CN116179657B - Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system - Google Patents

Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system Download PDF

Info

Publication number
CN116179657B
CN116179657B CN202211728879.2A CN202211728879A CN116179657B CN 116179657 B CN116179657 B CN 116179657B CN 202211728879 A CN202211728879 A CN 202211728879A CN 116179657 B CN116179657 B CN 116179657B
Authority
CN
China
Prior art keywords
microsatellite
primer
snakeheads
snakehead
combination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202211728879.2A
Other languages
Chinese (zh)
Other versions
CN116179657A (en
Inventor
欧密
赵建
刘海洋
夏威威
罗青
张新铖
陈昆慈
费树站
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pearl River Fisheries Research Institute CAFS
Original Assignee
Pearl River Fisheries Research Institute CAFS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pearl River Fisheries Research Institute CAFS filed Critical Pearl River Fisheries Research Institute CAFS
Priority to CN202211728879.2A priority Critical patent/CN116179657B/en
Publication of CN116179657A publication Critical patent/CN116179657A/en
Application granted granted Critical
Publication of CN116179657B publication Critical patent/CN116179657B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The application relates to the technical field of microsatellite markers and multiple PCR systems of snakeheads, in particular to a primer combination, microsatellite marker combination, multiple PCR system, a method and application thereof for identifying snakeheads. The method comprises the following steps: and (3) obtaining microsatellite primers of candidate satellite markers by using candidate microsatellite markers from the snakeheads and the snakeheads, screening the primers by using genome DNA of the hybridization offspring of the snakeheads and the snakeheads, sequentially obtaining a first primer group and a second primer group, and constructing a multiple PCR reaction system according to the second primer group. According to the primer combination, the microsatellite combination or the multiple fluorescent reaction system, microsatellite markers can be accurately amplified, the detection cost is saved, and the method can provide technical means for snakehead paternity test, individual identification, diversity analysis, genetic relationship identification, genetic map construction and germplasm resource protection effect evaluation, and has wide application prospect.

