CN113265471B - Method for detecting sheep FASN gene single nucleotide polymorphism and application of method in meat quality early screening - Google Patents
Method for detecting sheep FASN gene single nucleotide polymorphism and application of method in meat quality early screening Download PDFInfo
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Abstract
The invention discloses a method for detecting sheep FASN gene single nucleotide polymorphism and application thereof in meat quality early screening, comprising the following steps: extracting DNA samples of Hu sheep tissues and single nucleotide polymorphism typing of FASN gene mutation sites g.5157A & gtG and g.9413T & gtC; the relevance analysis of the single nucleotide polymorphism of the Hu sheep FASN gene and the intramuscular fat content shows that the molecular marker for early screening of the Hu sheep intramuscular fat content character can be obtained by typing and detecting the single nucleotide polymorphism sites of the Hu sheep FASN gene.
Description
Technical Field
The invention relates to screening and detection of a Single Nucleotide Polymorphism (SNP) molecular marker, in particular to a method for detecting the single nucleotide polymorphism of a sheep Fatty Acid Synthetase (FASN) gene by combining a DNA pool with an improved multiple high temperature ligase detection reaction (imLDR) technology and an application thereof.
Background
Morphological, cytological, biochemical and molecular markers are four types of genetic markers common today. The transmission of loci in pedigrees can be followed by genetic markers, which are themselves identifiable and inherited. Molecular markers are genetic variations at the DNA level. Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level, and is a genetic marker widely distributed in the genome. SNPs can be caused by single base transitions or transversions, or by base insertions or deletions. Coding region SNPs and non-coding region SNPs can be classified according to the positions of SNPs in a gene. Among them, SNPs (coding SNPs) located in coding regions of genes can be classified into synonymous mutations (amino acids encoded by genes are the same before and after base change, which do not change the sequence of proteins but may cause change in translation efficiency) and nonsynonymous mutations (amino acids encoded by genes are changed before and after base change, which affect the structure and biological functions of proteins). The detection and analysis of SNPs resulting from mutations in nucleotides of coding regions is therefore of great importance for breeding efforts.
At present, the detection methods of SNPs mainly comprise the following methods: DNA sequencing, Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) and DNA sequencing combination, Allele Specific PCR (AS-PCR), primer extension, oligonucleotide ligation, and the like. The DNA sequencing method has accurate result, but has strict requirements on instruments, equipment and personnel technology and higher detection cost; the PCR-SSCP has the problems of complex operation, long time consumption and the like; As-PCR in SNP locus detection needs to design primers aiming at specific gene loci, and the primer extension method and oligonucleotide ligation reaction technology have high requirements on instruments and personnel conditions and are not generally applied at present.
An improved multiple high temperature ligase detection reaction (imLDR) technology is a genotyping technology improved by a Ligase Detection Reaction (LDR) technology, and has the advantages of low cost, high speed and high accuracy, and 16-30 sites can be simultaneously detected by only one reaction. The principle of carrying out typing detection by imLDR is that the section where the target SNPs sites are located is amplified in a system by adopting multiple PCR reaction, and the product is used as a template for subsequent ligase reaction after being purified by exonuclease and shrimp alkali enzyme (Exol/SAP); in a ligation reaction, each site comprises two 5' allele-specific probes followed by a fluorescently-labeled specific probe for the Y-terminal site; ligation products were distinguished by capillary electrophoresis of ABI3730XL and the raw data files were analyzed using GeneMapper4.1 software. Compared with the LDR technology, the imLDR technology improves the accuracy and the success rate of typing.
Intramuscular fat (IMF) is mainly composed of phospholipids, triacylglycerides, semi-phosphates, diphosphoglycerides, cholesterol and sterol esters, and Fatty Acids (FA), and is deposited on muscle bundles and the intramuscular tunica adventitia to form marbling on the muscle surface, which is an important apparent content property in meat quality evaluation. IMF deposition is affected to varying degrees by gene, variety, management and nutrition. The IMF is in positive correlation with the tenderness, juiciness, fragrance, marbling score and eye muscle area of meat, and the meat quality, the tenderness and the flavor are improved along with the increase of the IMF content in a certain range. The deposition of IMF occurs at a later stage in ontogeny and later than the deposition of fat in other parts of the body, and its composition is also greatly different from that of fat in other parts of the body. Research shows that mutton with the IMF content of 4-5% has the best quality, and in order to improve the IMF content, the research has important research significance for carrying out variety improvement and breeding from the genetic aspect by utilizing candidate genes related to the IMF content besides paying attention to varieties, sexes, ages and feed nutrients.
