CN106755417A - A kind of method of closed colony WZSP Genetic monitoring - Google Patents
A kind of method of closed colony WZSP Genetic monitoring Download PDFInfo
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Abstract
The invention discloses a kind of method of closed colony WZSP Genetic monitoring, including：Wuzhi Mountain pig gathers 60 parts of ear skin histology samples, is placed in 1.5mL sterilization centrifuge tubes, and 20 DEG C of Refrigerator stores are standby；Wuzhi Mountain pig genomic DNA is extracted according to the method in Tiangeng DNA extraction kit, is dissolved in TE, 20 DEG C of preservations；With agarose gel electrophoresis and the purity and concentration of NanoDrop2000 double checks DNA；33 microsatellite marker primers carry out multiplexed PCR amplification；Multiple PCR products are carried out into genescan, using Genemapper software analysis SSR data, clip size and genotype is demarcated；Statistical analysis is carried out to data with Popgene32 softwares.The present invention is that the genetic breeding of colony and quality of heredity evaluation provide reference；Also for protection, seed selection and the utilization of the germ plasm resource of Wuzhi Mountain pig provide theoretical foundation.
Description
Technical field
The invention belongs to gene engineering technology field, more particularly to a kind of closed colony WZSP Genetic monitoring
Method.
Background technology
The very famous small-sized pig variety of Wuzhi Mountain Zhu Shi China, not only with meat is good, resistance to thick feeding, resistance and adaptation
Property it is strong, the features such as be easy to feeding and management, it is often more important that it is small, organ size and physiological property are approached with the mankind, is work
One of first-selected animal for mankind's test model, raising is being concentrated in suburb of Haikou City Lingshan town five after being introduced a fine variety from original producton location within 1998
Refer to that the self-closing breeding nearly 20 years (Sun Ruiping etc., 2015) of standard is bred in mountain Zhu Zhong pig farms according to closed colony.On July 11st, 2014, sea
Southern Shanxi Academy of Agricultural Sciences's animal and veterinary research institute obtains the experimental animal production licence that Hainan Department of Science and Technology of Shanxi Province issues, license
Card number is：SCXK (fine jade) 2014-0006.Microsatellite DNA is the section tandem sequence repeats sequence being distributed widely in eukaryotic gene group
Row, its core generally by 1-6 nucleotides for the multiple tandem sequence repeats of unit are constituted, with quantity is big, distribution extensively, polymorphism it is rich
The features such as richness, codominant inheritance.Microsatellite DNA mark is one of most-often used molecular labeling in population genetic study [Kim
T H,et.al,2005,Sahoo N R,et.al,2015].The analysis of microsatellite marker typically more uses polyacrylamide gel electrophoresis
Carry out Genotyping with reference to silver staining, easily influenceed by factors such as voltage, running gel and experimenters, it is impossible to accurately, on a large scale,
Aulomatizeted Detect, and multiplex PCR and fluorescence labeling-semi-automatic genotyping technique can be realized on DNA level it is fast and convenient
And the reliable and stable detection of result, at aspects such as the assignment of genes gene mapping, Germplasm Identification, analysis of genetic diversity, genetic map constructions
It is used widely [Sahoo, N.R., et, al, 2015.Cort é s O, et, al.2016].WZSP is wide at present
The general numerous areas for being applied to the biomedical researches such as tumour, angiocarpy, diabetes, skin burn, new drug safety evaluatio, by
The accuracy of Medical Biology result of study has been directly influenced in the quality problems of pig, and genetic monitoring is to evaluate and ensure small
The effective measures of type swinery body quality of heredity.With the development of Protocols in Molecular Biology, the horizontal genetic marker of DNA molecular goes out in succession
It is existing.Microsatellite DNA mark, with its information content (Tautz D, 1989) widely distributed, high polymorphism high, is easy to exchange, surveys
Fixed quick the features such as is considered as one kind of most worthy in all kinds of molecular genetic markers.However, micro- from current domestic and international application
Satellite seen to the report data that miniature pig population genetic is analyzed, the microsatellite locus pole disunity applied, and certain is defended slightly
Championship point polymorphism is poor, therefore selects suitable microsatellite locus and quantity, is the key for setting up Genetic Detection method.
Barker (1994) proposes that in order that result has certain accuracy the microsatellite number for Population Genetics detection should
It is not chain each other less than 26, each at least 4 allele of mark.