Description

Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system
Technical Field
The application relates to the technical field of microsatellite markers and multiple PCR systems of snakeheads, in particular to a primer combination, microsatellite marker combination, multiple PCR system, a method and application thereof for identifying snakeheads.
Background
Snakehead (Channa spp.) is an important freshwater aquaculture economic fish in China, and the annual yield is basically stabilized to be more than 50 ten thousand tons. At present, main breeding varieties are snakehead (Channa argus), macula maculata (Channa maculata) and hybrid snakehead, wherein the snakehead is mainly distributed in various water systems in north of Yangtze river, and the macula maculata is mainly distributed in water systems in south of China. The hybrid snakehead is a first generation obtained by hybridization of snakehead and macula maculata, and has the advantages of high survival rate of fries, high growth speed, easy domestication of artificial compound feed and the like. At present, the black-bone hybrid snakehead (snakehead female parent) and black-bone hybrid snakehead (snakehead female parent and snakehead female parent) with strong cold resistance are expected to replace fresh-frozen snakehead to become a northern dominant breed variety, and the breeding range of the hybrid snakehead is enlarged. More parents and fries are needed for the expansion of the cultivation scale, but in the breeding process, the parent sources are mixed, meanwhile, the operation of partial farmers is not standard, and the problems of inbreeding and the like exist in the breeding process, so that the snakehead germplasm is degraded, the genetic diversity is reduced, the disease resistance is reduced and the like are caused. In addition, when artificial selective breeding is carried out, parents with different qualities and different sources are difficult to distinguish through morphological characteristics and the like, so that genetic diversity analysis is carried out through a molecular marker method, the relation of pedigrees is clear, parents with different sources are identified, and the method plays an important role in sustainable development of snakehead genetic breeding.
Disclosure of Invention
Microsatellites (microsatellites), also known as simple repeats (Simple sequence repeats, SSRs), are widely distributed in eukaryotic genomes. The microsatellite mainly consists of two parts, namely a core sequence and flanking sequences, wherein the core sequence and the flanking sequences are repeatedly arranged in series, and the flanking sequences are single copy sequences positioned at two sides of the core sequence and have conservation. Microsatellites follow Mendelian co-dominant genetic law, and meanwhile, have the advantages of wide site distribution, high polymorphism, strong stability, convenient detection and the like. The inventor obtains a group of candidate microsatellite markers by analyzing the existing genome data of snakeheads and snakeheads, and carries out primer design and screening according to the candidate microsatellite markers, so as to screen a primer combination and a multiplex fluorescence PCR reaction system for identifying snakeheads, and correspondingly screen a group of microsatellite combinations capable of identifying or assisting in identifying the hybrid offspring individuals, paternity test or population genetic analysis of the snakeheads, the snakeheads or both. According to the primer combination, the microsatellite combination or the multiple fluorescent reaction system, microsatellite markers can be accurately amplified, the detection cost is saved, and the method can provide technical means for snakehead paternity test, individual identification, diversity analysis, genetic relationship identification, genetic map construction and germplasm resource protection effect evaluation, and has wide application prospect. Therefore, the embodiment of the application at least discloses the following technical scheme:
in a first aspect, embodiments of the present application disclose a screening method for identifying primer combinations for snakeheads, comprising:
obtaining a microsatellite marker sample set from snakeheads and snakeheads;
according to the sample combination, a microsatellite primer sample group is obtained, wherein a joint sequence is designed at the 5' end of the microsatellite primer sample;
performing PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the microsatellite primer sample set, and screening according to amplification products to obtain candidate primer pairs;
grouping and combining the candidate primer pairs, performing multiplex PCR amplification on genome DNA of the snakehead and the snakehead filial generation by utilizing the combined candidate primer pairs, and constructing the primer combination for identifying fluorescent multiplex PCR reaction of the snakehead according to the multiplex PCR amplification result.
In a second aspect, the embodiment of the application discloses a construction method of a multiplex fluorescence PCR reaction system for identifying snakeheads, which comprises the following steps:
obtaining a microsatellite marker sample set from snakeheads and snakeheads;
according to the sample combination, a microsatellite primer sample group is obtained, wherein a joint sequence is designed at the 5' end of the microsatellite primer sample;
performing PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the microsatellite primer sample set, and screening according to amplification products to obtain candidate primer pairs;
grouping and combining the candidate primer pairs, carrying out multiplex PCR amplification on genome DNA of the snakehead and the snakehead filial generation by utilizing the combined candidate primer pairs, and constructing a fluorescent multiplex PCR reaction system for identifying snakeheads according to the multiplex PCR amplification result.
In a third aspect, the embodiments of the present application disclose a screening method for identifying microsatellite marker combinations of snakes, comprising:
obtaining a microsatellite marker sample set from snakeheads and snakeheads;
according to the sample combination, a microsatellite primer sample group is obtained, wherein a joint sequence is designed at the 5' end of the microsatellite primer sample;
