CN109161601B - Method for auxiliary rapid detection of cattle growth traits by using SNP marker of PLAGL1 gene and application thereof - Google Patents
Method for auxiliary rapid detection of cattle growth traits by using SNP marker of PLAGL1 gene and application thereof Download PDFInfo
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Abstract
The invention discloses a method for auxiliary rapid detection of cattle growth traits by using a PLAGL1 gene SNP marker and application thereof. Taking cattle genome DNA as a template, and obtaining a partial fragment of cattle PLAGL1 gene through PCR amplification; digesting the PCR product by using restriction enzyme Nde1, and performing agarose gel electrophoresis; and identifying the SNP of 95141 th site of the cattle PLAGL1 gene according to an electrophoresis result. The genotype of the SNP locus of the cattle PLAGL1 gene detected by the invention can be used as a molecular genetic marker closely related to the growth traits of cattle, and is used for the molecular marker-assisted selection of cattle to promote the breeding process of fine cattle.
Description
Technical Field
The invention belongs to the field of molecular genetics, relates to screening and detection of Single Nucleotide Polymorphism (SNP) of a cattle gene as a molecular genetic marker, and particularly relates to a method for detecting the SNP of the cattle gene PLAGL1 and application thereof.
Background
Molecular genetic markers, i.e., markers of genetic polymorphisms at the DNA level, can directly reflect genetic variations in DNA sequences. The DNA sequence difference is originated from the DNA sequence difference, so the DNA sequence difference is called as a molecular marker, and the DNA sequence difference has the characteristics of high genetic diversity, strong stability and small environmental influence in biological populations. The molecular marker assisted selective breeding is a main research content of modern animal molecular breeding, and because the genotype of characters is directly selected on the DNA level, the accuracy of seed selection is greatly improved, and the defects of the traditional animal breeding method are overcome.
Single Nucleotide Polymorphism (SNP) is an important molecular genetic marker, mainly refers to sequence polymorphism at the genome level caused by a change of a certain nucleotide in a DNA sequence, and mainly takes the form of single nucleotide transition and transversion. Compared with other molecular markers, SNP has the advantages of large genome coverage, high density, high resolution, genetic stability, easy realization of analysis automation and the like.
Since the first proposal by Botestein et al of the concept relating to DNA Restriction Fragment Length Polymorphism (RFLP), the PCR-RFLP method has been used in large quantities for the detection of SNPs. The premise for this approach is that the site of the SNP must contain the appropriate restriction enzyme recognition site. The target fragment is cut by using restriction endonuclease, and then gel electrophoresis analysis is carried out, so that the genotype of the SNP site can be accurately identified.
The study shows that PLAGL1 is related to cell cycle, is a growth inhibitory gene and is involved in apoptosis and cell cycle arrest. Meanwhile, it has been confirmed to be a transcription regulator of type 1 pituitary adenylate cyclase activating polypeptide receptor, possibly involved in the regulation of pancreatic growth. At present, the research on the PLAGL1 gene polymorphism at home and abroad mainly focuses on the research on the relevance of the PLAGL1 gene polymorphism to neonatal diabetes, tumors and the like, and the research on the livestock PLAGL1 gene single nucleotide polymorphism is not reported yet.
Disclosure of Invention
The invention aims to provide a method for assisting in rapidly detecting the growth traits of cattle by using a PLAGL1 gene SNP marker and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting SNP of a cattle PLAGL1 gene comprises the following steps:
taking cattle genome DNA as a template and a primer pair P as primers, carrying out PCR amplification on partial fragments of cattle PLAGL1 gene, digesting the PCR amplification product by restriction enzyme NdeI, carrying out agarose gel electrophoresis, and identifying the genotype of 95141 th (reference sequence is NC-037336.1) SNP site of cattle PLAGL1 gene according to the electrophoresis result;
preferably, the sequence of the primer pair P is:
an upstream primer F: 5'-CAGAGGCAAGTCCTCAAGCA-3', respectively;
a downstream primer R: 5'-CTCACCGGTGTGCTTACTGT-3' are provided.
