CN106755417B - Method for detecting genetic quality of closed-herd Wuzhishan miniature pigs - Google Patents
Method for detecting genetic quality of closed-herd Wuzhishan miniature pigs Download PDFInfo
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Abstract
The invention discloses a method for detecting genetic quality of a closed group Wuzhishan miniature pig, which comprises the following steps: collecting 60 parts of ear skin tissue samples from Wuzhishan pigs, and placing the ear skin tissue samples in a 1.5mL disinfection centrifuge tube, and storing the ear skin tissue samples in a refrigerator at the temperature of-20 ℃ for later use; extracting genomic DNA of the Wuzhishan pig according to a method in a Tiangen DNA extraction kit, dissolving in TE, and storing at-20 ℃; double detection of the purity and concentration of DNA by agarose gel electrophoresis and NanoDrop 2000; carrying out multiplex PCR amplification on 33 microsatellite marker primers; performing gene scanning on the multiple PCR products, analyzing SSR data by using Genemapper software, and calibrating the size and the genotype of the fragments; statistical analysis of the data was performed using Popgene32 software. The invention provides reference data for the genetic breeding and the genetic quality evaluation of the population; also provides a theoretical basis for the protection, breeding and utilization of germplasm resources of the Wuzhishan pigs.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for detecting genetic quality of a closed herd Wuzhishan miniature pig.
Background
The Wuzhishan pig is a very famous miniature pig variety in China, not only has the characteristics of good meat quality, coarse feeding resistance, stress resistance, strong adaptability, easy feeding management and the like, but also has the characteristics of small size, organ size and physiological characteristics close to those of human beings, is one of the first-choice animals as a human experiment model, is intensively fed in a Wuzhishan pig breeding farm in Lingshan town in the suburb of Haikou after being introduced from an original production place in 1998, and is bred for nearly 20 years according to a closed group breeding standard (Daphna ruigensis and the like, 2015). 11 days 7 and 2014, experimental animal production licenses issued by the scientific and technical hall of Hainan province and obtained by animal husbandry and veterinary research of agricultural academy of Hainan province, and license numbers are as follows: SCXK (Qiong) 2014-. Microsatellite DNA is a segment tandem repeat sequence widely distributed in eukaryotic genomes, the core of the microsatellite DNA is usually composed of multiple tandem repeats taking 1-6 nucleotides as units, and the microsatellite DNA has the characteristics of large quantity, wide distribution, rich polymorphism, codominant inheritance and the like. Microsatellite DNA markers are one of the most commonly used molecular markers in population genetics studies [ KimT H, et. al,2005, Sahoo N R, et. al,2015 ]. The analysis of microsatellite markers is generally carried out by combining polyacrylamide electrophoresis with silver staining for genotyping, is easily influenced by a plurality of factors such as voltage, electrophoresis gel and experimenters, cannot be accurately detected in a large scale and automatically, and the multiplex PCR and fluorescence marker-semi-automatic genotyping technology can realize rapid, simple and reliable detection with stable and reliable results on the DNA level, and is widely applied to the aspects of gene positioning, germplasm identification, genetic diversity analysis, genetic map construction and the like [ Sahoo, N.R., et al,2015.Cort é s O, et al.2016 ]. The Wuzhishan miniature pig is widely applied to various fields of biomedical research such as tumors, cardiovascular diseases, diabetes, skin burns, new drug safety evaluation and the like at present, and because the quality problem of the pig directly influences the accuracy of medical and biological research results, genetic monitoring is an effective measure for evaluating and ensuring the genetic quality of miniature pig groups. With the development of molecular biology techniques, genetic markers appear at the DNA molecular level. The microsatellite DNA marker is considered to be the most valuable one of various molecular genetic markers by the characteristics of wide distribution, high polymorphism, easy communication, rapid determination and the like of the microsatellite DNA marker with high information content (Tautz D, 1989). However, from the reported data of the existing domestic and foreign microsatellite analysis of miniature pig population genetics, the applied microsatellite loci are not uniform, and the polymorphism of some microsatellite loci is poor, so that the selection of proper microsatellite loci and the number thereof are the key for establishing a genetic detection method. Barker (1994) suggested that in order to have a certain accuracy of the results, the number of microsatellites used for population genetics testing should not be less than 26, not linked to each other, with at least 4 alleles per marker.
