029 DNA samples passed the quality inspection, 165 DNA samples were randomly selected and uniformly diluted to 30 ng / μL. One mixed pool was constructed for every 15 DNA samples, and a total of 11 DNA mixed pools were constructed.

[0042] S3 PCR amplification

[0043] According to the NCBI published sheep LPL The genome sequence (NC_056055.1) was used as a reference, and Primer-Blast online software was used to design amplification LPL PCR specific primers for the gene exon 10 region and some intronic regions on both sides, the primer sequences are as follows:

[0044] Upstream primer LPL-F: 5'-TGTTCTGAACTTCTGGTTGACT -3'

[0045] Downstream primer LPL-R: 5'-CCAATGGCAAGAGGAGAGTAAT -3'

[0046] Reaction system (25 µL): 12.5 µL Mix, 10.5 µL ddH2O, 0.5 µL each of upstream primer (10 pmol / µL) and downstream primer (10 pmol / µL), and 1 µL template (30 ng / μL DNA pool).

[0047] The reaction procedure was as follows: pre-denaturation at 95 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 63.8 °C for 60 s, and extension at 72 °C for 90 s, for a total of 34 cycles; and extension at 72 °C for another 9 min.

[0048] The PCR products were subjected to electrophoresis, and the results were as follows: Figure 1 As shown, except for the Marker lane, all the DNA mixed pool lanes on the right side amplified the target fragment (size is 624 bp).

[0049] S4 PCR product sequencing and mutation site genotyping

[0050] The PCR products amplified from the DNA pool in S3 were sequenced bidirectionally according to Figure 2 The sequencing peak graph shown in the figure found a LPL The mutation site on the gene is g.23413T>C (the reference sequence is NC_056055.1);

[0051] The 1029 DNA samples that passed the quality inspection (uniformly diluted to 30 ng / μL) were divided into 200 μL centrifuge tubes, and the location information of the pending SNP site (i.e. g.23413T>C) on the genome sequence was provided. The company used iMLDR technology to perform genotyping on the 1029 DNA samples.

[0052] S5 LPL Frequency statistics of genotyping loci.

[0053] The formula for calculating allele frequency is as follows:

[0054] PF=(2NFF+NFf1+NFf2+NFf3+NFf4+……+NFfn) / 2N

[0055] In the formula, PF represents the frequency of allele F, NFF represents the number of individuals with genotype FF in the population, NFfi represents the number of individuals with genotype Ffi in the population, and f1, f2, ..., fn are n different multiple alleles of allele F respectively;

[0056] The formula for calculating genotype frequency is as follows:

[0057] PFF=NFF / N

[0058] In the formula, PFF represents the FF genotype frequency of a certain site, NFF represents the number of individuals with the FF genotype in the population, and N is the total number of individuals in the test population;

[0059] The calculation results of allele and genotype frequencies of 1029 individuals are shown in Table 1:

[0060] Table 1. Hu sheep LPL Frequency statistics of gene mutation site g.23413T>C

[0061]

[0062] Note: HWB stands for Hardy-Weinberg equilibrium; He is the expected heterozygosity; Ho is the observed heterozygosity; PIC is the polymorphic information content.

[0063] As can be seen from Table 1, C is the dominant allele of the mutation site g.23413T>C, and the allele frequency is 75.61%. And combined with genetic indicators, it can be determined that the mutation site is a SNP site.

[0064] 2. Huyang LPL Association analysis between gene SNP sites and fatty acid percentage

[0065] Genotype data: The above typing results obtained using iMLDR technology

[0066] Meat quality data: fatty acid percentage of longissimus dorsi muscle of Hu sheep (fatty acid percentage refers to the percentage of a certain fatty acid in the total fatty acid content)

[0067] R-4.0.5 software was used to analyze the correlation between gene loci and fatty acid percentages, and the analysis model was as follows:

[0068] Y=µ+G+T+e

[0069] Where Y is the percentage of fatty acids; μ is the overall mean; G is the fixed effect corresponding to the SNP; T is the batch effect; and e is the random error.