In sum, current domestic and international application microsatellite sees to the report data that miniature pig population genetic is analyzed, institute
The microsatellite locus pole disunity of application, some microsatellite locus polymorphisms are poor (commercial circles great waves, 2003, Zhang Guixiang, 2003).
The content of the invention
It is an object of the invention to provide a kind of method of closed colony WZSP Genetic monitoring, it is intended to solve
Current domestic and international application microsatellite sees to the report data that miniature pig population genetic is analyzed, the microsatellite locus pole applied
Disunity, the poor problem of some microsatellite locus polymorphisms.
The present invention is achieved in that a kind of method of closed colony WZSP Genetic monitoring, the closing
The method of group's WZSP Genetic monitoring is comprised the following steps：
Step one, Wuzhi Mountain pig gathers 60 parts of ear skin histology samples, and sample is placed in 1.5mL sterilization centrifuge tubes, takes back reality
Room is tested, -20 DEG C of Refrigerator stores are standby；
Step 2, Wuzhi Mountain pig genomic DNA is extracted according to the method in Tiangeng DNA extraction kit, is dissolved in TE ,-
20 DEG C of preservations；With agarose gel electrophoresis and the purity and concentration of NanoDrop2000 double checks DNA；
Step 3,33 microsatellite marker primers carry out multiplexed PCR amplification；
Multiple PCR products are carried out genescan by step 4, use Genemapper software analysis SSR data, demarcation piece
Duan great little and genotype；
Data are carried out statistical analysis by each microsatellite locus of all samples by step 5 with Popgene32 softwares
Genotype with ab, the form such as bb is input into the data file of Popgene32 softwares.
Further, the Wuzhi Mountain pig carries out closing and breeds nearly 20 years according to panmixia principle, bred to 3 generations with
On.
Further, the PCR reaction systems of the multiplexed PCR amplification are 20ul, DNA profiling 1.0ul, Taq Buffer
The μ l of 2.5ul, dNTP 10mM 0.5, on have primer 0.5ul, anti-sense primer 0.5ul, TaqaDNA polymerase 0.5ul, finally mend
H2O to 20ul；Reaction condition is：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, are carried out
10 circulations；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, carry out 20 circulations；Repair for last 72 DEG C and extend
6min。
Further, gene frequency, average observation equipotential base of the sample on each microsatellite locus are calculated in the step 5
Factor, average effective number of alleles, fragrant grand index, average effective heterozygosity；Colony P is calculated using the softwares of Cervus 3.0
Value.
The method of the closed colony WZSP Genetic monitoring that the present invention is provided, using multiplex PCR scan method
33 microsatellite locus to closed colony WZSP carry out analysis of genetic polymorphisms, have counted the heterozygosis of each locus
Degree and expectation heterozygosity, effective number of allele, polymorphism information content, fixation index and Shannon comentropies.33 microsatellites
Site average number of alleles in WZSP closed colonies is 6.42, and average heterozygosity is 0.66, expects that heterozygosity average value is
0.69.Additionally, total group is 0.65 in 33 average values of site polymorphism information content, average fixation index is 0.05.Show
Closed colony WZSP colony has certain genetic stability, meets the genetic structure feature of closed colony animal, selected
Microsatellite locus can be used for Wuzhi Mountain genetic diversity assessment.Also be simultaneously the protection of germ plasm resource of Wuzhi Mountain pig, seed selection and
Using providing theoretical foundation.