In summary, in the current report data of applying microsatellites at home and abroad to analyze the inheritance of the miniature pig population, the applied microsatellite loci are extremely non-uniform, and the polymorphism of some microsatellite loci is poor (Shanghai Tao, 2003, Zhang Gui Xiang, 2003).
Disclosure of Invention
The invention aims to provide a method for detecting the genetic quality of a miniature pig in a closed group Wuzhishan, which aims to solve the problems that the applied microsatellite loci are not uniform and the polymorphism of some microsatellite loci is poor in the current report data of analyzing the miniature pig group heredity by applying microsatellites at home and abroad.
The invention is realized in such a way that the method for detecting the genetic quality of the miniature pig in the closed group Wuzhishan comprises the following steps:
step one, collecting 60 ear skin tissue samples of Wuzhishan pigs, putting the samples into a 1.5mL disinfection centrifuge tube, and taking the samples back to a laboratory for storage in a refrigerator at minus 20 ℃ for later use;
extracting the genomic DNA of the Wuzhishan pig according to a method in a Tiangen DNA extraction kit, dissolving in TE, and storing at-20 ℃; double detection of the purity and concentration of DNA by agarose gel electrophoresis and NanoDrop 2000;
step three, carrying out multiple PCR amplification on 33 microsatellite marker primers;
performing gene scanning on the multiple PCR products, analyzing SSR data by using Genemapper software, and calibrating the size and the genotype of the fragments;
and fifthly, performing statistical analysis on the data by using Popgene32 software, and inputting the genotypes of all the microsatellite loci of all the samples into a data file of Popgene32 software in the forms of ab, bb and the like.
Further, the Wuzhishan pigs are bred in a closed mode for nearly 20 years according to a random mating principle and are bred to more than 3 generations.
Furthermore, the PCR reaction system for multiplex PCR amplification is 20 ul, 1.0 ul DNA template,2.5 μ l TaqBuffer, 0.5 μ l dNTP 10mM, 0.5 μ l primer, 0.5 μ l downstream primer, 0.5 μ l TaqaDNA polymerase, and finally supplementing H2O to 20 μ l; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 10 cycles; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles; finally, repairing and extending for 6min at 72 ℃.
Further, calculating the gene frequency, the average observed allele factor, the average effective allele factor, the bloom index and the average effective heterozygosity of the sample on each microsatellite locus in the fifth step; population P values were calculated using Cervus 3.0 software.
The method for detecting the genetic quality of the closed group Wuzhishan miniature pig adopts a multiple PCR scanning method to analyze genetic polymorphism of 33 microsatellite loci of the closed group Wuzhishan miniature pig, and statistics is carried out on heterozygosity and expected heterozygosity of each gene locus, effective allele factors, polymorphic information content, fixed index and Shannon information entropy. The mean allele factor for the 33 microsatellite loci in the WZSP closed group was 6.42, the mean heterozygosity was 0.66, and the mean heterozygosity was expected to be 0.69. In addition, the average value of polymorphic information content at 33 sites in the total population was 0.65, and the average fixed index was 0.05. The method shows that the enclosed group Wuzhishan miniature pig group has certain genetic stability, accords with the genetic structure characteristics of enclosed group animals, and the selected microsatellite locus can be used for Wuzhishan genetic diversity evaluation. Meanwhile, a theoretical basis is provided for the protection, breeding and utilization of germplasm resources of the Wuzhishan pigs.