Preferably, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 50s for 32 cycles; extension at 72 ℃ for 5 min.
Preferably, the agarose gel electrophoresis uses agarose gel with mass concentration of 3%.
Preferably, the electrophoresis result of the genotype of the 95141 th SNP site of the cattle PLAGL1 gene is as follows: the TT genotype is expressed as a strip of 821 bp; the TC gene type is represented by three bands of 821bp, 742bp and 79 bp; the CC genotype shows two bands of 742bp and 79 bp.
The application of the detection method of the cattle PLAGL1 gene SNP in cattle molecular marker-assisted selection breeding.
The application of the detection method of the cattle PLAGL1 gene SNP in auxiliary detection of cattle growth traits.
Preferably, the growth trait is ischiatal width or nojirimus length.
Preferably, the cattle is Qinchuan cattle or Jiaxian red cattle.
A kit for detecting SNP of a cattle PLAGL1 gene comprises the PCR amplification primer pair P for carrying out 95141 th SNP locus typing of the cattle PLAGL1 gene.
The invention has the beneficial effects that:
aiming at the 95141 th SNP of the cattle PLAGL1 gene, the invention can simply, quickly, low-cost and high-precision identify the genotype of the single nucleotide polymorphism site of the gene by designing a primer, amplifying a target fragment by PCR and then carrying out restriction enzyme digestion identification. The invention detects the gene type of the SNP locus and analyzes the gene frequency, and the locus and the growth traits of two cattle varieties are subjected to correlation analysis. The result shows that the genotype of the 95141 site single nucleotide polymorphism (T > C) site of the obtained cattle PLAGL1 gene can be used as a genetic marker for molecular marker assisted selection breeding. The invention not only can provide basic data for the molecular marker-assisted selective breeding of cattle, but also can promote the germplasm resource improvement process of cattle.
Drawings
FIG. 1 is a sequence diagram of 95141 th site (NC-037336.1) of cattle PLAGL1 gene: boxed positions indicate the mutation sites (T95141C).
FIG. 2 shows the electrophoresis of the PCR amplified fragment of cattle PLAGL1 gene.
FIG. 3 shows the electrophoresis result of NdeI digestion of the PCR amplification product of different genotypes (TT, TC and CC) at position 95141 of cattle PLAGL1 gene, wherein: m is D2000, which is 2000bp, 1000bp, 750bp, 500bp, 300bp and 200bp respectively.
Detailed Description
The invention is described in detail below with reference to the drawings and examples, which are illustrative and not restrictive of the invention.
The invention designs primers according to the sequence of the cattle PLAGL1 gene, respectively takes genome DNA pools of 2 cattle varieties as templates, performs PCR amplification, and obtains the partial sequence of the cattle PLAGL1 gene by product sequencing. Comparing with a reference sequence published on NCBI, finding that T > C mutation exists at 95141 th site of cattle PLAGL1 gene, and detecting the mutation site by using PCR-RFLP method. Finally, the single nucleotide polymorphism of the cattle PLAGL1 gene at the site is subjected to correlation analysis with the growth traits of cattle, so that the method can provide a basis for cattle molecular breeding.
Cloning of partial sequence of cattle PLAGL1 gene and polymorphism detection thereof
1. Sample collection and genomic DNA extraction
(1) Collection of blood samples
The invention adopts 2 cattle breeds, total 342 individuals are taken as detection objects, and the blood collection method is jugular vein blood collection. The area where the blood sample was collected and the data of each variety are shown in Table 1.
TABLE 1 Experimental animal Condition
(2) Extraction of genomic DNA from blood samples
Freezing blood sample (mainly blood cells) and unfreezing at room temperature, sucking 500 mu L of blood into a 1.5mL centrifuge tube, adding Phosphate Buffer Solution (PBS) with the same volume, uniformly mixing, gently shaking, centrifuging at 4 ℃ and 12000rpm for 5min, discarding supernatant, and repeating the steps until the supernatant is transparent.