Microsatellite locus PIC selected by the present invention is all higher than 0.25,29 site PIC and is all higher than 0.50, and average PIC is
0.65, belong to highly polymorphic, abundanter than former WZSP core group and inbred strais polymorphism information content (Huang Liguang,
2005) also superior to Fangshan closed colony miniature pig and Guangxi Bama Mini-pig closed colony (Huang Jianfang, 2015. Wei Jie, 2013).Compared with
Conventional research more meet the feature of closed colony, this also fully reflects Wuzhi Mountain pig closed colony miniature pig and is cultivated in long-term closing
During colony's gene frequency tended towards stability, basically formed a new closed colony.In addition, in genetic linkage analysis,
The microsatellite locus that can be marked as ideal chose, its PIC value should be greater than 0.7.What the size of PIC values can also show site can
With property and service efficiency, in a colony, PIC is bigger in certain site, and its heterozygote ratio is then higher, there is provided hereditary information just
More, Selection potentiaD is also bigger.The polymorphism information content of site SW886 is up to 0.81, shows Wuzhi Mountain pig closed colony at this
The hereditary information in site more horn of plenty, then has bigger Selection potentiaD compared to other sites.33 microsatellite positions in the present invention
Point shannon comentropies are distributed between 0.71~2.07, show WZSP closed colonies colony in some site genetic diversities
It is barren.In 33 microsatellite locus, heterozygosity in SWR186 and SW886 sites, expect heterozygosity, polymorphism information content and
The equal highest of Shannon comentropies, illustrates that WZSP closed colonies have diversity the abundantest in two sites.33 sites
In, 16 site fixation indexs are negative value, and other sites are on the occasion of average fixation index is 0.05, and this shows that WZSP is closed
Group colony is in heterozygosity miss status, there is different degrees of inbred phenomenon in colony.Prompting is to Wuzhi Mountain pig
During artificially breeding, it is still necessary to increase selection dynamics, strengthen gene exchange, so as to select more excellent high productivity energy
Kind.The shadow of the factors such as genic balance of the colony in certain site is mainly mutated, migrated, drift, selection and founder effect
Ring.In actual production, artificial selection is a key factor for influenceing colony's genic balance, and certain quantitative character is determined
During to artificial selection, its result necessarily causes the major gene resistance of the quantitative character and other genes for being attached thereto or genetic marker
Genic balance be broken.The comptibility tests of χ 2 show that Wuzhi Mountain pig reaches Hardy- on total group on 33 sites
Weinberg is balanced.
Brief description of the drawings
Fig. 1 is the method flow diagram of WZSP Genetic monitoring provided in an embodiment of the present invention.
Fig. 2 is microsatellite PCR amplified production Sw902 agarose gel electrophoresis schematic diagrames provided in an embodiment of the present invention；
In figure：1~6：The PCR primer of different samples；M：The marker of DL2000.
Fig. 3 is microsatellite PCR amplified production STR parting figures provided in an embodiment of the present invention；
In figure：A：S0062；B：S0068；C：S0070；D：S0091.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail below in conjunction with the accompanying drawings.
As shown in figure 1, the method for WZSP Genetic monitoring provided in an embodiment of the present invention includes following step
Suddenly：
S101：Wuzhi Mountain pig gathers 60 parts of ear skin histology samples, and sample is placed in 1.5mL sterilization centrifuge tubes, takes back experiment
Room, -20 DEG C of Refrigerator stores are standby；
S102：Wuzhi Mountain pig genomic DNA is extracted according to the method in Tiangeng DNA extraction kit, is dissolved in TE, -20
DEG C preserve；With agarose gel electrophoresis and the purity and concentration of NanoDrop2000 double checks DNA；
S103:33 microsatellite marker primers carry out multiplexed PCR amplification；
S104:Multiple PCR products are carried out into genescan, using Genemapper software analysis SSR data, fragment is demarcated
Size and genotype；
S105:Data are carried out with statistical analysis by each microsatellite locus of all samples with Popgene32 softwares
With ab, the form such as bb is input into the data file of Popgene32 softwares to genotype, calculates gene of the sample on each microsatellite locus
Frequency, average observation number of alleles, average effective number of alleles (Ne), fragrant grand index, average effective heterozygosity (H) etc.；
Colony's P values are calculated using the softwares of Cervus 3.0.
Application principle of the invention is further described with reference to experiment.
1 material and method
1.1 experimental animals
From national Wuzhi Mountain pig conservation Yongfa base experimental animal breeding field (the production licence number SCXK (fine jade) in Hainan
60 parts of ear skin histology samples 2014-0006) are gathered, sample is placed in 1.5mL sterilization centrifuge tubes, takes back laboratory, -20 DEG C of refrigerators
Save backup.The kind carries out closing and breeds nearly 20 years in breeding field according to panmixia principle, has bred to 3 more than generation.
1.2 main agents
Taq DNA polymerase, 10 × PCR Buffer (without Mg2+:100mM Tris-HCl pH 8.8 at25℃；
500mM KCl, 0.8% (v/v) Nonidet)；MgCl2(25mM)、dNTP(10mM)、Marker、6×DNA Loading Dye
(raw work)；HIDI, LIZ500 (American AB I companies).
The extraction of 1.3DNA
Wuzhi Mountain pig genomic DNA is extracted according to the method in Tiangeng DNA extraction kit, is dissolved in TE, -20 DEG C of guarantors
Deposit.With agarose gel electrophoresis and the purity and concentration of NanoDrop2000 double checks DNA.Genomic DNA portion sample
Electrophoresis result is as shown in Figure 2.