Fatty Acid Synthase (FASN) is one of the essential enzymes in the fatty acid synthesis process, and the main function is to catalyze the synthesis of fatty acids in the intercellular matrix. The FASN gene is located on sheep chromosome 11 and has a full length of 18795 bp. Through correlation analysis of the expression level of FASN gene of ruminants and fat content (Sanz et al, 2015), it was found that nutrition (when feeds with different nutritional ingredients are fed) stimulates the expression of FASN gene, thereby affecting the change of fat content.
At present, a great deal of research is carried out around the influence of FASN gene polymorphism on meat quality of ruminants, but most of the reports related to cattle are available, some goats are used as research objects, and few sheep-related reports are available, and meanwhile, the early screening of sheep individuals with meat quality trait superiority by using molecular markers still belongs to the technical problem.
Disclosure of Invention
The invention aims to provide a method for detecting sheep FASN gene single nucleotide polymorphism and application thereof in early screening of meat quality traits, to realize early screening of sheep excellent meat quality traits, and to be applied to sheep variety improvement and breeding.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting single nucleotide polymorphism of sheep FASN gene comprises the following steps:
the genomic DNA of sheep individuals is extracted as a nucleic acid sample, and the genotype of individual FASN gene single nucleotide polymorphism sites is determined by analyzing the extracted nucleic acid sample, wherein the single nucleotide polymorphism sites are FASN gene mutation sites g.5157A > G (reference sequence is NC-040262.1) and g.9413T > C (reference sequence is NC-040262.1).
Preferably, the analysis of the nucleic acid sample employs DNA sequencing, or the analysis of the nucleic acid sample employs imLDR techniques.
Preferably, the FASN gene mutation site is determined by mixing DNAs extracted from testis tissues of individual sheep (e.g., Hu sheep) and amplifying and sequencing a target fragment from the mixed DNA pool.
Preferably, the extraction method of the DNA of the testis tissue is a high-salt method, the extraction of the DNA of the testis tissue by the high-salt method has the advantages of simple and convenient operation, short time consumption and easy obtainment of extraction reagents, and the quality of the extracted DNA (genome DNA) can meet the typing requirement of the imLDR technology.
Preferably, the primer used for amplifying the target fragment is a primer pair P1 or a primer pair P2;
the primer pair P1 is specifically (the amplification product contains mutation sites g.5157A > G; namely, the single nucleotide polymorphism sites of the amplified FASN gene):
the upstream primer F1: 5'-GTCAGTAGAGCTGTCGGAGCC-3'
The downstream primer R1: 5'-CATGGGATGTGGGATGTGAGCA-3', respectively;
the primer pair P2 is specifically (the amplification product contains mutation sites g.9413T > C; namely, the single nucleotide polymorphism sites of the amplified FASN gene):
the upstream primer F2: 5'-GTCTAGGGGAGACGGGTTGA-3'
The downstream primer R2: 5'-GCCAAGGTCCATCCAGAGAC-3' are provided.
The method for detecting the single nucleotide polymorphism of the sheep FASN gene is applied to early screening of sheep traits and marker-assisted selection.
The sheep FASN gene mutation site g.5157A > G (reference sequence is NC-040262.1) and/or g.9413T > C (reference sequence is NC-040262.1) is/are applied to sheep character early-stage screening and marker-assisted selection.
Preferably, in different sheep (e.g., Hu sheep) populations, the meat quality of the individual with the AA genotype at the FASN gene mutation site g.5157A > G (reference sequence is NC-040262.1) is better, and the meat quality of the individual with the TT genotype at the FASN gene mutation site g.9413T > C (reference sequence is NC-040262.1) is better.
Preferably, the meat quality trait is selected from the group consisting of intramuscular fat (IMF) content.
A kit for detecting single nucleotide polymorphism of sheep FASN gene, which comprises necessary reagents for completing analysis of the nucleic acid sample.
Preferably, the necessary reagents specifically include the primer pair P1 and the primer pair P2.
The invention has the beneficial effects that:
according to the invention, by carrying out typing detection on newly discovered FASN gene SNP sites (specifically g.5157A > G and g.9413T > C), a candidate molecular marker is provided for the early screening of the sheep excellent meat quality traits, so that theoretical research and practical basis are provided for the breeding of sheep varieties with excellent meat quality by marker-assisted selection.
