A method for detecting SNP of PLIN2 gene in Hu sheep and its application in early screening of meat quality traits

Abstract

The present invention discloses a method for detecting SNP of PLIN2 gene of Hu sheep and its application in early screening of meat quality traits. The present invention extracts Hu sheep tissue DNA, performs typing by iMLDR technology, calculates allele frequency and genotype frequency, and performs association analysis between the fatty acid percentage of Hu sheep longissimus dorsi muscle and the mutation site of PLIN2 gene g.287G>A, reveals the polymorphism of the site and the molecular markers that are significantly correlated with the percentage of beneficial fatty acids, and provides a basis for breeding new sheep strains 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 specifically relates to Hu sheep detected by DNA mixed pool sequencing. PLIN2 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] Single nucleotide polymorphism (SNP) is the most common genetic variation in organisms and an important pathway in the process of biological evolution. SNPs occur more frequently in intronic regions. SNPs in intronic regions do not change the amino acid sequence of gene-encoded proteins, but may lead to alternative splicing or interact with genetic elements, thereby affecting the transcription and translation efficiency of genes and causing phenotype changes. Screening for SNPs associated with important traits plays an important role in genetic improvement. iMLDR technology is a new type of high-throughput SNP genotyping technology, which has the advantages of high typing accuracy, low cost, fast speed, and the ability to detect multiple SNP sites simultaneously.

[0003] Fatty acids are one of the most important properties that affect meat quality. Their content and composition determine the texture characteristics of fat and the oxidative stability of muscle, and affect the flavor, tenderness, shelf life, etc. of meat. Fatty acids are divided into long-chain fatty acids (more than 12 carbons), medium-chain fatty acids (6-12 carbons), and short-chain fatty acids (less than 6 carbon atoms) according to the length of the carbon chain. Long-chain fatty acids are more common in triglycerides in food and are an important component of cell membranes. Long-chain fatty acids affect the quality of livestock products and the growth performance of livestock. Most of the fat in milk is composed of long-chain fatty acids, which are mainly provided by the diet and are easily affected by the fatty acid composition of the diet. Long-chain polyunsaturated fatty acids are essential for growth and development, and the long-chain polyunsaturated fatty acids in the body can be improved through dietary supplementation.

[0004] Adipose differentiation-related protein (ADRP) belongs to the PAT protein family, also known as PLIN2 , which can specifically promote the uptake of long-chain fatty acids. GAO and other studies have found that PLIN2 The mRNA and protein expression levels of α-lactamase were affected by the dose and duration of stimulation with long-chain fatty acids, whereas those with short-chain fatty acids were PLIN2No effect. Tobin et al. found that in human placental choriocarcinoma cell line (BeWo) and primary human trophoblast cells PLIN2 The mRNA and protein expression levels of PLIN2 increased with the increase of long-chain polyunsaturated fatty acid concentration. Taïb et al. treated glioblastoma multiforme cells with C18:1n9c and found that the protein expression of PLIN2 increased.

[0005] although PLIN2 As a fatty acid binding protein, it can promote the uptake of long-chain fatty acids by acting as a carrier protein. PLIN2 Genes have been extensively studied mainly at the fatty acid induction cellular level, but not yet in sheep PLIN2 There are reports that gene SNPs affect fatty acid phenotypes, but the use of molecular markers for early screening of individuals with beneficial fatty acid phenotypes remains a technical challenge. Summary of the invention

[0006] The object of the present invention is to provide a kind of Hu sheep PLIN2 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 PLIN2 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 PLIN2 The mutation site on the gene is g.287A>G (the reference sequence is NC_056055.1).

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

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

[0012] Downstream primer: 5′-ATGAGAACCACGCCGAAGG -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 PLIN2 A kit for detecting single nucleotide polymorphism of a gene, the kit comprising a PLIN2 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 PLIN2 The mutation site on the gene is g.287A>G (the reference sequence is NC_056055.1).

[0018] The above detection of Huyang PLIN2 Application of single nucleotide polymorphism method in sheep marker-assisted selection breeding.

[0019] sheep PLIN2 Application of gene mutation site g.287A>G (reference sequence NC_056055.1) and its detection reagent in sheep marker-assisted selection breeding.

[0020] Preferably, individuals with the GG genotype have superior meat quality traits (for example, individuals with the GG genotype have higher C18:1n9c and MUFA percentages than those with the GA and AA genotypes).

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

[0022] The present invention is to PLIN2 A new SNP site on the gene (located at the gene mutation site g.287A>G) was genotyped, and the significant correlation between this site and the percentage of fatty acids revealed by association analysis revealed that PLIN2 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 flavor), 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 PLIN2 Electrophoresis of the PCR amplification products in the region near the gene mutation site g.287A>G.