1.4 micro-satellite primers
33 pairs of micro-satellite primers and fluorescence labeling of this experimental selection are all referred to (Xu Ling's tinkling of pieces of jade etc., 2009. Wei Jie, 2013)
By Shanghai, Sheng Gong Bioisystech Co., Ltd synthesizes.
The multiplexed PCR amplification of 1.5 microsatellite locus
33 microsatellite marker primers carry out multiplexed PCR amplification.PCR reaction systems are 20ul, DNA profiling 1.0ul, Taq
Buffer 2.5ul, the μ l of dNTP 10mM 0.5, on have primer 0.5ul, anti-sense primer 0.5ul, TaqaDNA polymerase 0.5ul,
Finally mend H2O to 20ul.Reaction condition is：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extensions
30s, carries out 10 circulations；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, carry out 20 circulations；Last 72 DEG C are repaiied
Extend 6min again.
1.6 microsatellite genescans
Multiple PCR products are served into the raw work biology Co., Ltd in sea carries out genescan, and the sequenator for using is 3730XL,
Using Genemapper software analysis SSR data, clip size and genotype are demarcated.
1.7 data statistics and analysis
Data are carried out with statistical analysis by the genotype of each microsatellite locus of all samples with Popgene32 softwares
With ab, the form such as bb is input into the data file of Popgene32 softwares, calculate gene frequency of the sample on each microsatellite locus,
Average observation number of alleles, average effective number of alleles (Ne), fragrant grand index, average effective heterozygosity (H) etc..Utilize
The softwares of Cervus 3.0 calculate colony's P values.
2 results and analysis
2.1PCR product detections
The agarose gel electrophoresis of the concentration of PCR primer 1.5%, partial results are as shown in Figure 2.PCR primer is in ABI3730XL
STR partings are as shown in Figure 3 after Genetic Analyzer sequencings.Each peak figure represents closed colony Wuzhi Mountain pig microsatellite locus etc.
The size and genotype of position gene.
2.2 gene frequencies and its allele distributions
The Wuzhi Mountain pig closed colony group 33 number of alleles of microsatellite locus, allele size and highest frequency equipotentials
Gene is shown in Table 1.As seen from Table 1,60 Wuzhi Mountain pigs detect 212 allele altogether on 33 microsatellite locus, put down
Equal 6.17, each site allele.Number of alleles is detected on SWR158 sites at most, is 12, range of variation is
The gene frequency highest of 164~216bp, 170bp, is 0.3667；Show that 33 microsatellite locus are deposited in the pig of the Wuzhi Mountain
In polymorphism, can be used for analyzing Wuzhi Mountain pig hereditary capacity.Gene frequency by 2 visible 33 microsatellite locus of table is high
Low inequality, each gene loci is in addition to several gene frequencies are higher, and other gene frequencies are relatively low.
Table 1 WZSP closed colonies 33 microsatellite locus number of alleles, effective number of allele, size, highest frequencies etc.
Position gene and its frequency
The Population variation analysis of 2.3 Wuzhi Mountain pig closed colonies
The genetic diversity index of 33 microsatellite locus is shown in Table 3.Table 3 shows that 33 microsatellite locus are closed in WZSP
Apparent homozygosity minimum is respectively 0.10 and 0.82 with peak in group, and average value is 0.34；Apparent heterozygosity be 0.22~
0.90, average out to 0.66.Expect that homozygosity minimum is respectively 0.16 and 0.69 with peak；Expect heterozygosity minimum with most
High level is respectively 0.31 and 0.84, and its average value is 0.67.In addition, total group is in 35 average values of site polymorphism information content
It is that 0.69,29 sites are in high polymorphism.In 33 sites, 16 site fixation indexs are negative value, and other sites are just
Value, average fixation index is 0.05；Heterozygosity in SWR186 and SW886 sites, expect heterozygosity, polymorphism information content and
The equal highest of Shannon comentropies, in 33 microsatellite locus, WZSP closed colonies have the abundantest in the site.In colony
Hardy-Weinberg balance checks in it has also been found that total group is in 33 P in site>0.05, meet the law of genetic equilibrium.