performing PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the microsatellite primer sample set, and screening according to amplification products to obtain candidate primer pairs;
grouping and combining the candidate primer pairs, performing multiplex PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the combined candidate primer pairs, and determining microsatellite marker combination for identifying the snakehead according to the result of the multiplex PCR amplification.
In a fourth aspect, embodiments of the present application disclose a microsatellite marker combination for identifying snakes, comprising:
opc001, opc004, opc017, opc029, opc036 and Opc044; or alternatively
Opc020, opc027 and Opc043; or alternatively
Opc032, opc033 and Opc039; or alternatively
Opc003, opc007 and Opc047; or alternatively
Opc006, opc024 and Opc046; or alternatively
Opc015 and Opc022; wherein,,
the nucleotide sequences of Opc001, opc003, opc004, opc006, opc007, opc015, opc017, opc020, opc022, opc024, opc027, opc029, opc032, opc033, opc036, opc039, opc043, opc044, opc046 and Opc047 are shown in SEQ ID NO. 42-61 in sequence.
In a fifth aspect, embodiments of the present application disclose a primer set for identifying snakeheads, comprising:
combinations of primer pairs for amplifying the microsatellite markers Opc001, opc004, opc017, opc029, opc036 and Opc044, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc020, opc027 and Opc043, respectively; or alternatively
Combinations of primer pairs for amplifying the microsatellite markers Opc032, opc033 and Opc039, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc003, opc007 and Opc047, respectively; or alternatively
Combinations of primer pairs for amplifying the microsatellite markers Opc006, opc024 and Opc046, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc015 and Opc022, respectively; wherein,,
the nucleotide sequences of the primer pairs used for amplifying the microsatellite markers Opc001, opc003, opc004, opc006, opc007, opc015, opc017, opc020, opc022, opc024, opc027, opc029, opc032, opc033, opc036, opc039, opc043, opc044, opc046 and Opc047, respectively, are shown in SEQ ID nos. 2 to 41 in sequence.
In a sixth aspect, the embodiment of the application discloses a multiplex fluorescence PCR detection kit for identifying snakeheads, which comprises the primer group in the fifth aspect.
In a seventh aspect, the embodiment of the application discloses an application of the microsatellite combination according to the fourth aspect, the primer combination according to the fifth aspect or the multiplex fluorescence PCR detection kit according to the sixth aspect in snakehead individual identification, paternity test and population genetic analysis.
The screening method for identifying the primer combination or the microsatellite marker combination of the snakehead, the construction method for identifying the multiplex fluorescence PCR reaction system of the snakehead, the identified primer combination or microsatellite marker combination, the multiplex fluorescence PCR detection kit and the application specific technical effects provided by the embodiment of the application are specifically described in the embodiment.
Drawings
FIG. 1 is a schematic flow chart of a screening method for identifying primer combinations for snakeheads according to an embodiment of the application.
FIG. 2 is a schematic flow chart of a construction method of a multiplex fluorescence PCR reaction system for identifying snakeheads, which is provided by the embodiment of the application.
FIG. 3 is a schematic flow chart of a screening method for identifying microsatellite marker combinations of snakeheads according to an embodiment of the present application.
Fig. 4 is a diagram of the results of capillary electrophoresis detection provided in the example of the present application for PCR amplification of 30 DNA hybrids of snakehead offspring using Opc001, opc003, opc004, opc006, opc007 and primer pairs thereof (top to bottom in the figure), respectively.
Fig. 5 is a diagram of a capillary electrophoresis detection result of PCR amplification of 30 filthy obscura progeny DNA using Opc015, opc017, opc020, opc022, opc024 and primer pairs thereof (top to bottom in the figure), respectively, provided in the present embodiment.
FIG. 6 is a diagram of the results of a capillary electrophoresis test for PCR amplification of 30 DNA progenitors of the hybrid snakehead using Opc027, opc029, opc032, opc033, opc036 and primer pairs thereof (top to bottom in the figure), respectively, as provided in the examples of the present application.
FIG. 7 is a graph of the results of a capillary electrophoresis test for PCR amplification of 30 DNA progenitors of hybridized snakehead using Opc039, opc043, opc044, opc046 and Opc047 and primer pairs thereof (top to bottom in the figure), respectively, as provided in the examples of the present application.
Fig. 8 is a diagram of a capillary electrophoresis detection result of PCR amplification of an individual 1 and an individual 2 (from top to bottom in the figure) using a microsatellite marker combination 1 or a multiplex PCR reaction system 1 according to an embodiment of the present application.
Fig. 9 is a diagram of a capillary electrophoresis detection result of PCR amplification of an individual 1 and an individual 2 (from top to bottom in the figure) using a microsatellite marker combination 2 or a multiplex PCR reaction system 2 according to an embodiment of the present application.
Fig. 10 is a diagram of a capillary electrophoresis detection result of PCR amplification of an individual 1 and an individual 2 (from top to bottom in the figure) using a microsatellite marker combination 3 or a multiplex PCR reaction system 3 according to an embodiment of the present application.
FIG. 11 is a diagram showing the results of PCR amplification of individuals 1 and 2 (from top to bottom in the figure) using microsatellite marker combination 4 or multiplex PCR reaction system 4 according to the present application.
Fig. 12 is a diagram of the result of capillary electrophoresis detection of PCR amplification of individual 1 and individual 2 (from top to bottom in the figure) using microsatellite marker combination 5 or multiplex PCR reaction system 5 according to an embodiment of the present application.
Fig. 13 is a diagram of the result of capillary electrophoresis detection of PCR amplification of individual 1 and individual 2 (from top to bottom in the figure) using microsatellite marker combination 6 or multiplex PCR reaction system 6 according to an embodiment of the present application.