[0025] Figure 2 For Huyang PLIN2 Sequencing peak diagram of gene mutation site g.287A>G. 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 PLIN2 Gene SNP site

[0028] S1 Sample selection

[0029] From August 2018 to August 2021, testicular tissue and longissimus dorsi muscle tissue were collected from 1041 (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 1041 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 in a -20 ℃ refrigerator, shake gently until precipitation occurs, place in a -20 ℃ refrigerator for 30 min, centrifuge at 12000 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°C 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. The DNA was stored in a -80 ℃ refrigerator.

[0041] b) After 1041 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 PLIN2 The genome sequence (NC_056055.1) was used as a reference, and Primer-Blast online software was used to design amplification PLIN2 PCR specific primers for the gene exon 1 region and some intron regions on both sides, the primer sequences are as follows:

[0044] Upstream primer PLIN2-F: 5'-GTGAACGCAGCACAGACTC -3'

[0045] Downstream primer PLIN2-R: 5'-ATGAGAACCACGCCGAAGG -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 398 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 PLIN2 The mutation site on the gene is g.287A>G (the reference sequence is NC_056055.1);

[0051] The 1041 DNA samples that passed the quality inspection were uniformly diluted to 30 ng / μL and dispensed into 200 μL centrifuge tubes. The location information of the pending SNP site (i.e. g.287A>G) on the genome sequence was provided, and the company used iMLDR technology to perform SNP typing on the 1041 DNA samples.

[0052] S5 PLIN2 Frequency statistics of genotyping loci

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

[0054] PE=(2NEE+NEe1+NEe2+NEe3+NEe4+……+NEen) / 2N

[0055] In the formula, PE represents the frequency of allele E, NEE represents the number of individuals with EE genotype in the population, NEei represents the number of individuals with Eei genotype in the population, e1, e2, …, en are n different multiple alleles of allele E respectively;

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

[0057] PEE=NEE / N

[0058] In the formula, PEE represents the frequency of the EE genotype at a certain site, NEE represents the number of individuals with the EE 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 1041 individuals are shown in Table 1:

[0060] Table 1. Hu sheepPLIN2 Gene mutation site g.287A>G frequency statistics

[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, G is the dominant allele of the g.287A>G site, and the allele frequency is 55.43%. And combined with genetic indicators, it can be determined that the mutation site is a SNP site.

[0064] 2. Huyang PLIN2 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 PLIN2 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 PLIN2 Association analysis between gene g.287A>G and fatty acid percentage of longissimus dorsi muscle (Mean ± SD)

[0072]

[0073] Note: Different capital letters in the same row indicate significant differences (P<0.05).

[0074] The results showed that the g.287A>G site had a significant effect on C18:1n9c and MUFA, among which the C18:1n9c and MUFA percentages of GG genotype individuals were significantly higher than those of AA genotype individuals.

[0075] C18:1n9c is a long-chain fatty acid and one of the MUFAs. MUFA is positively correlated with meat flavor and overall acceptability. Therefore, by selecting and establishing a group with a genotype of GG (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 breeds with excellent meat quality. <110> Gansu Runmu Bioengineering Co., Ltd. <120> A method for detecting SNP of PLIN2 gene in Hu sheep and its application in early screening of meat quality traits <160> 2 <210> 1 <211> 19 <212> DNA <213> PLIN2-F <400> 1 gtgaacgcag cacagactc 19 <210> 2 <211> 19 <212> DNA <213> PLIN2-R <400> 2 atgagaacca cgccgaagg 19

Claims

1. Detection of Huyang PLIN2 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 PLIN2 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 PLIN2 The mutation site on the gene is g.287A>G, and the reference sequence is NC_056055.1; among them, the Hu sheep individuals with the GG genotype are superior to the Hu sheep individuals with the AA genotype in meat quality traits, and the meat quality traits are C18:1n9c and MUFA percentage.

2. The use according to claim 1, characterized in that: The primers for the PCR amplification are: Upstream primer: 5'-GTGAACGCAGCACAGACTC -3' Downstream primer: 5′-ATGAGAACCACGCCGAAGG -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 PLIN2 The application of the gene mutation site g.287A>G detection reagent in the assisted selection breeding of Hu sheep meat quality traits is characterized by: The Hu sheep individuals with GG genotype are superior to the Hu sheep individuals with AA genotype in meat quality traits, wherein the meat quality traits are C18:1n9c, MUFA percentage, PLIN2 The reference sequence of the gene is NC_056055.1.

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