The selected microsatellite loci PIC are all larger than 0.25, the PICs of 29 loci are all larger than 0.50, the average PIC is 0.65, the high-polymorphism is high, and the polymorphism information content of the microsatellite loci PIC is more abundant than that of the core group and the inbred line of the original Wuzhishan miniature pigs (yellow etiquette, 2005), and the microsatellite loci PIC is also superior to the closed group of the small pigs in the real estate and the small pigs in the Guangxi Bama (yellow Jianfang, 2015 Weijie, 2013). Compared with the previous research, the method is more suitable for the characteristics of the closed group, and fully reflects that the gene frequency of the group of the miniature pigs of the closed group of the Wuzhishan pigs tends to be stable in the long-term closed breeding process, and a new closed group is basically formed. In addition, in genetic linkage analysis, the PIC value of the microsatellite locus which can be used as an ideal selection marker is more than 0.7. The size of the PIC value can also show the availability and use efficiency of the locus, and in a population, the greater the PIC of a locus, the higher the heterozygote proportion, the more genetic information is provided, and the greater the selection potential is. The content of polymorphic information of the SW886 locus is 0.81 at most, which indicates that the genetic information of the Wuzhishan pig closed group at the locus is richer and has larger selection potential compared with other loci. The information entropies of 33 microsatellite loci shannon are all distributed between 0.71 and 2.07, which indicates that WZSP closed group populations are poor in genetic diversity at some loci. The heterozygosity, the expected heterozygosity, the polymorphic information content and the Shannon information entropy of the SWR186 and SW886 sites are highest in 33 microsatellite sites, which indicates that the WZSP closed group has the most abundant diversity in the two sites. And in the 33 sites, the fixing index of 16 sites is negative, the fixing index of other sites is positive, and the average fixing index is 0.05, which indicates that the WZSP closed group population is in a heterozygosity deletion state, and the inbreeding phenomenon of different degrees exists in the population. The suggestion is that in the artificial breeding process of the Wuzhishan pigs, the selection strength still needs to be increased, and the gene exchange needs to be enhanced, so that more excellent varieties with high production performance are bred. The gene balance of a population at a certain site is mainly influenced by factors such as mutation, migration, drift, selection, and founder effect. In practical production, artificial selection is an important factor influencing the gene balance of a population, and when a quantitative trait is subjected to targeted artificial selection, the result of the targeted artificial selection inevitably causes the gene balance of the major gene of the quantitative trait and other genes or genetic markers connected with the major gene to be broken. Chi 2 fitness tests showed that the Wuzhishan pigs reached Hardy-Weinberg equilibrium at 33 loci in the total population.
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FIG. 1 is a flow chart of a method for detecting the genetic quality of a Wuzhishan miniature pig provided by an embodiment of the invention.
FIG. 2 is a schematic diagram of the Sw902 agarose gel electrophoresis of the microsatellite PCR amplification product provided by the embodiment of the invention;
in the figure: 1-6: PCR products of different samples; m: marker of DL 2000.
FIG. 3 is a typing diagram of a microsatellite PCR amplification product STR provided by an embodiment of the invention;
in the figure: a: s0062; b: s0068; c: s0070; d: s0091.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
As shown in fig. 1, the method for detecting the genetic quality of the minipig in Wuzhishan provided by the embodiment of the present invention includes the following steps:
s101: collecting 60 ear skin tissue samples from Wuzhishan pigs, placing the samples in a 1.5mL disinfection centrifuge tube, and taking the samples back to a laboratory for storage in a refrigerator at-20 ℃ for later use;
s102: extracting genomic DNA of the Wuzhishan pig according to a method in a Tiangen DNA extraction kit, dissolving in TE, and storing at-20 ℃; double detection of the purity and concentration of DNA by agarose gel electrophoresis and NanoDrop 2000;
s103, performing multiplex PCR amplification on 33 microsatellite marker primers;
s104, performing gene scanning on the multiple PCR products, analyzing SSR data by using Genemapper software, and calibrating the size and the genotype of the fragments;
s105, performing statistical analysis on the data by using Popgene32 software, inputting the genotype of each microsatellite locus of all samples into a data file of Popgene32 software in the forms of ab, bb and the like, and calculating the gene frequency, the average observed allele factor, the average effective allele factor (Ne), the bloom index, the average effective heterozygosity (H) and the like of the samples on each microsatellite locus; population P values were calculated using Cervus 3.0 software.