In fig. 4 to 13, HEX: green; ROX: red; TAMRA: black; FAM: blue.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present application and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the application described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
Microsatellites (microsatellites), also known as simple repeats (Simple sequence repeats, SSRs), are widely distributed in eukaryotic genomes. The microsatellite mainly consists of two parts, namely a core sequence and flanking sequences, wherein the core sequence and the flanking sequences are repeatedly arranged in series, and the flanking sequences are single copy sequences positioned at two sides of the core sequence and have conservation. Microsatellites follow Mendelian co-dominant genetic law, and meanwhile, have the advantages of wide site distribution, high polymorphism, strong stability, convenient detection and the like. The microsatellite is used for constructing a multiplex PCR reaction system or a kit, 3 pairs or 4 pairs of primers with overlapped fragment sizes can be distinguished through fluorescent markers with different colors, and the primer number of the multiplex PCR can be increased. The microsatellite primers which are subjected to multiplex PCR and need to be amplified in advance are subjected to amplification efficiency and combination matching degree, once the primer combination is determined, the application efficiency of microsatellite markers can be greatly improved, the genotyping cost is reduced, and the microsatellite marker is an important guarantee for smoothly implementing molecular marker-assisted breeding of aquatic animals. Therefore, the primer combination, the microsatellite combination and the multiplex PCR reaction kit which can be obtained by screening can be widely applied to researches such as genetic diversity analysis, genetic relationship identification, genetic map construction, effect evaluation of germplasm resource protection and the like.
In one aspect, as shown in FIG. 1, the embodiment of the application discloses a screening method for identifying primer combinations for snakeheads, which comprises the following steps:
s101, acquiring microsatellite marker sample sets from snakeheads and snakeheads;
s102, combining the samples to obtain a microsatellite primer sample group, wherein a joint sequence is designed at the 5' end of the microsatellite primer sample;
s103, carrying out PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the microsatellite primer sample set, and screening according to amplification products to obtain candidate primer pairs;
s104, grouping and combining the candidate primer pairs, carrying out multiplex PCR amplification on genome DNA of the snakehead and the snakehead hybrid progeny by using the combined candidate primer pairs, and constructing the primer combination for identifying fluorescent multiplex PCR reaction of snakeheads according to the multiplex PCR amplification result.
On the one hand, as shown in fig. 2, the embodiment of the application discloses a construction method of a multiplex fluorescence PCR reaction system for identifying snakeheads, which comprises the following steps:
s201, acquiring microsatellite marker sample sets from snakeheads and snakeheads;
s202, combining the samples to obtain a microsatellite primer sample group, wherein a joint sequence is designed at the 5' end of the microsatellite primer sample;
s203, carrying out PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the microsatellite primer sample group, and screening according to amplification products to obtain candidate primer pairs;
s204, grouping and combining the candidate primer pairs, carrying out multiplex PCR amplification on genome DNA of the snakehead and the snakehead hybrid progeny by using the combined candidate primer pairs, and constructing a fluorescent multiplex PCR reaction system for identifying snakeheads according to the multiplex PCR amplification result.
In one aspect, as shown in fig. 3, the embodiment of the application discloses a screening method for identifying microsatellite marker combinations of snakes, which comprises the following steps:
s301, acquiring microsatellite marker sample sets from snakeheads and snakeheads;
s302, combining the samples to obtain a microsatellite primer sample group, wherein a joint sequence is designed at the 5' end of the microsatellite primer sample;
s303, carrying out PCR amplification on genome DNA of the snakehead and the snakehead filial generation by using the microsatellite primer sample set, and screening according to amplification products to obtain candidate primer pairs;
s304, grouping and combining the candidate primer pairs, performing multiplex PCR amplification on genome DNA of the snakehead and the snakehead hybrid offspring by using the combined candidate primer pairs, and determining and identifying microsatellite marker combinations of the snakeheads according to the multiplex PCR amplification result.
In some embodiments, the nucleotide sequence of the linker sequence is set forth in SEQ ID NO.1,
in some embodiments, in step S103, S203 or S303, "screening for candidate primer pairs based on amplification products" comprises: and (3) subjecting the amplified product to agarose gel electrophoresis, and screening primers which can amplify single, clear, bright bands and have high polymorphism into the first primer group. In these examples, multiple passes of primers can be made using different sample amounts of hybridized snakehead progeny DNA as templates to obtain candidate primer pairs.
In some embodiments, the steps S104, S204 or S304 specifically include:
grouping and combining the candidate primer pairs according to the annealing temperature and the size of PCR amplified products so as to perform multiplex PCR reaction, and connecting different fluorescent groups (FAM, HEX, TEMRA, ROX) at the 5' end of the forward primer of the candidate primer pair so as to perform fluorescence detection;
constructing a multiple PCR reaction system by utilizing the recombined candidate primer pairs with the fluorescent groups, and determining an optimal multiple PCR reaction system and a reaction program according to the concentration proportion of the primers in the reaction system, the annealing temperature in the reaction program and the circulation times, so as to determine the corresponding optimal primer combination, and further determine the microsatellite marker combination optimally used for identifying snakeheads.