② adding 500 mu L of DNA extraction buffer solution into a centrifuge tube, gently blowing and beating to separate the blood cell sediment from the wall of the centrifuge tube, and carrying out water bath at 37 ℃ for 1 h.
③ adding protease K to 3 mu L (20mg/mL), mixing, digesting in water bath at 55 ℃ overnight (about 16 h) until no flocculent precipitate is seen, clarifying the solution, adding 1 mu L protease K, mixing, and continuing digesting until the solution is clarified.
Fourthly, the sample is taken out and added with 200 mu L of 6mol/L NaCl, the mouth bottom is shaken for 15 times to be fully and evenly mixed, the mixture is centrifuged for 10min at 12000rpm at 4 ℃, and the supernatant is taken out and put into a 2.0mL centrifuge tube.
Adding 1mL of Tris-saturated phenol, placing on ice, and gently shaking for 20min to fully mix; centrifuge at 12000rpm for 10min at 4 deg.C, transfer the upper aqueous phase to another sterilized 2.0mL centrifuge tube with a pipette.
Sixthly, adding 0.5mL of Tris-saturated phenol and 0.5mL of chloroform, and gently shaking on ice for 20 min; centrifuging at 12000rpm for 10min at 4 deg.C; the upper aqueous phase was pipetted into another sterilized 2.0mL centrifuge tube.
Seventhly, adding 1mL of chloroform, placing on ice, and gently shaking for 20 min; centrifuging at 12000rpm for 10min at 4 deg.C; the upper aqueous phase was pipetted into another sterile 1.5mL centrifuge tube.
Adding 1mL of precooled absolute ethyl alcohol (-20 ℃), shaking the mixture for many times at the bottom of a mouth until DNA is separated out, and then placing the mixture for 30min at the temperature of minus 20 ℃; after taking out, the mixture was centrifuged at 12000rpm for 10min at 4 ℃ and ethanol was discarded.
Ninthly, adding 1mL of 70% ethanol, and gently shaking for 10 min; the tube was then centrifuged at 12000rpm for 10min at 4 ℃ and the ethanol was discarded, and the rinsing was repeated once (pipette off the remaining alcohol from the bottom of the tube).
Placing the red body at room temperature for 15min, and then placing the red body in a 60 ℃ oven for 30s to completely volatilize ethanol; add ultra pure water 50 u L, 4 degrees C storage until DNA completely dissolved, spectrophotometer determination concentration, the-80 degrees C storage.
(3) Construction of DNA pools
And measuring the OD values of the DNA samples at 260nm and 280nm and the DNA content of the DNA samples by using an ultraviolet spectrophotometer. If OD is greater than the total260/OD280The ratio is less than 1.6, which indicates that the sample contains more protein or phenol, and purification is required; if the ratio is greater than 1.8, then RNA purification removal should be considered. After the DNA detection is finished, a certain amount of the DNA is taken out and diluted to 10 ng/. mu.L, then 50 diluted samples are randomly selected from 2 cattle groups, and 2.5. mu.L of each sample is uniformly mixed to construct a DNA pool.
2. Amplification primer design (primer design completion time 2017 years 9 months)
By taking a bovine PLAGL1 gene sequence (NC _037336.1) published by NCBI as reference, the Oligo 7.0 software is used for designing a PCR primer pair P capable of amplifying a 8 th exon region of a bovine PLAGL1 gene, and the primer sequences are as follows:
an upstream primer F: 5'-CAGAGGCAAGTCCTCAAGCA-3' (20nt)
A downstream primer R: 5'-CTCACCGGTGTGCTTACTGT-3' (20nt)
PCR amplification
(1) The PCR reaction system is shown in Table 2.
TABLE 2 PCR reaction System
(2) The PCR reaction procedure is shown in Table 3.
TABLE 3 PCR reaction procedure
Sequencing of PCR products
The PCR product amplified by using the mixed DNA pool as a template is sent to Beijing Okkomy Biotech limited for sequencing. The sequencing result of the target fragment of the cattle PLAGL1 gene is compared with a reference sequence, and a T > C mutation exists in the 8 th exon region (FIG. 1, NC-037336.1: T95141C).
