CN112831573B - SNP primer for sturgeon germplasm identification and detection method - Google Patents
SNP primer for sturgeon germplasm identification and detection method Download PDFInfo
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Abstract
The invention discloses SNP primers and a detection method for sturgeon germplasm identification. According to the invention, the germplasm specificity SNP loci are developed by comparing the mitochondrial genomes of the Acipenser sinensis, the Siberian sturgeon and the huso dauricus, the peripheral primer 2 pairs and the middle primer 7 are invented, so that germplasm identification can be carried out on the Acipenser sinensis, the Siberian sturgeon and the huso dauricus, and the method is applied to sturgeon germplasm resource identification. The SNP primer is suitable for SNaPshot detection technology, has the characteristics of high efficiency, accuracy and low cost, not only can accurately detect mutation sites, but also can realize high-flux detection requirements.
Description
Technical Field
The invention belongs to the technical field of sturgeon identification, and particularly relates to a SNP primer for sturgeon germplasm identification and a detection method.
Background
Sturgeon is one of the earliest vertebrates of existing origin. Sturgeons belong to the orders teleostoma, the subdivision of the radial fin, the general order of cartilage scale, and the order sturgeon. The term sturgeon is commonly referred to as a sturgeon-like fish. Sturgeon fish is a kind of fish with polyploid origin, has a large number of chromosomes, and has a plurality of natural hybrid seeds, and the hybrid seeds of sturgeon fish have high fertility biological characteristics. Sturgeons, particularly hybrid sturgeons, are difficult to morphologically distinguish, have strong morphological subjectivity, and are prone to erroneous judgment. The fish germplasm molecular identification technology is a new technology developed by utilizing molecular biology technology and aiming at researching biological species identification and systematic evolution relation, and the variety is identified by identifying the difference in DNA level. A single nucleotide polymorphism is a sequence polymorphism caused by variation of a single nucleotide at a specific site in a genomic DNA sequence. The SNP is distributed in the whole genome, human beings have an SNP of 1900bp on average, compared with microsatellites, the SNP has higher genetic stability, can realize detection automation, and is very suitable for high-speed quantitative detection for germplasm identification. Sequencing is the most accepted and accurate genotyping technique, which is the most intuitive, efficient and pollution-free mutation detection technique, but has higher cost. The SNaPshot detection technology adopted in the research is also called micro-sequencing, has the characteristics of high efficiency, accuracy and low cost, not only can accurately detect mutation sites, but also can realize the high-flux detection requirement.
Disclosure of Invention
The invention aims to provide an SNP primer and a detection method for sturgeon germplasm identification, which solve the existing problems: aiming at the technical defects in sturgeon germplasm identification, a visual, accurate and efficient germplasm identification method for sturgeon, acipenser schrenki and huso dauricus is provided. And provides application of the SNP primer to identification of sturgeon germplasm.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the invention relates to a SNP primer and a detection method for sturgeon germplasm identification, which adopts a primer combination of AF+AR+SNP1/SNP2/SNP3/SNP4 or BF+BR+SNP5/SNP6/SNP7 to identify sturgeon germplasm. The detection method comprises the steps of sample collection and DNA extraction, primer amplification SNP locus and SNaPshot detection SNP locus, wherein the sample collection and genomic DNA extraction comprises the following steps:
step one: collecting a sturgeon tail fin sample to be tested;
step two: fixing the tail fin sample in absolute ethyl alcohol, and preserving at 4 ℃ for later use.
Further, a SNP primer for sturgeon germplasm identification consists of 2 pairs of peripheral primers and 7 intermediate primer sets, wherein the nucleotide sequences of the primers are shown in the following table:
further, the SNaPshot detection SNP site includes the steps of:
step one: peripheral primer amplification fragment conditions: the total volume of the PCR reaction was 50. Mu.L, in which: 100ng of template DNA, I-5 TM High-Fidelity Master Mix (MCLAB) 25. Mu.L, 2. Mu.L each of peripheral primers (10. Mu. Mol/L), and sterile double distilled water was supplemented to 50. Mu.L;
step two: peripheral primer amplification fragment program: 98 ℃ for 2min;98 ℃ for 10s,57 ℃ for 10s,72 ℃ for 10s,30 cycles; extending at 72℃for 5min.