Furthermore, the invention can rapidly establish sheep population with excellent meat quality character by adopting imLDR technology to carry out effective genotyping on FASN gene SNP sites, thereby accelerating the breeding process of fine sheep breeds.
Drawings
FIG. 1 is the electrophoresis diagram of the PCR amplification product (753bp fragment) at the 5157 th mutation site (g.5157A > G) of the Hu sheep Fatty Acid Synthetase (FASN) gene.
FIG. 2 is the electrophoresis diagram of the PCR amplification product (749bp fragment) at the 9413 th mutation site (g.9413T > C) of the Hu sheep Fatty Acid Synthetase (FASN) gene.
FIG. 3 is a diagram showing sequencing peaks of DNA pool amplification products of different types of mononucleotide of Hu sheep (arrow indicates 5157 th mutation site of fatty acid synthetase gene).
FIG. 4 is a diagram showing sequencing peaks of DNA pool amplified products of different types of mononucleotide of Hu sheep (arrow indicates the 9413 th mutation site of fatty acid synthetase gene).
Detailed Description
The invention is described in further detail below with reference to the figures and examples. The examples are only used to explain the technical solution of the present invention, and do not limit the protection scope of the present invention.
Screening new Hu sheep Fatty Acid Synthetase (FASN) gene single nucleotide mutation sites by DNA pool sequencing
Selection of S1 sample:
in 2018, 8-2020, 1, 921 (four batches) of testis tissues and longisimus dorsi tissues of 6-month-old healthy Hu sheep from the same breeding condition of the Ministry of Denfu agriculture technology Limited company are collected, and the intramuscular fat content of the longisimus dorsi of the individual is measured.
S2, construction of a DNA pool:
a) the gDNA in the testis tissues of 921 Hu sheep is extracted by a high-salt method, and the method comprises the following specific steps:
i) grinding a tissue sample under liquid nitrogen, and taking 0.1g of tissue powder into a 1.5 centrifugal tube;
II) adding 20% SDS (100 mu L) and DNA extraction solution (400 mu L) as lysis solution, standing for 5min, whirling to completely dissolve precipitate (tissue powder) in the lysis solution, adding 20 mu L proteinase K, and reversing and mixing;
III) placing the uniformly mixed sample in a 56 ℃ water bath kettle, and digesting overnight;
IV) adding saturated saline solution (300 mu L) into the digested sample, reversing for 3min, and standing at 4 ℃ for 10 min;
v) centrifuging at 12000r for 10min, and transferring the supernatant into a new centrifuge tube;
VI) adding precooled absolute ethyl alcohol (1mL), reversing and uniformly mixing for 1min, centrifuging at 12000r for 2min, removing supernatant, and keeping DNA precipitate;
VII) adding 75% ethanol (500 mu L) into the centrifuge tube, shaking gently for mixing uniformly, centrifuging at 12000r for 1min, discarding the supernatant, retaining the DNA precipitate, adding 75% ethanol (500 mu L) into the centrifuge tube again, shaking gently for mixing uniformly, centrifuging at 12000r for 1min, discarding the supernatant, retaining the DNA precipitate, uncovering the cover and standing for 5h to completely volatilize the residual ethanol;
VIII) Add 56 ℃ TE buffer (200. mu.L) to the centrifuge tubes, uncap for 10min at 56 ℃ (dissolve the ethanol-evaporated precipitate), and dissolve DNA overnight at 4 ℃.
b) For DNA dissolved overnight at 4 ℃, the concentration and purity of the DNA were measured using Nanodrop 2000, and the samples with the concentration of more than 50 ng/. mu.L and the OD 260/280 of 1.6 to 1.8 were judged as qualified samples.
c) After the DNA samples of 921 Hu sheep pass the quality test, 100 DNA samples are randomly selected from the DNA samples, and 10 groups of DNA samples are combined to prepare 10 groups of mixed pools (wherein, one group of mixed pools is a DNA pool formed by mixing 10 DNA samples).