In one embodiment, the screening method for identifying primer combinations or microsatellite combinations of snakes or the construction method for identifying multiple fluorescent PCR reaction systems of snakes comprises the following steps:
(1) And (3) analyzing the existing genome data of the snakehead and the snakehead by using MISA software to obtain a microsatellite marker sample group.
(2) According to the microsatellite marker sample set, designing and synthesizing a microsatellite primer sample set, wherein the 5' end of the microsatellite primer is connected with a joint sequence, and the nucleotide sequence of the joint sequence is shown as SEQ ID NO. 1;
(3) The microsatellite primer sample group is used for carrying out PCR amplification on 10 randomly selected hybridized snakehead offspring DNA, and amplified PCR products are detected by agarose gel electrophoresis, and primers with single, clear and bright bands and high polymorphism are selected.
(4) And (3) connecting different fluorophores (FAM, HEX, TEMRA, ROX) at the 5' end of the forward primer obtained in the step (3), carrying out PCR amplification on randomly selected 30 hybridized snakehead offspring DNA again, carrying out capillary electrophoresis detection on amplified products by using an ABI3730 genetic analyzer, carrying out statistical analysis on each microsatellite genetic parameter, and finally screening out primers with good polymorphism, namely 20 candidate primer pairs, wherein the candidate primer pairs are shown in table 1.
Fig. 4 to 13 show capillary electrophoresis patterns of the 20 pairs of candidate primer pairs, respectively, and as a result, it was found that the 20 pairs of candidate primer pairs can have good polymorphisms.
Table 1 screening to obtain 20 candidate primer pairs (adaptor sequences not shown)
(5) And (3) regrouping and combining the candidate primer pairs screened in the step (4) according to the respective annealing temperature and the size of the PCR amplification products so as to carry out multiple PCR reactions.
Mixing the grouped candidate primer pairs to prepare a multiplex PCR primer working solution, taking 30 hybridized snakehead offspring DNA as a template, establishing a multiplex fluorescence PCR reaction system and carrying out PCR reaction, adjusting the proportion of each grouped primer, annealing temperature and cycle times to achieve the optimal amplification effect, and determining the optimal identification of the primer combination of the snakehead. And according to the primer combination for optimally identifying the snakehead, a microsatellite combination of the primer combination for optimally identifying the snakehead and a multiplex PCR reaction system for optimally identifying the snakehead can be correspondingly determined.
In the embodiment of the application, 6 groups of primer combinations for optimally identifying snakeheads, microsatellite marker combinations for optimally identifying snakeheads and optimal multiple PCR systems are established together, as shown in the table 2, wherein the primer ratio is the concentration ratio of the primers in the multiple PCR reaction system.
TABLE 2
In this embodiment, the multiplex PCR reaction system comprises: the primers for any of the six combinations described above, each primer concentration being shown in Table 2, each reaction system comprising, in 10 μl: 2. Mu.L of 10 XBuffer (Mg2+), 0.5. Mu.L of dNTP mix (2.5 mM each), 0.2. Mu.L of rTaq (5U/. Mu.L), 50-100ng of template DNA, 0.5. Mu.L of upstream primer mix, 0.5. Mu.L of downstream primer mix, deionized water was made up to 10. Mu.L. The concentration ratios of the primers and the fluorophores are shown in Table 2. In this example, the amplification procedure for the multiplex PCR reaction is: pre-denaturation at 95℃for 1min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 30s, 25 cycles in total, and extension at 72℃for 20min.
According to the screening result, the embodiment of the application also discloses a microsatellite marker combination for identifying snakeheads, which comprises the following steps:
opc001, opc004, opc017, opc029, opc036 and Opc044; or alternatively
Opc020, opc027 and Opc043; or alternatively
Opc032, opc033 and Opc039; or alternatively
Opc003, opc007 and Opc047; or alternatively
Opc006, opc024 and Opc046; or alternatively
Opc015 and Opc022; wherein,,
the nucleotide sequences of Opc001, opc003, opc004, opc006, opc007, opc015, opc017, opc020, opc022, opc024, opc027, opc029, opc032, opc033, opc036, opc039, opc043, opc044, opc046 and Opc047 are shown in SEQ ID NO. 42-61 in sequence.
According to the screening result, the embodiment of the application discloses a primer group for identifying snakeheads, which comprises the following steps:
combinations of primer pairs for amplifying the microsatellite markers Opc001, opc004, opc017, opc029, opc036 and Opc044, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc020, opc027 and Opc043, respectively; or alternatively
Combinations of primer pairs for amplifying the microsatellite markers Opc032, opc033 and Opc039, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc003, opc007 and Opc047, respectively; or alternatively
Combinations of primer pairs for amplifying the microsatellite markers Opc006, opc024 and Opc046, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc015 and Opc022, respectively; wherein,,
the nucleotide sequences of the primer pairs used for amplifying the microsatellite markers Opc001, opc003, opc004, opc006, opc007, opc015, opc017, opc020, opc022, opc024, opc027, opc029, opc032, opc033, opc036, opc039, opc043, opc044, opc046 and Opc047, respectively, are shown in SEQ ID nos. 2 to 41 in sequence.
According to the screening result, the embodiment of the application discloses a multiplex fluorescence PCR detection kit for identifying snakeheads, which comprises a multiplex fluorescence PCR detection mixed solution, wherein the multiplex fluorescence PCR detection mixed solution comprises the following components:
combinations of primer pairs for amplifying the microsatellite markers Opc001, opc004, opc017, opc029, opc036 and Opc044, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc020, opc027 and Opc043, respectively; or alternatively
Combinations of primer pairs for amplifying the microsatellite markers Opc032, opc033 and Opc039, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc003, opc007 and Opc047, respectively; or alternatively