Step three: the PCR product is digested by SAP enzyme to remove the rest dNTPs and primers in the PCR system, and the reaction conditions are as follows: 37 ℃ for 1h,75 ℃ for 15min,4 ℃ for infinity;
step four: intermediate primer amplification SNP site, single strand extension:
the reaction system: 5 mu L
Template 2. Mu.L after SAP digestion
ABI SnapShot multiplex Mix 2μL
1 μl of intermediate primer;
step five: reaction conditions:
the SNP primer is used to obtain the corresponding genotypes of 30 individuals of Siberian sturgeon, acipenser schrenki and huso dauricus, and the genotypes are shown in the following table:
the invention is proved by experiments: primer combinations of AF+AR+SNP1/SNP2/SNP3/SNP4 or BF+BR+SNP5/SNP5/SNP7 can be used for identifying sturgeon germplasm.
The invention has the following beneficial effects:
1. according to the developed special SNP loci of the germplasm of the Acipenser sinensis, the Siberian sturgeon and the huso dauricus, 2 pairs of peripheral primers and 7 middle primers are designed, and the germplasm identification of the Acipenser sinensis, the Siberian sturgeon and the huso dauricus can be carried out through the collocation of the peripheral primers and the middle primers, so that the method can be applied to the germplasm resource identification of the Acipenser sinensis.
2. The SNP primer is suitable for SNaPshot detection technology, has the characteristics of high efficiency, accuracy and low cost, not only can accurately detect mutation sites, but also can realize high-flux detection requirements.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a table of information on peripheral primers according to the present invention;
FIG. 2 is a table of intermediate primer information according to the present invention;
FIG. 3 shows germplasm-specific SNP loci of Acipenser sibiricus, acipenser schrenki and huso dauricus of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-3, the invention discloses an SNP primer and a detection method for sturgeon germplasm identification, which comprise sample collection, genomic DNA extraction, primer design and SNaPshot detection of SNP loci. Sample collection and genomic DNA extraction included the following steps:
step one: collecting tail fin samples of the parent fish 30 tails of Siberian sturgeon, the parent fish 30 tails of Acipenser sinensis and the parent fish 30 tails of huso dauricus respectively;
step two: fixing the tail fin sample in absolute ethyl alcohol, and preserving at 4 ℃ for later use.
Step three: about 50mg of sturgeon fin sample is cut, ground into powder by liquid nitrogen, and DNA is extracted by a magnetic bead method genome DNA (animal) extraction kit (Shanghai Biotechnology Co., ltd.) and the quality and concentration of the DNA are measured by agarose gel electrophoresis and a NanoDrop lite (Thermo Scientific) ultraviolet spectrophotometer.
The primer design comprises the following steps:
mitochondrial genome sequences of sturgeon sibiricum, sturgeon and huso dauricus were obtained from GenBank databases, and the GenBank accession numbers thereof are JQ045341.1, KC820796.1 and KJ402277.1, respectively. Genome sequence comparison is carried out by using MEGA 6.0 software, two sequence fragments of 46-849bp and 1725-2383bp are selected according to comparison results, 10 SNP loci are contained, and Primer premier5 software is used for designing peripheral primers to amplify gene fragments, wherein the information of the peripheral primers is shown in table 1. SNaPshot detection technology is adopted to detect SNP loci, so 10 intermediate primers are designed to detect SNP loci, and the information of the intermediate primers is shown in Table 2.
