CN108588231B - Chinese sturgeon genetic relationship identification kit and application thereof - Google Patents
Chinese sturgeon genetic relationship identification kit and application thereof Download PDFInfo
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Abstract
The invention discloses an Acipenser sinensis genetic relationship identification kit and an application method thereof, belonging to the field of animal molecular genetics. The kit provides 5 groups of Chinese sturgeon microsatellite marked dual PCR reaction systems. The kit can be used for rapidly identifying the genetic relationship among individuals and avoiding the close reproduction of Chinese sturgeons. The amplification result of the kit has high polymorphism and stability, and the financial and manpower consumed by the kit is reduced by half compared with the traditional PCR reaction, so that the kit has great practical value.
Description
Technical Field
The invention relates to an Acipenser sinensis genetic relationship identification kit and application thereof, belonging to the field of animal molecular genetics.
Background
Chinese sturgeons are fish with the lowest distribution latitude and the largest body type in more than 20 sturgeons in the world and are the specific species in China. In the last 70 th century, the breeding population in Yangtze river can reach more than 1 thousand tails, and Chinese sturgeons are endangered and died due to a plurality of factors such as environmental pollution, excessive fishing, loss of habitat and the like. The protection of the endangered species not only protects individuals of the endangered species, but also knows and protects genes of the endangered species, so that a good breeding policy is made, and close breeding is prevented. However, the genetic studies of Chinese sturgeons up to now have been limited, and there is an urgent need to study Chinese sturgeons from the molecular direction.
Microsatellite markers are the most ideal mode in the current research of endangered animal protection genetics, and the microsatellite has the advantages of high polymorphism, codominant inheritance, distribution throughout the whole genome and the like, and is widely applied to animal genetics research. The microsatellite marker multiplex PCR is a PCR reaction in which more than two pairs of primers are added in the same PCR reaction system and a plurality of nucleic acid fragments are amplified simultaneously. The multiplex PCR has the advantages of high efficiency, systematicness, economy, simplicity and the like. The microsatellite marker multiplex PCR of the Chinese sturgeon is not reported at home and abroad. The invention provides a kit for identifying the genetic relationship between individuals by using an Acipenser sinensis microsatellite dual PCR system, which realizes the measurement of the genetic relationship between the Acipenser sinensis individuals so as to determine the genetic relationship between the individuals, and provides solid technical support for genetic management, artificial propagation breeding formulation and genetic background analysis of the Acipenser sinensis.
Disclosure of Invention
The invention aims to provide a kit for identifying the genetic relationship between individuals by using an Acipenser sinensis microsatellite dual PCR system and an identification method for identifying the genetic relationship between the individuals of the Acipenser sinensis by using the kit, so as to determine the genetic relationship between the individuals and avoid the close breeding between the Acipenser sinensis individuals.
Technical scheme
The microsatellite primers in the 5 groups of Chinese sturgeon microsatellite marked dual PCR reaction systems in the kit for measuring the genetic relationship between individuals by using the Chinese sturgeon microsatellite dual PCR system are as follows:
the Chinese sturgeon genetic relationship identification kit and the application thereof, the kit further comprises a PCR reagent, and the reagent is as follows: 10 times PCR Buffer 2-3ul, 2.5mmol/L dNTP 0.1-1ul, MgCl21-2ul, 0.5-2ul of upstream and downstream primers of the two pairs of primers in each group of reaction system, 0.1-0.5ul of Taq enzyme, 1-5ul of DNA template and 10-15ul of ultrapure water.
More preferably, the kit comprises a PCR reagent, and the reagent is: 10 times PCR Buffer 2.5ul, 2.5mmol/L dNTP 0.5ul, MgCl21.5ul, 1ul of each of the upstream and downstream primers of the two pairs of primers in each reaction system, 0.3ul of Taq enzyme, 3ul of DNA template and 13.2ul of ultrapure water.