PCR product restriction enzyme and RFLP detection
And (3) PCR reaction system: referring to table 2, genomic DNA extracted from individual blood samples was used as a template.
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 50s, for 32 cycles in total; extension at 72 ℃ for 5 min.
As shown in FIG. 2, the PCR amplification product showed an amplified fragment size of about 821 bp.
The PCR amplification products are firstly cut by restriction enzymes NdeI respectively, and then the SNP polymorphism is judged according to the electrophoresis result.
10 mu L of NdeI enzyme cutting system is as follows: mu.L of PCR product, 10 XBuffer Tango 1. mu.L, NdeI (10U/. mu.L) 1. mu.L, 3. mu.L of sterile double distilled water. Digesting the enzyme digestion system in a constant temperature incubator at 37 ℃ for 12-16 h, carrying out electrophoresis on 3% agarose gel at 110V and 55mA for 40min at room temperature, and imaging by using a Bio-RAD gel imager. And judging the genotype according to the images of the electrophoresis bands.
Referring to FIG. 3, since the amplification product of primer pair P does not contain other NdeI cleavage sites, the PCR amplification product fragment containing "C" will be cut into two fragments of 742bp and 79 bp; the PCR product containing "T" was not recognized by NdeI and remained an 821bp fragment. Therefore, the result of TT genotype electrophoresis shows a 821bp strip; the TC gene type is represented by three bands of 821bp, 742bp and 79bp, and the CC gene type is represented by two bands of 742bp and 79 bp. However, since 79bp is too short to be seen in the electrophoretogram, only two bands at most were observed in the actual electrophoretic analysis, but the typing was not affected.
Second, frequency statistics of SNP loci of cattle PLAGL1 gene and correlation analysis of frequency statistics and growth traits
1. Gene and genotype frequency
Genotype frequency refers to the ratio between the various genotypes of a trait in a population. The calculation formula is as follows:
PYY＝NYY/N
wherein P isYYRepresents the YY genotype frequency of a certain locus; n is a radical ofYYRepresenting the number of individuals in the population having a YY genotype; and N is the total number of individuals in the detection population.
Gene frequency refers to the relative ratio of a gene to its allele in a population. The calculation formula can be written as:
Py＝(2Nyy+Nyy1+Nyy2+Nyy3+Nyy4+……+Nyyn)/2N
in the formula, PyIndicates allele y frequency, NyyRepresenting the number of individuals in the population with yy genotype, NyyiIndicating having yy in the populationiNumber of genotyped individuals, y1～ynIs n mutually different multiple alleles of the allele y.
The statistical results of the gene frequency and genotype frequency in each cattle variety are shown in table 4.
TABLE 4 genotype and allele frequencies of the PLAGL1 gene in two cattle breeds
2. Correlation analysis statistical model
The association of gene loci with growth traits was analyzed using SPSS (18.0) software. Firstly, performing descriptive statistical analysis on data to determine whether outliers exist, and analyzing the genotype effect by using t analysis, variance analysis or a multivariate linear model according to the data characteristics. In the data processing, according to different factors influencing growth and development indexes such as body size, weight and the like, considering environmental effects, age, genotype effects and related interaction effects, a fixed model is adopted for analysis, and meanwhile, selection is carried out according to actual conditions. The complete model is as follows:
Yijk＝μ+Gj+Eijk
wherein: y isijk(ii) recording the phenotype of the individual; μ is the population mean; gjThe genotype effect for each site; eijkIs a random error.
The method is used for counting the related data of each cattle variety group, and the statistical results of the related data of Qinchuan cattle and Jiaxian county red cattle are shown in tables 5 and 6.