TABLE 1 peripheral primer information
TABLE 2 intermediate primer information
| Primer name | Primer sequences | Primer extension direction |
| SNP1 | TTTTTTTTTAAGCATAACACTGAAGATGTTAAGATG | Forward direction |
| SNP2 | TTTTTTTTTTTTTTTTTTTTTTTAACTCTGGCTGAGTCGAGCTT | Reverse direction |
| SNP3 | TTTTTTTTTTTTTTTTTTTTTTTTTTGCGTAAAGCGTGATTAAAGGAT | Forward direction |
| SNP4 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAACCACGAAGGTAGCTCTACC | Forward direction |
| SNP5 | TTTTTTTTTTTGATTGTTTTATCGGGATAATAGGGT | Reverse direction |
| SNP6 | TTTTTTTTTTTTTTTTTTTAGGGTCTTCTCGTCTTATGGG | Reverse direction |
| SNP7 | TTTTTTTTTTTTTTTTTTTTTTTTTTCGACCACGGAGGACAAAA | Forward direction |
| SNP8 | TTTTTTTTTTTTTTTTTTTTGGGATTAAGGGCATTCTCAC | Reverse direction |
| SNP9 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACGGCGGTATATAGGCGG | Reverse direction |
| SNP10 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTGCTAAATCCACCTTCAA | Reverse direction |
The SNaPshot detection of SNP sites comprises the following steps:
step one: peripheral primer amplification fragment conditions: the total volume of the PCR reaction was 50. Mu.L, in which: 100ng of template DNA, I-5 TM High-Fidelity Master Mix (MCLAB) 25. Mu.L, 2. Mu.L each of peripheral primers (10. Mu. Mol/L), and sterile double distilled water was supplemented to 50. Mu.L;
step two: peripheral primer amplification fragment program: 98 ℃ for 2min;98 ℃ for 10s,57 ℃ for 10s,72 ℃ for 10s,30 cycles; extending at 72℃for 5min. Sequencing the PCR amplified product by the qinghao biological company after agarose gel electrophoresis detection, and verifying the authenticity of the mutation site;
step three: the PCR product is digested by SAP enzyme to remove the rest dNTPs and primers in the PCR system, and the reaction conditions are as follows: 37 ℃ for 1h,75 ℃ for 15min,4 ℃ for infinity;
step four: intermediate primer amplification SNP site, single strand extension:
the reaction system: 5 mu L
Template 2. Mu.L after SAP digestion
ABI SnapShot multiplex Mix 2μL
SNP Primer 1μL；
Step five: reaction conditions:
SNaPshot detection
Step five: the product obtained after single strand extension was plate-mixed with HIDI added as an internal standard.
Mixing plate conditions: template 1. Mu.L; HIDI 10. Mu.L.
And (3) carrying out heat denaturation at 95 ℃ for 5min after mixing the plates, and then putting the plates into a refrigerator at-20 ℃ to cool for 2-3min and then putting the plates into a machine.
Step six: raw data analysis was performed with Genemapper4.0 software after 50min on-machine Applied Biosystems 3730 XL.
The results show that: primer combinations of AF+AR+SNP1/SNP2/SNP3/SNP4 or BF+BR+SNP5/SNP5/SNP7 can be used for identifying sturgeon germplasm.
One specific application of this embodiment is: collecting tail fin samples of 20 unknown sturgeons, and extracting DNA. And (3) performing germplasm identification of the unknown sturgeon by adopting the combination of the two primers AF+AR+SNP1/SNP2 and BF+BR+SNP 6.
Peripheral primer amplification fragment conditions: the total volume of the PCR reaction was 50. Mu.L, in which: 100ng of template DNA, I-5 TM 25. Mu.L of High-Fidelity Master Mix (MCLAB), 2. Mu.L of each peripheral primer (10. Mu. Mol/L), and the procedure for amplifying the target fragment by supplementing the peripheral primers with sterile double distilled water to 50. Mu.L: 98 ℃ for 2min;98 ℃ for 10s,57 ℃ for 10s,72 ℃ for 10s,30 cycles; extending at 72℃for 5min.