The method for measuring the genetic relationship between the Chinese sturgeon individuals by using the kit for measuring the genetic relationship between the individuals by using the Chinese sturgeon microsatellite dual PCR system comprises the following steps: taking an Acipenser sinensis tissue sample; extracting DNA of the Chinese sturgeon sample; carrying out PCR amplification on the Chinese sturgeon sample by using the microsatellite primers in the 5 groups of Chinese sturgeon microsatellite marked dual PCR reaction system shown in claim 1; carrying out electrophoretic separation on the PCR amplification product on 10% polyacrylamide gel; counting the genotype of the PCR amplification product according to the separation result; and (3) according to the genotyping result of each individual, drawing a clustering analysis graph among the individuals by using NTsys software, and determining the genetic relationship among the individuals. The PCR reaction program is as follows: pre-denaturation at 94 deg.C for 1-5 min; denaturation at 94 deg.C for 25-35s, annealing at 56 deg.C for 25-35s, extension at 72 deg.C for 25-35s, and 25-35 cycles; extending for 8-15min at 72 ℃; storing at 4 ℃. The PCR reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
Compared with the prior art, the invention has the following advantages:
the Chinese sturgeon genetic relationship identification kit and the application thereof provided by the invention can realize genetic diversity detection and individual identification of the Chinese sturgeons, can also identify the distance of genetic relationship among individuals, and avoid close breeding among the Chinese sturgeon individuals. And the financial resources and the manpower consumed by the kit provided by the invention are reduced by half compared with the traditional PCR, so that the kit has great popularization value.
Drawings
FIG. 1 is a graph of cluster analysis between individuals.
Detailed Description
Example 1
48 Chinese sturgeons are taken from the research institute of Chinese sturgeons of the three gorges of Yangtze river, China, the fin rays of the Chinese sturgeons are collected, the genome DNA of the Chinese sturgeons is extracted, and the extraction mode adopts the traditional phenol-chloroform extraction way.
Each individual was placed 0.1g of tail fin in a 1.5ml centrifuge tube, minced, and added 450. mu.L of LSTE extraction buffer (10mmol/L Tris-HCl, pH 8.0; 1mmol/L EDTA, pH8.0), 35. mu.L SDS (10%), 15. mu.L proteinase K (0.2%).
Putting the centrifuge tube into a water bath kettle at 55 ℃ for 1 hour until the centrifuge tube is clear and transparent.
Add 700ul Tris saturated phenol to the centrifuge tube, mix on a shaker for 30 minutes, centrifuge at 12000 rpm for 10 minutes at 4 ℃ and transfer the supernatant to another clean eppendorf tube (note take the supernatant with a 1mL tip cut flat to avoid confounding the underlying pellet).
Adding equal volume of phenol-ethanol mixture (phenol, chloroform and isoamylol in a ratio of 25: 24: 1) into the supernatant, shaking and mixing uniformly for 15min, centrifuging at 12000 r/min at 4 ℃ for 10min, and sucking the supernatant into another new Eppendorf tube.
Adding equal volume of chloroform into the supernatant, shaking and mixing for 15min, separating at 4 deg.C at 12000 rpm for 10min, and collecting the supernatant.
1mL of dehydrated ethanol precooled at-20 ℃ was added to precipitate the DNA, and the precipitate was collected.
The precipitate was washed twice with 70% ethanol, dried, and then added with 200. mu.L of TE (10mmol/L of LTris-HCl, pH 8.0; 0.1mmol/L of EDTA, pH8.0) and sufficiently dissolved at room temperature.
The PCR reaction is carried out on 48 Acipenser sinensis individuals by utilizing the Acipenser sinensis genetic relationship identification kit and the application thereof, wherein the PCR reaction system is 25 ul: 10 times PCR Buffer 2.5ul, 2.5mmol/L dNTP 0.5ul, MgCl21.5ul, 1ul of each of the upstream and downstream primers of the two pairs of primers in each reaction system, 0.3ul of Taq enzyme, 3ul of DNA template and 13.2ul of ultrapure water. The PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing temperature renaturation for 30s, extension at 72 ℃ for 30s, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃. The PCR products were electrophoresed and silver stained with 10% polyacrylamide gel.
