CN108913784B - Detection method of EC12 SNP marker of exopalaemon carinicauda - Google Patents
Detection method of EC12 SNP marker of exopalaemon carinicauda Download PDFInfo
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Abstract
The invention provides a primer for detecting EC12 locus SNP marker of palaemon carinicauda. In addition, the invention also provides a detection method of the EC12 site SNP marker of the exopalaemon carinicauda, which comprises the following steps: firstly, extracting genome DNA of individuals in different geographical groups or the palaemon carinicauda groups for later use; designing specific primers at two ends of a core sequence containing an EC12 SNP marker in a palaemon carinicauda transcriptome library; then, carrying out PCR amplification on the genomic DNA of individuals in different geographical groups or groups of the palaemon carinicauda by using the primer, and detecting a PCR product; and carrying out restriction enzyme digestion reaction by using the PCR product, analyzing according to the appeared bands, determining the genotype of each individual, and obtaining the genetic polymorphism map of the palaemon carinicauda. The invention can quickly obtain the genetic variation map of the EC12 genetic marker gene locus of the palaemon carinicauda, and the obtained result can intuitively detect the genotype of each individual of the palaemon carinicauda, thereby distinguishing homozygote and heterozygote individuals.
Description
Technical Field
The invention belongs to a DNA molecular genetic marker technology of exopalaemon carinicauda, and relates to an SNP marker detection method of exopalaemon carinicauda genetic polymorphism at EC12 site.
Background
Before the research reports of microsatellite genetic markers of Exopalaemon carinicauda are made at home and abroad, the universality of microsatellite markers of related species of Exopalaemon chinensis to Exopalaemon carinicauda is reported in China, and the like, and 2 universal markers are screened out. JashuWen et al (2011, 2012) adopt artificially synthesized biotin tag (AG)15 probe and magnetic bead enrichment method to construct a palaemon carinicauda genome microsatellite enrichment library, and screen out 26 polymorphic microsatellite markers; and analyzing the genetic diversity of the wild population of the Laizhou gulf, the Haizhou gulf and the Xiangshan mountain tail white shrimps by using 12 microsatellite markers. Microsatellite markers can also be used to reveal genetic changes in different pedigree populations. Evaluation of 3 inbred lines of palaemon carinicauda using 33 microsatellite markers as in Wang Rifang (2016) revealed the genetic nature of the inbred lines. Liu Jiu Mei et al (2017) analyzed the genetic variation rules of the backcross families of palaemon carinicauda by using 25 microsatellite markers. Wangkaja et al (2017) developed 60 polymorphic microsatellite markers of Exopalaemon carinicauda based on high throughput sequencing. The only Reports on SNP markers in Exopalaemon carinicauda are the use of Exopalaemon carinicauda transcriptome (Molecular Biology Reports, 2015) published in Lijintao. At present, registered palaemon carinicauda SNP markers are not found in GenBank, the number of SNP markers which can be used for genetic linkage map construction and pedigree identification is extremely short, and research reports on the application of the palaemon carinicauda SNP polymorphism map construction, specific genetic markers and the like are not found at home and abroad.
Disclosure of Invention
The invention aims to provide a method for detecting DNA molecular genetic markers of palaemon carinicauda, which mainly utilizes a core sequence containing SNP markers in an established palaemon carinicauda transcriptome library, designs specific primers at two ends of the core sequence for PCR amplification, and carries out enzyme digestion reaction on PCR products through restriction enzymes, thereby rapidly detecting the genetic variation of each individual of the palaemon carinicauda at the SNP site, obtaining a polymorphic map of the palaemon carinicauda at the site, and intuitively detecting the genotype of each individual through the map.
Firstly, the invention provides a primer for detecting the SNP marker at the EC12 locus of palaemon carinicauda, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.
In addition, the invention also provides a detection method of the EC12 site SNP marker of the exopalaemon carinicauda, which comprises the following steps: firstly, extracting genome DNA of individuals in different geographical groups or the palaemon carinicauda groups for later use; designing specific primers at two ends of a core sequence containing an EC12 SNP marker in a palaemon carinicauda transcriptome library; then, carrying out PCR amplification on the genomic DNA of individuals in different geographical groups or groups of the palaemon carinicauda by using the primer, and detecting a PCR product; carrying out restriction enzyme digestion reaction by using the PCR product, analyzing according to the appeared bands, determining the genotype of each individual, and obtaining the genetic polymorphism map of the palaemon carinicauda; the specific primer sequences are respectively as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.