[0070] Determined by analyzing data LPL The influence of the SNP sites of the gene on the percentage of fatty acids was studied to find potential molecular markers that affect the percentage of fatty acids. The results are shown in Table 2:

[0071] Table 2. Hu sheep LPL Association analysis between gene g. 23413T>C and fatty acid percentage of longissimus dorsi muscle (Mean ± SD)

[0072]

[0073]

[0074] Note: Different capital letters in the same row indicate significant differences (P<0.05), and different lowercase letters in the same row indicate extremely significant differences (P<0.01).

[0075] The results showed that the g. 23413T>C site had a very significant effect on C18:2n6c, C20:1, and n-6 ​​PUFA, among which the percentages of C18:2n6c, C20:1, and n-6 ​​PUFA in individuals with TT genotype were significantly higher than those in individuals with CT genotype; the g. 23413T>C site had a significant effect on C18:3n6, PUFA, and P / S, among which the percentages of C18:3n6, PUFA, and P / S in individuals with TT genotype were significantly higher than those in individuals with CT genotype.

[0076] Since the beneficial fatty acids (C18:2n6c, n-6 PUFA, C18:3n6, PUFA) and P / S of individuals with TT genotype are significantly higher than those with CT genotype, and PUFA and n-6 ​​PUFA have the functions of regulating blood pressure, blood lipids, and preventing cardiovascular diseases, the closer the P / S is to or exceeds 0.4, the higher the nutritional value of mutton. Therefore, by selecting and establishing a group with TT genotype (as a molecular marker), we can conduct early screening of Hu sheep meat quality traits (fatty acid composition and content) and accelerate the breeding of new sheep strains with excellent meat quality. <110> Gansu Runmu Bioengineering Co., Ltd. <120> A method for detecting SNP of LPL gene of Hu sheep and its application in early screening of meat quality traits <160> 2 <210> 1 <211> twenty two <212> DNA <213> LPL-F <400> 1 tgttctgaac ttctggttga ct 22 <210> 2 <211> twenty two <212> DNA <213> LPL-R <400> 2 ccaatggcaa gaggagagta at 22

Claims

1. Detection of Huyang LPL The application of the gene single nucleotide polymorphism method in the assisted selection breeding of Hu sheep meat quality traits is characterized by: The detection of Huyang LPL The method for detecting single nucleotide polymorphism of a gene comprises the following steps: The extracted Hu sheep genomic DNA was used as a template for PCR amplification, and the single nucleotide polymorphism sites contained in the amplified products were genotyped. The single nucleotide polymorphism sites were located at LPL The mutation site on the gene is g.23413T>C, and the reference sequence is NC_056055.1; among them, the Hu sheep individuals with TT genotype are superior to the Hu sheep individuals with CT genotype in meat quality traits, and the meat quality traits are C18:2n6c, C20:1, n-6 PUFA, C18:3n6, PUFA content percentage and P / S.

2. The use according to claim 1, characterized in that: The primers for the PCR amplification are: Upstream primer: 5'-TGTTCTGAACTTCTGGTTGACT -3' Downstream primer: 5′-CCAATGGCAAGAGGAGAGTAAT -3′.

3. The use according to claim 1 or 2, characterized in that: The PCR amplification reaction system includes 1 µL of 30 ng / µL template and 0.5 µL of 10 pmol / µL upstream and downstream primers respectively; the PCR amplification reaction program is: 95°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 63.8°C annealing for 60 s, 72°C extension for 90 s, for a total of 34 cycles; 72°C extension for another 9 min.

4. The use according to claim 1, characterized in that: The genotyping was performed using iMLDR technology.

5. The use according to claim 1, characterized in that: The genomic DNA was extracted using the phenol-chloroform method.

6. The use according to claim 1, characterized in that: The mutation site was determined by DNA pool sequencing.

7. Hu Sheep LPL The application of the gene mutation site g.23413T>C detection reagent in the assisted selection breeding of Hu sheep meat quality traits is characterized by: The meat quality traits of Hu sheep individuals with TT genotype are superior to those of Hu sheep individuals with CT genotype, wherein the meat quality traits are C18:2n6c, C20:1, n-6 PUFA, C18:3n6, PUFA content percentage and P / S. LPL The reference sequence of the gene is NC_056055.1.

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