The application principle of the present invention will be further described with reference to experiments.
1 materials and methods
1.1 Experimental animals
60 ear skin tissue samples are collected from a permanent hair base experimental animal breeding field (production permit number SCXK 2014-. The variety is bred in a closed breeding field for nearly 20 years according to a random mating principle and is bred to more than 3 generations.
1.2 Primary reagents
TaqDNA polymerase, 10 × PCR Buffer (without Mg)2+:100mM Tris-HCl pH 8.8at25℃；500mM KCl,0.8％(v/v)Nonidet)；MgCl2(25mM), dNTP (10mM), Marker, 6 × DNA Loading Dye (Bio/Rad), HIDI, LIZ500 (ABI, USA).
1.3 extraction of DNA
Extracting genomic DNA of Wuzhishan pig according to the method in the Tiangen DNA extraction kit, dissolving in TE, and storing at-20 ℃. The purity and concentration of the DNA were determined by agarose gel electrophoresis and NanoDrop 2000. The electrophoresis results of the samples of the genomic DNA fractions are shown in FIG. 2.
1.4 microsatellite primers
The 33 pairs of microsatellite primers and fluorescent markers selected in this experiment were all referenced (xu ling et al, 2009. Weijie, 2013) synthesized by Shanghai Biotechnology Ltd.
1.5 multiplex PCR amplification of microsatellite loci
Multiplex PCR amplification was performed with 33 microsatellite tagged primers. The PCR reaction system is 20 mul, DNA template is 1.0 mul, TaqBuffer is 2.5 mul, dNTP is 10mM is 0.5 mul, primer is 0.5 mul, downstream primer is 0.5 mul, TaqaDNA polymerase is 0.5 mul, and H is supplemented finally2O to 20. mu.l. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 10 cycles; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles; finally, repairing and extending for 6min at 72 ℃.
1.6 microsatellite gene scanning
And (3) sending the multiple PCR products to Shanghai Biometrics limited company for gene scanning, using a 3730XL sequencer, analyzing SSR data by using Genemapper software, and calibrating the size and the genotype of the fragment.
1.7 data statistics and analysis
Statistical analysis of the data is carried out by using Popgene32 software, the genotype of each microsatellite locus of all samples is input into a data file of Popgene32 software in the form of ab, bb and the like, and the gene frequency, the average observed allele factor, the average effective allele factor (Ne), the bloom index, the average effective heterozygosity (H) and the like of the samples on each microsatellite locus are calculated. Population P values were calculated using Cervus 3.0 software.
2 results and analysis
2.1PCR product detection
The PCR product was electrophoresed on a 1.5% agarose gel, and the results are shown in FIG. 2. STR typing of PCR products after ABI3730 XLNedic Analyzer sequencing is shown in FIG. 3. Each peak represents the size and genotype of closed group microsatellite locus alleles of the Wuzhishan pigs.
2.2 allele frequencies and allelic distributions
The allele factors, allele sizes and highest frequency alleles of the 33 microsatellite loci of the Wuzhishan pig closed herd are shown in Table 1. As can be seen from Table 1, 212 alleles were detected in a total of 33 microsatellite loci from 60 Wuzhishan pigs, averaging 6.42 alleles per locus. The number of detected alleles on the SWR158 site is 12, the variation range is 164-216 bp, and the allele frequency of 170bp is 0.3667; the 33 microsatellite loci have polymorphism in the Wuzhishan pigs, and can be used for analyzing the genetic characteristics of the Wuzhishan pigs. As can be seen from Table 2, the allele frequencies of 33 microsatellite loci are different, and the allele frequencies of each locus are lower except for a few alleles.