S3, PCR amplification: the Primer-Blast design was used to amplify the DNA sequence of the FASN gene (NC-040262.1) already published in NCBI:
a) primer pair P1 for the complete fragments of exons 5, 6 and 7 and introns 5 and 6 of the FASN gene, and for the partial fragments of introns 4 and 7:
the upstream primer F1: 5'-GTCAGTAGAGCTGTCGGAGCC-3' 21nt
The downstream primer R1: 5'-CATGGGATGTGGGATGTGAGCA-3' 22 nt;
b) primer pair P2 for the complete fragments of exons 16, 17, 18 and introns 16, 17 of the FASN gene, and for the partial fragments of introns 15, 18:
the upstream primer F2: 5'-GTCTAGGGGAGACGGGTTGA-3' 20nt
The downstream primer R2: 5'-GCCAAGGTCCATCCAGAGAC-3' 20 nt.
Carrying out PCR amplification by taking the DNA mixed pool as a template;
the reaction system is as follows: mu.L of 2 × EasyTagSuperMix (2.5U/. mu.L), 0.5. mu.L of forward primer (10 pmol/. mu.L), 0.5. mu.L of reverse primer (10 pmol/. mu.L), 10.5. mu.L of sterilized double distilled water, 1.5. mu.L of DNA template (50 ng/. mu.L), and 25.5. mu.L of total PCR reaction system.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 30s, annealing at 62.6 deg.C for 30s, extension at 72 deg.C for 1min, and performing 34 cycles; further extension was carried out at 72 ℃ for 5 min.
Referring to fig. 1 and 2, agarose gel electrophoresis is respectively performed on products amplified by using the primer pair P1 and the primer pair P2 to obtain 753bp and 749bp target fragments respectively, and the electrophoresis results show that the primer pairs have specificity and can be used for conveniently obtaining enough target DNA fragments through PCR amplification.
Sequencing of S4 amplification products:
the PCR products amplified using the pool-mixed in step S3 were subjected to bidirectional sequencing. Referring to FIG. 3, the 5157 th single nucleotide site of the Hu sheep fatty acid synthetase gene was found to be a mutation site (g.5157A > G) by sequencing the target fragment amplified using primer pair P1. Referring to FIG. 4, the 9413 rd single nucleotide site of the Hu sheep fatty acid synthetase gene was found to be a mutation site (g.9413T > C) by sequencing the target fragment amplified using primer pair P2.
(II) typing candidate SNP sites by imLDR technology
Diluting 921 Hu sheep DNA samples qualified in quality detection to 50 ng/. mu.L respectively, subpackaging into 1mL centrifuge tubes, taking the mutation sites as FASN gene candidate single nucleotide polymorphism sites, taking 15bp DNA sequence information at the upper and lower stream of the sites to provide to Shenzhen Tianhao science and technology Limited company, and typing two candidate single nucleotide polymorphism sites simultaneously by utilizing imLDR technology on the diluted and subpackaged DNA samples by the company.
(III) Hu sheep fatty acid synthetase gene (FASN gene) typing and intramuscular fat content data analysis
3.1 frequency statistics of Hu sheep FASN Gene mutation sites
Gene frequency calculation formula:
PB＝(2NBB+NBb1+NBb2+NBb3+NBb4+……+NBbn)/2N，
in the formula, PBIndicates allele B frequency, NBBRepresenting the number of individuals in the population with the BB genotype, NBbiIn the presentation groupHaving a structure of BbiThe genotype individual number, B1-bn is the N different multiple alleles of allele B, and N is the total individual number of the detection population.
Genotype frequency calculation formula:
PBB＝NBB/N
in the formula, PBBRepresenting the BB genotype frequency, N, of a certain siteBBRepresenting the number of individuals having a BB genotype in the population, N being the total number of individuals in the test population;
after the analysis of the alleles and genotypes of different Hu sheep individuals is completed, the frequencies of the alleles and genotypes of FASN gene mutation sites of the Hu sheep population are calculated according to the formula, and the results are shown in Table 1:
TABLE 1 Hu sheep FASN Gene mutation site allele frequency and genotype frequency
According to the results in Table 1, it can be determined that g.5157A > G (G is dominant allele) and g.9413T > C (T is dominant allele) belong to the mutation sites of FASN gene in Hu sheep.