Combinations of primer pairs for amplifying the microsatellite markers Opc006, opc024 and Opc046, respectively; or alternatively
A combination of primer pairs for amplifying the microsatellite markers Opc015 and Opc022, respectively;
the nucleotide sequences of the primer pairs used for amplifying the microsatellite markers Opc001, opc003, opc004, opc006, opc007, opc015, opc017, opc020, opc022, opc024, opc027, opc029, opc032, opc033, opc036, opc039, opc043, opc044, opc046 and Opc047, respectively, are shown in SEQ ID nos. 2 to 41 in sequence.
In one embodiment, the reaction system of the multiplex PCR detection kit comprises, in 10. Mu.l: 2 mu L10 XBuffer (Mg) 2+ ) 0.5. Mu.L dNTP mix (2.5 mM each), 0.2. Mu.L rTaq (5U/. Mu.L), 50-100ng template DNA, 0.5. Mu.L upstream primer mix, 0.5. Mu.L downstream primer mix, deionized water make up to 10. Mu.L.
In one embodiment, the reaction procedure of the multiplex PCR detection kit comprises: pre-denaturation at 95℃for 1min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 30s, 25 cycles in total, and extension at 72℃for 20min.
In some embodiments, the method of using the multiplex PCR detection kit comprises:
preparing a sample nucleic acid extracting solution;
preparation of the reagent: preparing a multiplex fluorescence PCR detection mixed solution, adding RT-PCR enzyme into the mixed solution, oscillating and centrifuging to obtain a reaction mixed solution;
sample adding: placing the reaction mixed solution in a PCR tube, and respectively adding a sample nucleic acid extracting solution, a positive reference substance and a negative reference substance into different PCR tubes filled with the reaction mixed solution to obtain a sample reaction tube, a negative reference reaction tube and a positive reaction tube;
and (3) PCR amplification: the reaction tube is arranged on a quantitative fluorescent PCR instrument, an amplification program is set, and PCR amplification is carried out;
after the PCR reaction is finished, detecting a fluorescence signal value of a PCR reaction system, wherein a fluorescence channel is selected from FAM, HEX and CalRed 610 channels.
In some embodiments, the amplification procedure of the PCR reaction is: pre-denaturation at 95℃for 1min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 30s, 25 cycles in total, and extension at 72℃for 20min.
Therefore, the embodiment of the application discloses the application of the microsatellite combination provided by the embodiment, the primer combination provided by the embodiment or the multiplex fluorescence PCR detection kit provided by the embodiment in snakehead individual identification, paternity test and population genetic analysis.
In one embodiment, the microsatellite combination, the primer combination and the multiplex fluorescence PCR detection kit provided by the embodiment of the application are adopted for parent-child identification of the hybridized snakehead. The method specifically comprises the following steps:
(1) Construction of pedigree and sampling
Constructing a mixed family of two hybrid snakeheads, wherein the family one is a mixed family of 3 YY snakeheads (M1-M3) and 9 XX snakeheads (F1-F9) from Shandong, and 334 offspring individuals are provided; the family two is a mixed family of 3 YY snakeheads (M1-M3) and 1 complete male hybrid snakeheads (F10) constructed by XX snakeheads from Hunan, and the offspring individuals are 343. Cutting off fin strips of tail fins of parent and offspring individuals, soaking in absolute ethyl alcohol, and preserving at-20 ℃ for later use.
(2) Genomic DNA extraction
About 10-20mg of sample fin was taken, genomic DNA was extracted using a MicroElute Genomic DNA Kit (OMEGA, USA) kit, and the integrity of the DNA was checked by 1% agarose gel electrophoresis.
(3) Construction of multiplex PCR System
Parental and progeny NA were amplified using a multiplex PCR system. The total multiplex PCR reaction system was 10. Mu.L: 2 mu L10 XBuffer (Mg) 2+ ) 0.5. Mu.L dNTP mix (2.5 mM each), 0.2. Mu.L rTaq (5U/. Mu.L), 50-100ng template DNA, 0.5. Mu.L upstream primer mix, 0.5. Mu.L downstream primer mix, deionized water make up to 10. Mu.L. PCR amplification procedure: pre-denaturation at 95℃for 1min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 30s, 25 cycles in total, and extension at 72℃for 20min. The amplified PCR products were subjected to capillary electrophoresis using an ABI3730 genetic analyzer.
(4) Genetic diversity and paternity analysis
Based on the results of capillary electrophoresis detection, the genotypes were analyzed using Peak Scanner Software V1.0 software. Genotyping data was introduced into Cervus v.3.0, and genetic parameters such as allele (Number of alleles, na), observed heterozygosity (Observed heterozygosity, ho), expected heterozygosity (Expected heterozygosity, he), polymorphic information content (Polymorphism information content, PIC) were obtained after running the analysis. The genetic diversity parameters of two hybrid snakeheads are shown in Table 3. family-PIC is 0.602-0.879, all show high polymorphism (p > 0.5), average PIC is 0.7654, and the experimental population is shown to be at high polymorphism level. Family two PICs are 0.465-0.879, most of the family two PICs are high polymorphism (p is more than or equal to 0.5), and average PICs are 0.7695, so that the experimental population is at a high polymorphism level.
TABLE 3 genetic diversity parameters of two hybrid snakehead families
TABLE 4 family one-paternity test results
F1 F2 F3 F4 F5 F6 F7 F8 F9 Total
M1 98 0 0 22 15 12 7 18 13 185
M2 0 8 0 7 2 1 0 7 11 36
M3 0 0 55 9 6 3 36 4 0 113
total 98 8 55 38 23 16 43 29 24 334
TABLE 5 family two identification results
Simulation identification of 10000 offspring was performed with 95% confidence using the Cervus 3.0 software Simulation of Parentage Analysis function, and assignment of offspring of two families under different parental combinations is shown in table 4 and table 5. The results showed that 9979 offspring were successfully assigned to the parental controls with an identification accuracy of 99.99%. And then, using the function of Cervus 3.0 software Parentage Analysis, performing paternity test on the first and second families by using 20 sites to show that all offspring of the two families are successfully matched with respective parents, and the success rate of the test reaches 100%.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims (3)