The PCR product is digested by SAP enzyme to remove the rest dNTPs and primers in the PCR system, and the reaction conditions are as follows: 37 ℃ for 1h,75 ℃ for 15min,4 ℃ for infinity, reaction system: 5. Mu.L, template 2. Mu.L, ABI SnapShot multiplex Mix. Mu.L, intermediate primer 1. Mu.L after SAP digestion; the reaction conditions were 96℃for 10s,50℃for 5s,60℃for 30s, and 4℃for infinity.
SNaPshot detection: the product obtained after single strand extension was mixed with HIDI added as an internal standard, and was subjected to analysis of raw data using Genemapper4.0 software after 50min on-line Applied Biosystems 3730XL. Referring to FIG. 3, SNP loci and germplasm identification results of 20 unknown sturgeons are shown in Table 3:
TABLE 3 germplasm identification results of 20 sturgeons
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
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Claims (2)
1. A SNP primer for sturgeon germplasm identification, characterized in that: consists of 2 pairs of peripheral primers and 7 intermediate primers, wherein the nucleotide sequences of the primers are as follows:
2. a method for identifying sturgeon germplasm by using the SNP primer for identifying sturgeon germplasm according to claim 1, which is characterized by comprising the following steps:
step one: peripheral primer amplification fragment conditions: the total volume of the PCR reaction was 50. Mu.L, in which: 100ng of template DNA, I-5 TM High-Fidelity Master Mix MCLAB. Mu.L, 10. Mu. Mol/L peripheral primer each 2. Mu.L, and sterile double distilled water was supplemented to 50. Mu.L;
step two: peripheral primer amplification fragment program: 98 ℃ for 2min;98 ℃ for 10s,57 ℃ for 10s,72 ℃ for 10s,30 cycles; extending at 72 ℃ for 5min;
step three: the PCR product is digested by SAP enzyme to remove the rest dNTPs and primers in the PCR system, and the reaction conditions are as follows: 37 ℃ for 1h,75 ℃ for 15min,4 ℃ for infinity;
step four: intermediate primer amplification SNP site, single strand extension:
the reaction system: 5 mu L
Template 2. Mu.L after SAP digestion
ABI SnapShot multiplex Mix 2μL
1 μl of intermediate primer;
step five: reaction conditions:
96℃10s
50℃5s
60℃30s
4℃∞。
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| CN105002170A (en) * | 2015-07-31 | 2015-10-28 | 中国长江三峡集团公司 | Amur sturgeon germplasm molecular marker identification kit and application thereof |
| CN110747279A (en) * | 2019-11-21 | 2020-02-04 | 南京师范大学 | Fugu obscurus SNP molecular marker and application thereof in genetic breeding |
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| CN105063192B (en) * | 2015-07-31 | 2017-08-25 | 中国长江三峡集团公司 | Molecular labeling and its application for identifying Amur Sturgeon germplasm |
| CZ307525B6 (en) * | 2015-12-31 | 2018-11-07 | Ăšstav biologie obratlovcĹŻ AV ÄŚR, v. v. i. | A method of identifying European freshwater fish and hybrids in biological materials using the S7iCAPS method |
| CN108660220B (en) * | 2018-05-21 | 2021-07-13 | 中国水产科学研究院 | Sturgeon SNP molecular marker set, sturgeon seed production evaluation method and sturgeon genetic evaluation method |
| CN110923336A (en) * | 2019-12-27 | 2020-03-27 | 四川省农业科学院水产研究所(四川省水产研究所) | Primer, molecular marker and method for identifying germplasm of Acipenser dabryanus and Acipenser sinensis |
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| CN105002170A (en) * | 2015-07-31 | 2015-10-28 | 中国长江三峡集团公司 | Amur sturgeon germplasm molecular marker identification kit and application thereof |
| CN110747279A (en) * | 2019-11-21 | 2020-02-04 | 南京师范大学 | Fugu obscurus SNP molecular marker and application thereof in genetic breeding |
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| Title |
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| Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing;R Ogden等;Mol Ecol;第22卷(第22期);3112-3123 * |
| 鲟鱼种质鉴定技术的发展及应用;董颖等;中国水产(第undefined期);70-72 * |
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