Table 2 WZSP closed colonies, 33 gene frequencies of microsatellite locus
The genetic diversity index of table 3 WZSP closed colonies, 33 microsatellite locus
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (5)
1. a kind of method of closed colony WZSP Genetic monitoring, it is characterised in that the closed colony Wuzhi Mountain is small
The method of type pig Genetic monitoring is comprised the following steps：
Step one, Wuzhi Mountain pig gathers 60 parts of ear skin histology samples, and sample is placed in 1.5mL sterilization centrifuge tubes, takes back experiment
Room, -20 DEG C of Refrigerator stores are standby；
Step 2, Wuzhi Mountain pig genomic DNA is extracted according to the method in Tiangeng DNA extraction kit, is dissolved in TE, -20 DEG C
Preserve；With agarose gel electrophoresis and the purity and concentration of NanoDrop2000 double checks DNA；
Step 3,33 microsatellite marker primers carry out multiplexed PCR amplification；
Multiple PCR products are carried out genescan by step 4, using Genemapper software analysis SSR data, demarcate fragment big
Small and genotype；
Data are carried out statistical analysis by the base of each microsatellite locus of all samples by step 5 with Popgene32 softwares
Because type is with ab, bb forms are input into the data file of Popgene32 softwares.
2. the method for closed colony WZSP Genetic monitoring as claimed in claim 1, it is characterised in that described five
Refer to that mountain pig carries out closing and breeds nearly 20 years according to panmixia principle, bred to 3 more than generation.
3. the method for closed colony WZSP Genetic monitoring as claimed in claim 1, it is characterised in that described many
Weight PCR amplification PCR reaction systems be 20ul, DNA profiling 1.0ul, Taq Buffer 2.5ul, the μ l of dNTP 10mM 0.5, on
There are primer 0.5ul, anti-sense primer 0.5ul, TaqaDNA polymerase 0.5ul, finally mend H2O to 20ul；Reaction condition is：95℃
Predegeneration 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, carry out 10 circulations；95 DEG C are denatured 30s, 55 DEG C
Annealing 30s, 72 DEG C of extension 30s, carry out 20 circulations；Repair for last 72 DEG C and extend 6min.
4. the method for closed colony WZSP Genetic monitoring as claimed in claim 1, it is characterised in that the step
Gene frequency, average observation number of alleles, average effective allele of the sample on each microsatellite locus are calculated in rapid five
Several, fragrant grand index, average effective heterozygosity；Colony's P values are calculated using the softwares of Cervus 3.0.
5. the method inspection of closed colony WZSP Genetic monitoring described in a kind of utilization claim 1~4 any one
The pig of survey.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110122415A (en) * | 2019-05-28 | 2019-08-16 | 上海市农业科学院 | The mating system of height inbreeding Experimental Miniature Pig closed colony |
| CN110951894A (en) * | 2020-01-01 | 2020-04-03 | 华中农业大学 | SNP molecular marker related to pig initial-mating day-age character |
| CN111363830A (en) * | 2020-04-10 | 2020-07-03 | 上海市农业科学院 | Specific microsatellite primer composition for identifying experimental miniature pig breeds and method for identifying experimental miniature pig breeds |
| CN116287301A (en) * | 2023-02-22 | 2023-06-23 | 海南省农业科学院畜牧兽医研究所 | SNPs molecular marker combination and method for five-finger mountain pig variety identification |
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2016
- 2016-12-23 CN CN201611209526.6A patent/CN106755417B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110122415A (en) * | 2019-05-28 | 2019-08-16 | 上海市农业科学院 | The mating system of height inbreeding Experimental Miniature Pig closed colony |
| CN110122415B (en) * | 2019-05-28 | 2021-06-08 | 上海市农业科学院 | Breeding method of small pig closed group for high inbred experiment |
| CN110951894A (en) * | 2020-01-01 | 2020-04-03 | 华中农业大学 | SNP molecular marker related to pig initial-mating day-age character |
| CN111363830A (en) * | 2020-04-10 | 2020-07-03 | 上海市农业科学院 | Specific microsatellite primer composition for identifying experimental miniature pig breeds and method for identifying experimental miniature pig breeds |
| CN111363830B (en) * | 2020-04-10 | 2022-06-21 | 上海市农业科学院 | Specific microsatellite primer composition for identifying experimental miniature pig breeds and method for identifying experimental miniature pig breeds |
| CN116287301A (en) * | 2023-02-22 | 2023-06-23 | 海南省农业科学院畜牧兽医研究所 | SNPs molecular marker combination and method for five-finger mountain pig variety identification |
| CN116287301B (en) * | 2023-02-22 | 2023-10-10 | 海南省农业科学院畜牧兽医研究所 | SNPs molecular marker combination and method for five-finger mountain pig variety identification |
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