1. A primer set for identifying microsatellite markers of snakes, comprising:
the microsatellite marker consists of Opc001, opc004, opc017, opc029, opc036 and Opc044, wherein the nucleotide sequence of a primer pair for amplifying the microsatellite marker Opc001 is shown as SEQ ID NO. 2-3, the nucleotide sequence of a primer pair for amplifying the microsatellite marker Opc004 is shown as SEQ ID NO. 6-7,
the nucleotide sequence of the primer pair for amplifying the microsatellite marker Opc017 is shown as SEQ ID NO. 14-15,
the nucleotide sequence of the primer pair for amplifying the microsatellite marker Opc029 is shown as SEQ ID NO. 24-25,
the nucleotide sequence of the primer pair for amplifying the microsatellite marker Opc036 is shown as SEQ ID NO. 30-31,
the nucleotide sequence of the primer pair for amplifying the microsatellite marker Opc044 is shown as SEQ ID NO. 36-37.
2. A multiplex fluorescent PCR test kit for identifying snakeheads comprising the primer combination of claim 1.
3. Use of the primer set of claim 1 or the multiplex fluorescence PCR detection kit of claim 2 in snakehead individual identification, paternity test and population genetic analysis.
CN202211728879.2A 2022-12-30 2022-12-30 Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system Active CN116179657B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211728879.2A CN116179657B (en) 2022-12-30 2022-12-30 Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211728879.2A CN116179657B (en) 2022-12-30 2022-12-30 Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system