TABLE 5 correlation analysis of different genotypes and growth traits of Qinchuan cattle PLAGL1 gene
Note: having the same letter indicates that the difference is not significant (P >0.05) and the letter is different indicates that the difference is significant (P <0.05)
TABLE 6 correlation analysis of different genotypes of the Jiaxian Red bull PLAGL1 Gene with growth traits
Note: having the same letter indicates that the difference is not significant (P >0.05) and the letter is different indicates that the difference is significant (P <0.05)
The results show that the correlation analysis of different genotypes at the 95141 site of single nucleotide polymorphism of the cattle PLAGL1 gene sequence and growth and development indexes such as the length and width of the ischium of Qinchuan cattle and Jiaxian cattle shows that: the single nucleotide polymorphism site has obvious influence on the width of the ischium and the length of the nojiri. In Qinchuan cattle, the width of the ischium of individuals with CC genotype and TC genotype is obviously narrower than that of individuals with TT genotype, and the length of the nojiri of individuals with CC genotype and TC genotype is obviously longer than that of individuals with TT genotype. In the red cattle from Jiaxian county, individuals with the TC genotype had significantly narrower sitz width than those with the TT genotype, and individuals with the TC genotype had significantly longer nojiri length than those with the TT genotype. It can be seen that allele C is closely related to the growth traits (e.g., ischium width and nojiri length) of Qinchuan cattle and Jiaxian red cattle, and the corresponding genotype can be used as a molecular genetic marker for early selection of ischium width and nojiri length of cattle.
<110> northwest agriculture and forestry science and technology university
<120> method for rapidly detecting growth traits of cattle assisted by SNP marker of PLAGL1 gene and application thereof
<160> 2
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 1
cagaggcaag tcctcaagca 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 2
ctcaccggtg tgcttactgt 20
Claims (6)
1. A method for detecting SNP of a cattle PLAGL1 gene is characterized by comprising the following steps: the method comprises the following steps:
taking cattle genome DNA as a template, amplifying partial fragments of cattle PLAGL1 gene by PCR, digesting the fragments obtained by PCR amplification by restriction enzyme NdeI, performing agarose gel electrophoresis, and identifying the genotype of 95141 th SNP site in cattle PLAGL1 gene reference sequence NC _037336.1 according to the result of the agarose gel electrophoresis;
the electrophoresis result of the genotype of the SNP locus is as follows: the TT genotype is expressed as a strip of 821 bp; the TC gene type is represented by three bands of 821bp, 742bp and 79 bp; the CC genotype is represented by two bands of 742bp and 79 bp; allele C is closely related to growth traits of Qinchuan cattle and Jiaxian red cattle, and the corresponding genotype can be used as a molecular genetic marker for early selection of the hip end width and the nojiri length of cattle.
2. The method for detecting SNP of a PLAGL1 gene of cattle according to claim 1, which is characterized in that: the primer pair adopted by the PCR amplification is as follows:
an upstream primer F: 5'-CAGAGGCAAGTCCTCAAGCA-3'
A downstream primer R: 5'-CTCACCGGTGTGCTTACTGT-3' are provided.
3. The method for detecting SNP of a PLAGL1 gene of cattle according to claim 1, which is characterized in that: the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 50s for 32 cycles; extension at 72 ℃ for 5 min.
4. The method for detecting SNP of a PLAGL1 gene of cattle according to claim 1, which is characterized in that: the agarose gel electrophoresis was performed using agarose gel with a mass concentration of 3%.
5. The use of the method for detecting SNP of a PLAGL1 gene of cattle of claim 1 in molecular marker assisted selection breeding of cattle.
6. The application of the method for detecting the SNP of the PLAGL1 gene of cattle as claimed in claim 1 in auxiliary detection of the growth traits of cattle.
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| Title |
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| Emerging role of PLAG1 as a regulator of growth and reproduction;Almas R Juma等;《Journal of Endocrinology》;20161231;第228卷(第2期);R45–R56 * |
| rs110111117;佚名;《Ensenmbl》;20141031;第1页 * |
| rs110111117;佚名;《UCSC》;20140131;第1页 * |
| 中国三个黄牛品种ASB-3基因多态性及其遗传效应与mRNA表达研究;阎建宇;《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》;20161115(第11期);D050-23 * |
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