3.2 Association analysis of SNP site of FASN gene of Hu sheep and intramuscular fat (IMF) content
Correlation analysis data: the typing results of the imLDR technique; intramuscular fat content phenotype data
The data are analyzed by adopting SPSS 25 software, and the correlation analysis of the SNP locus typing result and the intramuscular fat content phenotype data specifically adopts the following analysis model:
Yijk＝μ+Gj+Eijk
in the formula, Yijk(ii) recording the phenotype of the individual; μ is the population mean; gjThe genotype effect for each site; eijkIs a random error. Further, the gene locus is deduced through the constructed model, and the single nucleotide polymorphism generated by the mutation of the FASN gene of the Hu sheep at the corresponding locus of the A & gtG at the 5157 th site and the T & gtC at the 9413 th site is proved to have the content of the single nucleotide polymorphism in the intramuscular fat of the Hu sheepInfluence of the quantitative early trait. The analytical results are shown in table 2:
TABLE 2 Association analysis of single nucleotide polymorphism of Hu sheep FASN gene and intramuscular fat content
Note: different shoulder symbols indicate significant differences (P <0.05), the same shoulder symbols indicate insignificant differences (P > 0.05).
The IMF content is the data after using R3.6.0 to remove the batch effect, and the negative sign is caused by the removal of the batch effect.
The correlation analysis result shows (table 2), for the single nucleotide polymorphism site at the 5157 th site (namely g.5157A & gtG) typed by the imLDR technology, the intramuscular fat content of the GG genotype individual is obviously lower than that of the AA genotype individual; for the single nucleotide polymorphism site at position 9413 (namely g.9413T & gt C) typed by the imLDR technology, the intramuscular fat content of the TT genotype individual is obviously higher than that of the CC genotype individual. According to the analysis results, the Hu sheep population with meat quality character superiority can be established by selecting individuals with the 5157 th single nucleotide polymorphism site of the FASN gene as AA homozygote and the 9413 th single nucleotide polymorphism site as TT homozygote.
In conclusion, the invention discovers that the FASN gene has important influence on the deposition of the intramuscular fat of the Hu sheep by performing typing, gene and genotype frequency calculation of the imLDR technology on the mononucleotide mutation site of the fatty acid synthetase gene and correlation analysis on intramuscular fat content phenotype data, develops further research on the gene function and genetic variation of the gene and is beneficial to improving the meat quality character of sheep. Meanwhile, the invention also establishes the relationship between the intramuscular fat content phenotype and the single nucleotide polymorphism generated by the mutation of the fatty acid synthetase gene with the 5157 th site A > G and the 9413 th site T > C, provides a screening molecular marker (SNP marker) for early screening of the Hu sheep intramuscular fat content character and breeding of sheep varieties with good meat quality and flavor, and has important significance for sheep molecular breeding.
<110> Lanzhou university
<120> method for detecting sheep FASN gene single nucleotide polymorphism and application thereof in early screening of meat quality traits
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gtcagtagag ctgtcggagc c 21
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catgggatgt gggatgtgag ca 22
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Claims (6)
1. Detection sheepFASNThe application of the gene single nucleotide polymorphism method in sheep meat quality character early screening and/or marker-assisted selection is characterized in that:
detecting sheepFASNA method for gene single nucleotide polymorphism comprising the steps of:
extracting sheep genome DNA for analysis and determinationFASNThe genotype of a single nucleotide polymorphic site of a gene, said single nucleotide polymorphic site comprisingFASNThe gene mutation site g.9413T is more than C;
FASNthe genotype of the gene mutation site g.9413T > C is TTThe meat quality is better;
the meat quality character is better, namely the intramuscular fat content is high;
the sheep is selected from Hu sheep.
2. Use according to claim 1, characterized in that: the above-mentionedFASNThe genotype of the gene single nucleotide polymorphism site is determined by adopting a DNA sequencing method or an imLDR technology.
3. Use according to claim 1, characterized in that: the above-mentionedFASNThe gene mutation sites are determined by mixing DNA extracted from different sheep individuals, and amplifying and sequencing target fragments of the mixed DNA pools.
4. Use according to claim 3, characterized in that: the extraction method of the DNA is a high-salt method.
5. Use according to claim 3, characterized in that: the primer adopted by the target fragment amplification is a primer pair P2;
the primer pair P2 is:
the upstream primer F2: 5'-GTCTAGGGGAGACGGGTTGA-3'
The downstream primer R2: 5'-GCCAAGGTCCATCCAGAGAC-3' are provided.
6. SheepFASNThe application of gene mutation site g.9413T > C in sheep meat quality character early screening and sheep marker auxiliary selection is characterized in that:FASNindividuals with the genotype TT with the gene mutation site g.9413T & gtC are superior in meat quality traits;
the meat quality character is better, namely the intramuscular fat content is high;
the sheep is selected from Hu sheep.
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