The size of the allele fragments of each individual shown by the polyacrylamide gel was judged by using BIO-PROFIL software, and a cluster analysis chart among the individuals was drawn by using NTsys software according to the size of the allele at each microsatellite locus of each individual, as shown in the first figure. According to the clustering analysis result, the genetic relationship among the individuals can be judged, and all the Acipenser sinensis individuals can be identified, so that the identification of the genetic relationship on the molecular genetics level of the Acipenser sinensis is realized. As can be seen from the first figure, individuals No. 1, 3, 14, 43, 32, 34, 35, 37, 27 and 41 are family members, and have relatively close relationships, and are not suitable for pairing propagation. Individuals No. 4, 30, 8 and 33 are a family, the relativity is relatively close, and the individuals are not suitable for pairing propagation. The rest individuals are a family, the relationship is relatively close, and the pairing propagation among the individuals is not suitable.
SEQUENCE LISTING
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Claims (5)
1. The application of the Chinese sturgeon genetic relationship identification kit in identifying the genetic relationship among Chinese sturgeon individuals comprises the following steps: taking an Acipenser sinensis tissue sample; extracting DNA of the Chinese sturgeon sample; performing PCR amplification on the Chinese sturgeon sample by using the microsatellite primers in the 5 groups of Chinese sturgeon microsatellite marked dual PCR reaction systems provided by the kit; carrying out electrophoretic separation on the PCR amplification product on polyacrylamide gel; counting the genotype of the PCR amplification product according to the separation result; according to the genotyping result of each individual, a cluster analysis graph among the individuals is drawn by using NTsys software to determine the genetic relationship among the individuals, and the kit comprises 10 pairs of microsatellite primers as follows:
2. the use of claim 1, wherein the kit comprises PCR reagents comprising: 10 times PCR Buffer 2-3ul, 2.5mmol/L dNTP 0.1-1ul, MgCl21-2ul, 0.5-2ul of upstream and downstream primers of the two pairs of primers in each group of reaction system, 0.1-0.5ul of Taq enzyme, 1-5ul of DNA template and 10-15ul of ultrapure water.
3. The use of claim 1, wherein the kit comprises PCR reagents comprising: 10 times PCR Buffer 2.5ul, 2.5mmol/L dNTP 0.5ul, MgCl21.5ul, 1ul of each of the upstream and downstream primers of the two pairs of primers in each reaction system, 0.3ul of Taq enzyme, 3ul of DNA template and 13.2ul of ultrapure water.
4. The use of claim 1, wherein the PCR reaction procedure is: pre-denaturation at 94 deg.C for 1-5 min; denaturation at 94 deg.C for 25-35s, annealing at 56 deg.C for 25-35s, extension at 72 deg.C for 25-35s, and 25-35 cycles; extending for 8-15min at 72 ℃; storing at 4 ℃.
5. The use of claim 1, wherein the PCR reaction procedure is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
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| CN201810312499.8A CN108588231B (en) | 2018-04-09 | 2018-04-09 | Chinese sturgeon genetic relationship identification kit and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498613A (en) * | 2014-12-31 | 2015-04-08 | 中国水产科学研究院长江水产研究所 | SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application |
| CN106811540A (en) * | 2017-03-22 | 2017-06-09 | 淮阴师范学院 | It is a kind of to identify female ussuriensis, male individual microsatellite marker and specific primer and application |
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2018
- 2018-04-09 CN CN201810312499.8A patent/CN108588231B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498613A (en) * | 2014-12-31 | 2015-04-08 | 中国水产科学研究院长江水产研究所 | SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application |
| CN106811540A (en) * | 2017-03-22 | 2017-06-09 | 淮阴师范学院 | It is a kind of to identify female ussuriensis, male individual microsatellite marker and specific primer and application |
Non-Patent Citations (2)
| Title |
|---|
| 中华鲟资源及其遗传多样性研究进展;孙丽婷等;《渔业信息与战略》;20170525;第112-117页 * |
| 基于SSR的中华鲟亲子鉴定和遗传特性研究;辛苗苗;《中国优秀硕士学位论文全文数据库 农业科技辑》;20151215;第18页2.2.4部分-第24页2.3.3部分、第43页第四章、第45页表4.2、第49-51页4.3.4部分 * |
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