Publications (2)

Publication Number Publication Date
CN116179657A CN116179657A (en) 2023-05-30
CN116179657B true CN116179657B (en) 2023-09-05

Family

ID=86441551

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211728879.2A Active CN116179657B (en) 2022-12-30 2022-12-30 Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system

Country Status (1)

Country Link
CN (1) CN116179657B (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102134586A (en) * 2010-01-22 2011-07-27 海南大学 High-efficiency screening method for microsatellite marker of Marbled Sand Goby and application of same
CN103276068A (en) * 2013-05-20 2013-09-04 中国水产科学研究院珠江水产研究所 Marking primer and method for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by channa argus and channa maculate
CN106947816A (en) * 2016-10-28 2017-07-14 中山大学 A kind of method of Epinephelus coioides paternity test microsatellite Multiplex fluorescent PCR
JP2018000116A (en) * 2016-07-04 2018-01-11 国立大学法人東京海洋大学 Discrimination method of epinephelus fuscoguttatus having growth heritage
KR20190053351A (en) * 2017-11-10 2019-05-20 제주대학교 산학협력단 Method for identification and parentage using marker in Ephinephelus akaara
CN114592070A (en) * 2022-03-07 2022-06-07 珠海市现代农业发展中心(珠海市金湾区台湾农民创业园管理委员会、珠海市农渔业科研与推广中心) Method for identifying microsatellite families of sparus latus and application of sparus latus

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102134586A (en) * 2010-01-22 2011-07-27 海南大学 High-efficiency screening method for microsatellite marker of Marbled Sand Goby and application of same
CN103276068A (en) * 2013-05-20 2013-09-04 中国水产科学研究院珠江水产研究所 Marking primer and method for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by channa argus and channa maculate
JP2018000116A (en) * 2016-07-04 2018-01-11 国立大学法人東京海洋大学 Discrimination method of epinephelus fuscoguttatus having growth heritage
CN106947816A (en) * 2016-10-28 2017-07-14 中山大学 A kind of method of Epinephelus coioides paternity test microsatellite Multiplex fluorescent PCR
KR20190053351A (en) * 2017-11-10 2019-05-20 제주대학교 산학협력단 Method for identification and parentage using marker in Ephinephelus akaara
CN114592070A (en) * 2022-03-07 2022-06-07 珠海市现代农业发展中心(珠海市金湾区台湾农民创业园管理委员会、珠海市农渔业科研与推广中心) Method for identifying microsatellite families of sparus latus and application of sparus latus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Characterization, expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead (Channa maculata);Mi Ou et al.;Comp Biochem Physiol B Biochem Mol Biol;第257卷;第1-10页 *

Also Published As

Publication number Publication date
CN116179657A (en) 2023-05-30

Similar Documents

Publication Publication Date Title
CN106947816B (en) Microsatellite fluorescent multiplex PCR (polymerase chain reaction) method for parent-child identification of epinephelus coioides
CN101880719B (en) Microsatellite multi-PCR (Polymerase Chain Reaction) method for turbot paternity test
CN112280881B (en) SNP (Single nucleotide polymorphism) marker combination for identifying broccoli germplasm resources and varieties and application
CN107760789B (en) Genotyping detection kit for parent-child identification and individual identification of yaks
CN109706231B (en) High-throughput SNP (single nucleotide polymorphism) typing method for molecular breeding of litopenaeus vannamei
KR102301324B1 (en) Genetic marker for parentage and thereof in Limanda yokohamae
CN114350841B (en) Polymorphic molecular marker based on whole genome sequencing, preparation method and application
CN111500762B (en) Sagittaria trifolia SSR primer group and application thereof
CN114836543A (en) Female molecular marker primer of channa maculata, application thereof and method for identifying sex of channa maculata
CN109182546B (en) SSR fluorescence labeling primer for paternity test of pangolin scales and application thereof
CN108611405B (en) Microsatellite fluorescent multiplex PCR (polymerase chain reaction) method for parent-child identification of culter alburnus
CN114774558A (en) Cloud black goat SNP marker and application thereof in identification of cloud black goat variety
KR102064039B1 (en) Method for identification and parentage using marker in Ephinephelus akaara
CN102321752B (en) Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof
CN112575094B (en) InDel marker for identifying northern subspecies, florida subspecies and hybrids thereof of micropterus salmoides and application thereof
CN114292925A (en) SSR molecular marker primer related to growth traits of procambarus clarkii and application of SSR molecular marker primer in auxiliary selection
CN116179657B (en) Primer combination, microsatellite marker combination, multiplex PCR system, method for identifying snakehead, and application of multiplex PCR system
CN115125326A (en) Development and application of corn S-type cytoplasmic male sterility gene KASP molecular marker
JP4982746B2 (en) Pig parent-child determination method using DNA marker
CN114959056A (en) SSR marker for identifying female procambarus clarkii and application thereof
CN113981103A (en) Microsatellite primer pair for parent-child identification of macrobrachium rosenbergii microsatellites, detection kit and identification method
CN114134237A (en) SRAP molecular marker for identifying northern American subspecies and Youtipe No. 3 parents of micropterus salmoides and application thereof
CN111793699A (en) Efficient matching and breeding method for procypris merus
KR102530342B1 (en) Genetic maker for parentage and thereof in Stichopus japonicus
KR102530346B1 (en) Genetic maker for parentage and thereof in Stichopus japonicus

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant