CN111363830B - Specific microsatellite primer composition for identifying experimental miniature pig breeds and method for identifying experimental miniature pig breeds - Google Patents
Specific microsatellite primer composition for identifying experimental miniature pig breeds and method for identifying experimental miniature pig breeds Download PDFInfo
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Abstract
The invention discloses a specific microsatellite primer composition for identifying a miniature pig variety for experiments and a method for identifying the miniature pig variety for experiments, wherein the primer composition comprises 3 pairs of primers. According to the invention, based on the microsatellite sequence in the minipig genome, the specific microsatellite sequence and primers among different varieties of the minipig for experiments are obtained through group test verification and screening, the test conditions and the use method of the primers are optimized, and the identification of the minipig variety for experiments is solved.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a specific microsatellite primer composition for identifying a miniature pig variety for an experiment and a method for identifying the miniature pig variety for the experiment.
Background
The experimental miniature pig is one of the most widely used non-rodent large-scale experimental animals in biomedical research, has irreplaceable superiority of other experimental animals, becomes a research hotspot as the most possible donor for xenogeneic organ transplantation, and is generally concerned by the biomedical field in research, development and utilization of the miniature pig. The variety and the strain of the experimental miniature pig are various, and the typical pig in China is Wuzhishan pig, Bama pig, inbred Luchuan pig, Tibetan miniature pig, Banna miniature pig, Jianbaixiang pig, potentilla anserina miniature pig, black-gold pig, cooperative pig and the like. However, the appearance of some miniature pig breeds is similar, or the phenomenon of cross breeding occurs in the breeding process, so that the breed identification based on the phenotype by naked eyes is difficult, and scientific identification means is urgently needed.
At present, many researches for identifying plant or animal varieties from the gene perspective are carried out, wherein the most effective method is to design specific primers for variety identification based on the conservation of sequences at two ends of a microsatellite. However, as the excavation of the microsatellite in the pig genome is not comprehensive enough, the microsatellite sequences of other species can only be used for carrying out homologous comparison analysis, the designed corresponding primers have no specificity among breeds, the time and labor are wasted during the breed identification, the result is inaccurate, and the identification of the miniature pig breeds for experiments is limited.
Disclosure of Invention
The invention aims to solve the technical problems that the appearance of the experimental miniature pig is similar and the identification by naked eyes is difficult in the prior art, and aims to provide a method for identifying the variety of the experimental miniature pig by using a gene technology means.
One of the purposes of the invention is to provide a specific microsatellite primer composition for identifying experimental miniature pig breeds, wherein the primer composition comprises 3 pairs of primers, and specifically comprises the following components:
the forward fluorescent primer F7 shown as SEQ ID NO: 1:
ATCCCCCACTCATTCTCACA；
the reverse common primer R7 shown in SEQ ID NO. 2:
GCAAAACTGCACACAGCCTA；
3, and a forward fluorescent primer F9 shown as SEQ ID NO:
TCCAGGCCCAGGTTGTATTA；
the reverse common primer R9 shown in SEQ ID NO. 4:
AGGATCTTTTTCGGCAGGAT；
forward fluorescent primer F19 shown in SEQ ID NO. 5:
CAGCTTACAGCAAAAGTAGCCA；
the reverse common primer R19 shown in SEQ ID NO. 6:
AAGATGTTTCATCAGACTATATCCGT。
wherein, F7 and R7 refer to a forward primer and a reverse primer of the No. 7 microsatellite locus; f9 and R9 refer to a forward primer and a reverse primer of the microsatellite seat No. 9; f19 and R19 refer to the forward primer and the reverse primer of the microsatellite locus No. 19. That is, the nucleotide sequence of microsatellite locus No. 7 is a nucleotide sequence located between the nucleotide sequence of forward primer F7 and the nucleotide sequence of reverse primer R7 on minipigs; the nucleotide sequence of the microsatellite seat No. 9 is a nucleotide sequence which is positioned between a forward primer F9 nucleotide sequence and a reverse primer R9 nucleotide sequence on the minipig; the nucleotide sequence of the microsatellite locus No. 19 is a nucleotide sequence located between a nucleotide sequence of a forward primer F19 and a nucleotide sequence of a reverse primer R19 on a miniature pig.
Wherein the experimental miniature pig breed is as follows: wuzhishan pig, Bama pig, inbred Luchuan pig or Tibet pig.
The method of the invention can identify the experimental miniature pig breed at any growth stage, and mainly comprises the following steps: starting from the gene, 3 pairs of species-specific microsatellite primers are used for carrying out processes such as fluorescent primer PCR amplification, purification, capillary electrophoresis, data analysis and the like, so that the experimental miniature pig variety is accurately identified.
The invention also aims to provide a method for identifying the miniature pig breed for experiment, which comprises the following steps:
step S1: collecting a tissue sample of a miniature pig individual for an experiment to be identified, and carrying out DNA extraction and quality detection;
step S2: carrying out PCR amplification by using the specific microsatellite primer composition for identifying the experimental miniature pig breed as claimed in claim 1 to obtain a PCR amplification product;
step S3: taking a part of PCR amplification products to perform capillary electrophoresis, and determining the fragment size and the signal value of a corresponding site according to the molecular internal standard and HiDi;
step S4: according to the sizes of the gene fragments of the experimental miniature pig individuals to be identified on the microsatellite loci No. 7, 9 and 19, the corresponding alleles and genotypes are counted, and the varieties of the experimental miniature pig individuals to be identified are calculated according to the alleles and the genotypes.
Wherein, the allele and genotype obtained in step S4 are used for identifying the breed of the experimental miniature pig individual according to the following standards:
if the individual to be identified has the 7G or 7K allele at microsatellite locus No. 7, or the specific genotype 7F7K at microsatellite locus No. 7, or the specific genotype 9C9H or 9E9H at microsatellite locus No. 9, or the specific genotype 19A19D or 19C19D at microsatellite locus No. 19, the individual to be identified is a Wuzhishan pig;
if the individual to be identified has a 7D or 7J allele at microsatellite locus No. 7, or a specific genotype 7C7J or 7F7J at microsatellite locus No. 7, or a specific genotype 19C19H at microsatellite locus No. 19, then the individual to be identified is a boar;
if the individual to be identified has a 7L allele at the microsatellite locus No. 7, or a 9I allele at the microsatellite locus No. 9, or a specific genotype 9B9I, 9H9I or 9I9I at the microsatellite locus No. 9, or a specific genotype 19A19G, 19A19H, 19G19H or 19H19H at the microsatellite locus No. 19, the individual to be identified is an inbred Luchwa pig;
if the individual to be identified has the 7A allele at the microsatellite locus 7 or the specific genotype 9A9F, 9E9F or 9F9F at the microsatellite locus 9, the individual to be identified is a Tibetan pig.
Wherein, the sizes of the corresponding fragments of the microsatellite loci No. 7, No. 9 and No. 19 and the allele numbers
The tissue sample is a porcine ear tissue sample.
The invention has the advantages that the method has high accuracy and reliability for identifying the variety based on 3 pairs of variety-specific microsatellite primers in the genome of the experimental miniature pig, and can identify the experimental miniature pig variety at any growth stage from a molecular level. In addition, the optimized test conditions for identifying the specific microsatellite primer provide a detailed operation process and corresponding reagents and instruments, and the problem of variety identification of the miniature pig for experiments is solved. The invention obtains the specific microsatellite sequence and primers among different breeds of the miniature pig for experiment through group test verification and screening based on the microsatellite sequence in the genome of the miniature pig breed, optimizes the test condition and the using method of the primers and solves the identification of the miniature pig breed for experiment.
The conception, the specific structure, and the technical effects produced by the present invention will be further described below to fully understand the objects, the features, and the effects of the present invention.
Detailed Description
Example 1
160 miniature pig breeds (respectively 48 Wuzhishan pigs, 15 Bama pigs, 50 inbred Luchuan pigs and 47 Tibet fragrant pigs) for 4 experiments are selected, ear tissue samples of each individual are respectively collected and placed in an EP (enhanced EP) tube filled with 75% ethanol, a phenol chloroform method is adopted for genome DNA extraction, NanoDrop 2000 (Thermo Fisher Scientific Co., USA), 1% agarose gel electrophoresis and gel imaging (Beijing Zhongluan biological science and technology Co., Ltd., Newbio Gi-1) are respectively used for detecting DNA concentration, OD value and DNA quality, and the following operations are carried out on DNA with the DNA concentration of more than or equal to 50 ng/mu L, OD260/280 of 1.6-1.8 and imaging of the agarose gel electrophoresis as a highlight strip.
The fluorescent primers and the common primers were synthesized by Shanghai Senno Biotechnology GmbH, the primer nucleic acid sequences are shown in Table 1, the PCR reaction system is shown in Table 2, wherein the DNA concentration is 50ng/μ L (concentration after dilution), 10 XBuffer, dNTP and Taq enzyme are purchased from Bao bioengineering (Dalian) GmbH, and the product numbers are respectively: 9153A, D4030A and DRR20AM were subjected to fluorescent primer PCR amplification using a PCR instrument (ABI-2720, Applied Biosystems, USA) using 3 pairs of primers, respectively, and the reaction procedures are shown in Table 3.
TABLE 1 Mini pig breed specific 3 pairs of microsatellite primer information for experiments
Wherein F7 starts at 263106613bp of chromosome 1 and R7 starts at 263106417bp of chromosome 1; f9 starts at 4160895bp on chromosome 1, and R9 starts at 4160775bp on chromosome 1; f19 starts at 111498734bp on chromosome 13 and R9 starts at 111498933bp on chromosome 13.
TABLE 2 PCR reaction System
TABLE 3 PCR reaction procedure
After the PCR reaction is finished, the PCR product is instantaneously separated to remove the tube wall sample, 10 mu L of the PCR product is taken to two new 96-well plates, then cold 70% ethanol is added to the final volume of 50 mu L, and the mixture is shaken and fully mixed. Samples were purified using a bench top high speed refrigerated centrifuge (shanghai science instruments ltd, neogouge 13R) under the following centrifugation conditions: centrifuging at 3700r/min at 4 deg.C for 30min, inverting, instantly removing ethanol, standing for 15min, and volatilizing ethanol.
To the plates where ethanol had evaporated completely, GS-500LIZ molecular internal standard (correct molecular weight range) and HiDi (highly deionized formamide to dissolve DNA), both purchased from ABI, were added, with the respective code numbers: 4322682 and 4311320, shaking, fully mixing uniformly, removing a pipe wall sample instantly, placing the pipe wall sample into the PCR instrument for denaturation at 95 ℃ for 4min, placing the pipe wall sample into ABI 3730XL purchased from Applied Biosystems of America for capillary electrophoresis, and analyzing the obtained electrophoresis result by using GeneMapper software to obtain the fragment size and signal value of 3 microsatellite loci in 160 samples;
counting the segment sizes of the alleles of 160 individuals at microsatellite loci No. 7, 9 and 19, and respectively calculating allele frequencies and genotype frequencies by using formula 1 and formula 2, wherein the results are shown in a table 4;
equation 1:
allele frequency is the ratio of a certain gene in a population relative to the total number of alleles. Formula 1 p (A) A allele frequency; AA: the number of individuals homozygous for the a allele; AB: the number of individuals with the B allele co-occurring with A; AC: the number of individuals with the C allele co-occurring with A; AZ: the number of individuals with the Z allele co-occurring with A; n: the total number of individuals in the population;
equation 2:
genotype frequency is the ratio of a certain genotype in a population to all genotypes in the population. P (AA) in equation 2: AA genotype frequency; AA: number of individuals of AA genotype; n: the total number of individuals in the population;
and (3) displaying a statistical result: (1) among 48 wuzhishan pigs: specific alleles 7G (3 heads), 7K (9 heads) and specific genotype 7F7K (8 heads) were present at microsatellite locus No. 7, specific genotypes 9C9H (6 heads) and 9E9H (8 heads) were present at microsatellite locus No. 9, and specific genotypes 19a19D (6 heads) and 19C19D (21 heads) were present at microsatellite locus No. 19; (2)15 Bama pigs: the presence of the specific alleles 7D (2 heads), 7J (13 heads) and the specific genotypes 7C7J (5 heads), 7F7J (6 heads) at the microsatellite locus No. 7 and the presence of the specific genotype 19C19H (2 heads) at the microsatellite locus No. 19; (3) in 50 inbred Luchuan pigs: specific allele 7L (3 heads) at microsatellite locus No. 7, specific allele 9I (50 heads) and specific genotypes 9B9I (19 heads), 9H9I (20 heads) and 9I9I (9 heads) at microsatellite locus No. 9, and specific genotypes 19a19G (9 heads), 19a19H (16 heads), 19G19H (8 heads) and 19H19H (8 heads) at microsatellite locus No. 19; (4)47 Tibetan pigs: at microsatellite locus No. 7, there is a specific allele 7A (11 heads), and at microsatellite locus No. 9, there are specific genotypes 9A9F (8 heads), 9E9F (11 heads) and 9F9F (8 heads). Microsatellite fragment sizes and corresponding allele frequencies, specific alleles and genotype frequencies are shown in Table 4.
TABLE 44 alleles, allele frequencies, idiotypic and genotype frequencies at 3 microsatellite loci in experimental piglet species
Specific alleles and specific genotypes were obtained at 3 pairs of microsatellite loci based on 160 experimental minipig individuals, and the following criteria were developed for identifying 4 experimental minipig breeds: if the individual to be identified has the 7G or 7K allele at microsatellite locus No. 7, or the specific genotype 7F7K at microsatellite locus No. 7, or the specific genotype 9C9H or 9E9H at microsatellite locus No. 9, or the specific genotype 19A19D or 19C19D at microsatellite locus No. 19, the individual to be identified is a Wuzhishan pig;
if the individual to be identified has a 7D or 7J allele at microsatellite locus No. 7, or a specific genotype 7C7J or 7F7J at microsatellite locus No. 7, or a specific genotype 19C19H at microsatellite locus No. 19, then the individual to be identified is a boar;
if the individual to be identified has a 7L allele at the microsatellite locus No. 7, or a 9I allele at the microsatellite locus No. 9, or a specific genotype 9B9I, 9H9I or 9I9I at the microsatellite locus No. 9, or a specific genotype 19A19G, 19A19H, 19G19H or 19H19H at the microsatellite locus No. 19, the individual to be identified is an inbred Luchwa pig;
if the individual to be identified has the 7A allele at the microsatellite locus 7 or the specific genotype 9A9F, 9E9F or 9F9F at the microsatellite locus 9, the individual to be identified is a Tibetan pig.
Example 2
Ear tissues of 10 experimental miniature pig individuals are collected, the 10 pigs are used as individuals to be detected for breed identification, the individuals are numbered as #1 to #10, and breed information (including that #1 to #3 are Wuzhishan pigs, #4 are Bama pigs, #5 to #7 are inbred Luchuan pigs and #8 to #10 are Tibetan pigs) corresponding to each individual number is recorded so as to be compared with the identification result of the microsatellite. Each individual ear tissue sample is collected and placed in an EP tube filled with 75% ethanol, a phenol chloroform method is adopted for extracting genome DNA, the concentration, OD value and DNA quality of DNA are detected by using NanoDrop 2000 (Thermo Fisher Scientific Co., U.S.A.), 1% agarose gel electrophoresis and gel imaging (Beijing Zhonglilan Bio-technology Co., Ltd., Newbio Gi-1), and the following operations are carried out on the DNA with the DNA concentration of more than or equal to 50 ng/mu L, OD260/280 being between 1.6 and 1.8 and the DNA with the imaging of agarose gel electrophoresis being a highlight strip.
The fluorescent primers and the common primers were synthesized by Shanghai Senno Biotechnology GmbH, the primer nucleic acid sequences are shown in Table 1, the PCR reaction system is shown in Table 2, wherein the DNA concentration is 50ng/μ L (concentration after dilution), 10 XBuffer, dNTP and Taq enzyme are purchased from Bao bioengineering (Dalian) GmbH, and the product numbers are respectively: 9153A, D4030A and DRR20AM were subjected to fluorescent primer PCR amplification using a PCR instrument (ABI-2720, Applied Biosystems, USA) using 3 pairs of primers, respectively, and the reaction procedures are shown in Table 3.
After the PCR reaction, the PCR product was centrifuged to remove the tube wall sample, 10. mu.L of the PCR product was transferred to two new 96-well plates, then cold 70% ethanol was added to a final volume of 50. mu.L, and the mixture was shaken and mixed well. Samples were purified using a bench top high speed refrigerated centrifuge (shanghai science instruments ltd, neogouge 13R) under the following centrifugation conditions: centrifuging at 3700r/min at 4 deg.C for 30min, inverting, instantly removing ethanol, standing for 15min, and volatilizing ethanol.
Adding GS-500LIZ molecular internal standard and HiDi into a plate with completely volatilized ethanol, wherein the internal standard and HiDi are purchased from ABI and the product numbers are respectively as follows: 4322682 and 4311320, shaking, mixing well, removing the sample of the tube wall instantly, putting the sample into the PCR instrument for denaturation at 95 ℃ for 4min, putting the sample into ABI 3730XL purchased from Applied Biosystems of America for capillary electrophoresis, analyzing the electrophoresis result by GeneMapper software to obtain the fragment size and signal value of 3 microsatellite loci in 10 samples, and obtaining the alleles corresponding to the microsatellite loci corresponding to the Table 4.
The results showed that individual #1 had allele 7K at microsatellite locus No. 7; individual #2 presented genotype 9E9H at the microsatellite locus No. 9; individual #3 had genotype 19C19D at the microsatellite locus No. 19; #4 allele 7J at microsatellite locus No. 7; individual #5 presented allele 9I and genotype 9B9I at microsatellite locus No. 9; individual #6 presented allele 9I and genotype 9H9I at microsatellite locus No. 9; individual #7 presented allele 9I and genotype 9I9I at microsatellite locus No. 9; individual #8 had allele 7A at microsatellite locus No. 7; individual #9 presented genotype 9E9F at the microsatellite locus No. 9; individual #10 presented genotype 9A9F at the microsatellite locus No. 9.
Finally, in this experimental minipig, #1- #3 was Wuzhishan pig, #4 was Bama pig, #5- #7 was inbred Luchuan pig and #8- #10 was Tibetan pig.
Sequence listing
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Claims (1)
1. A method for identifying a miniature pig breed for experiment is characterized by comprising the following steps:
step S1: collecting a tissue sample of a minipig individual to be identified for experiment, and carrying out DNA extraction and quality detection, wherein the breed range of the minipig individual to be identified for experiment is Wuzhishan pig, Bama pig, inbred Luchuan pig or Tibet pig;
step S2: carrying out PCR amplification by using a specific microsatellite primer composition of a miniature pig variety for identification experiments to obtain a PCR amplification product;
the primer composition comprises 3 pairs of primers which respectively correspond to microsatellites No. 7, 9 and 19, and specifically comprises the following components:
the forward fluorescent primer F7 shown as SEQ ID NO: 1: ATCCCCCACTCATTCTCACA, respectively;
the reverse common primer R7 shown in SEQ ID NO. 2: GCAAAACTGCACACAGCCTA, respectively;
a forward fluorescent primer F9: TCCAGGCCCAGGTTGTATTA as shown in SEQ ID NO. 3;
a reverse common primer R9: AGGATCTTTTTCGGCAGGAT shown as SEQ ID NO. 4;
forward fluorescent primer F19: CAGCTTACAGCAAAAGTAGCCA as shown in SEQ ID NO. 5;
a reverse common primer R19: AAGATGTTTCATCAGACTATATCCGT as shown in SEQ ID NO 6;
step S3: taking part of PCR amplification products to perform capillary electrophoresis, and determining the fragment size and the signal value of a corresponding site according to the molecular internal standard and HiDi;
step S4: counting corresponding alleles and genotypes according to the sizes of the gene fragments of the experimental miniature pig individuals to be identified on the microsatellite loci No. 7, 9 and 19, and calculating the varieties of the experimental miniature pig individuals to be identified according to the alleles and the genotypes;
wherein, the allele sizes corresponding to the allele numbers on the microsatellite loci No. 7, No. 9 and No. 19 are shown as above;
and (5) identifying the varieties of the experimental miniature pig individuals according to the alleles and genotypes obtained in the step S4 according to the following standards:
if the individual to be identified has the 7G or 7K allele at microsatellite locus No. 7, or the specific genotype 7F7K at microsatellite locus No. 7, or the specific allele 9D at microsatellite locus No. 9, or the specific genotype 9C9H or 9E9H at microsatellite locus No. 9, or the specific allele 19F at microsatellite locus No. 19, or the specific genotype 19A19D or 19C19D at microsatellite locus No. 19, the individual to be identified is a five-finger mountain pig;
if the individual to be identified has a 7D or 7J allele at the microsatellite locus No. 7, or a specific genotype 7C7J or 7F7J at the microsatellite locus No. 7, or a specific genotype 19C19H at the microsatellite locus No. 19, then the individual to be identified is a babassu pig;
if the individual to be identified has a 7L allele at the microsatellite locus No. 7, or a 9I allele at the microsatellite locus No. 9, or a specific genotype 9B9I, 9H9I or 9I9I at the microsatellite locus No. 9, or a specific genotype 19A19G, 19A19H, 19G19H or 19H19H at the microsatellite locus No. 19, the individual to be identified is an inbred Luchwa pig;
if the individual to be identified has the 7A allele at the microsatellite locus 7 or the specific genotype 9A9F, 9E9F or 9F9F at the microsatellite locus 9, the individual to be identified is a Tibetan pig.
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