TABLE 1WZSP closed group 33 microsatellite locus allelic factors, effective allelic factors, sizes, highest frequency alleles and frequencies thereof
2.3 analysis of genetic variation in the population of closed herds of Wuzhishan pigs
The genetic diversity indices for the 33 microsatellite loci are shown in Table 3. Table 3 shows that the lowest value and the highest value of the apparent purity of the 33 microsatellite loci in the WZSP closed group are 0.10 and 0.82 respectively, and the average value is 0.34; the apparent heterozygosity is 0.22-0.90, and the average value is 0.66. The lowest and highest values of the desired degree of purity are 0.16 and 0.69, respectively; the expected heterozygosity lowest and highest values are 0.31 and 0.84, respectively, with an average value of 0.67. In addition, the total population had an average of polymorphic information content at 35 sites of 0.69, and was highly polymorphic at 29 sites. In the 33 sites, the fixed index of 16 sites is a negative value, the fixed index of other sites is a positive value, and the average fixed index is 0.05; the heterozygosity, expected heterozygosity, polymorphic information content and Shannon information entropy of the SWR186 and SW886 sites are all the highest, and the WZSP closed group is most abundant in the 33 microsatellite sites. P >0.05 was also found at 33 sites in the total population in Hardy-Weinberg equilibrium test of the population, according to the laws of genetic equilibrium.
TABLE 2 allele frequencies of 33 microsatellite loci of WZSP closed group
TABLE 3 genetic diversity index for WZSP closed group 33 microsatellite loci
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A method for detecting genetic quality of a closed group Wuzhishan miniature pig is characterized by comprising the following steps:
step one, collecting 60 ear skin tissue samples of Wuzhishan pigs, putting the samples into a 1.5mL disinfection centrifuge tube, and taking the samples back to a laboratory for storage in a refrigerator at minus 20 ℃ for later use;
extracting the genomic DNA of the Wuzhishan pig according to a method in a Tiangen DNA extraction kit, dissolving in TE, and storing at-20 ℃; double detection of the purity and concentration of DNA by agarose gel electrophoresis and NanoDrop 2000;
step three, carrying out multiple PCR amplification on 33 microsatellite marker primers; the 33 microsatellite markers OPEN, S0005, S0007, S0036, S0062, S0068, S0070, S0091, S0218, SW2, SW29, SW61, SW81, SW205, SW240, SW271, SW787, SW857, SW886, SW902, SW911, SW919, SW936, SW974, SW1066, SW1089, SW1092, SW1119, SW1374, SW2409, SWR158, SWR1008, SWR 1120;
performing gene scanning on the multiple PCR products, analyzing SSR data by using Genemapper software, and calibrating the size and the genotype of the fragments;
step five, performing statistical analysis on the data by using Popgene32 software, and inputting the genotype of each microsatellite locus of all samples into a data file of Popgene32 software in the form of ab and bb;
the Wuzhishan pigs are bred in a closed mode for nearly 20 years according to a random mating principle and are bred to more than 3 generations.
2. The method for detecting the genetic quality of a closed-group Wuzhishan miniature pig as claimed in claim 1, wherein the PCR reaction system for multiplex PCR amplification is 20 μ l, the DNA template is 1.0 μ l, the Taq Buffer is 2.5 μ l, the dNTP is 10mM 0.5 μ l, the upstream primer is 0.5 μ l, the downstream primer is 0.5 μ l, the Taqa DNA polymerase is 0.5 μ l, and finally H is supplemented2O to 20 μ l; reaction ofThe conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 10 cycles; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles; finally, repairing and extending for 6min at 72 ℃.
3. The method for genetic quality testing of a closed herd of Wuzhishan miniature pigs according to claim 1, wherein in step five the genetic frequency, the mean observed allele factor, the mean effective allele factor, the Conlon index, the mean effective heterozygosity of the sample at each microsatellite locus are calculated; population P values were calculated using Cervus 3.0 software.
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