Further, the PCR amplification system is: 100ng of exopalaemon carinicauda genomic DNA; 10 XPCR Buffer, 2.0. mu.L; 25mmol/L Mg 2+2 μ L; taq enzyme, 1U; dNTP, 2. mu.L; 2 mu L of each positive primer and negative primer; sterile water was added to 20. mu.L.
Further, detection and enzyme digestion of PCR products: carrying out electrophoresis on the PCR product in 1% agarose gel, and carrying out 160V constant voltage electrophoresis for 20-30 minutes for detection; after the PCR product is detected to be qualified, restriction endonuclease is utilizedHindIII, carrying out enzyme digestion reaction, wherein the enzyme digestion reaction system is 20 mu L and comprises 5 mu L of PCR product and 2.0 mu L of 10X Buffer, Hind III endonuclease 1. mu.L, add ddH2O to 20 μ L; and (3) preserving the temperature of the enzyme digestion reaction at 37 ℃ for 1 hour, then carrying out agarose gel electrophoresis on the enzyme digestion product, carrying out 160V constant voltage electrophoresis for 25 minutes for detection, and finally taking a picture by using a full-automatic gel imager to obtain the polymorphic map of the palaemon carinicauda at the EC12 SNP site.
Further, the core sequence of the EC12 SNP marker is shown as SEQ ID NO. 3.
The construction method of the exopalaemon carinicauda EC12 site SNP marker genetic polymorphism map comprises the following steps: designing specific primers at two ends of a core sequence containing an EC12 SNP marker in a palaemon carinicauda transcriptome library; the primers are used for carrying out PCR amplification on the genome DNA of individuals in different geographical groups or groups of the palaemon carinicauda, and PCR products are detected; carrying out restriction enzyme digestion reaction by using the PCR product, analyzing according to the appeared bands, determining the genotype of each individual, and obtaining the genetic polymorphism map of the palaemon carinicauda; the specific primer sequences are respectively as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.
Compared with the prior art, the invention has the advantages that:
1. the invention can quickly obtain the genetic variation map of the EC12 genetic marker gene locus of the palaemon carinicauda, the method is simple and convenient, and the obtained result can intuitively detect the genotype of each individual of the palaemon carinicauda so as to distinguish homozygote individuals from heterozygote individuals.
2. The method is mainly applied to genetic marking, genealogy authentication, genetic map construction and the like among palaemon carinicauda groups. PCR primers and restriction enzymes are the core of the invention, and show polymorphism in the detection of the palaemon carinicauda population.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1: the EC12 primer and the restriction enzyme of the invention have a detection map of 20 individuals of the palaemon carinicauda, the number of the EC12 primer and the restriction enzyme is 1-20, the number of the EC12 primer and the restriction enzyme of the restriction enzyme are 20 individuals of the palaemon carinicauda, and M is DL 2000 standard molecular weight.
Detailed Description
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.
The technical method for the DNA molecular genetic marker of the EC12 SNP core sequence of palaemon carinicauda is described in detail in the following by combining the attached drawings.
In the case of the example 1, the following examples are given,
the detection method of the EC12 site SNP marker of the exopalaemon carinicauda comprises the following steps: the method comprises the following steps:
1. firstly, extracting individual genome DNA in different geographical groups or the white shrimp groups and diluting for later use;
2. then, designing specific primers at two ends of a core sequence containing EC12 SNP in the transcriptome library of the exopalaemon carinicauda;
3. then, carrying out PCR amplification on the genomic DNA of individuals in different geographical groups or groups of the palaemon carinicauda by using the primer, and detecting a PCR product;
4. and carrying out restriction enzyme digestion reaction by using the PCR product, analyzing the appeared bands, determining the genotype of each individual and obtaining the genetic polymorphism map of the palaemon carinicauda.
Example 2, test method:
1. extracting genomic DNA of exopalaemon carinicauda:
taking 100mg of muscle tissue of palaemon carinicauda, cutting into pieces, putting into a 1.5mL centrifuge tube, adding 475 mu L of TE solution (10mmol/L Tris-Cl, 10mmol/L EDTA) with pH of 8.0, and grinding with a grinding rod; adding 25 μ L of 10% SDS solution, and mixing; adding protease K4 μ L20 mg/mL, mixing, and digesting at 55 deg.C for 2.5-3 h. Redistilled phenol is extracted twice, each time is 10min, the centrifugation is carried out for 5min at 12000 r/min, and the supernatant is taken; extracting with chloroform (1:1) for 10min, centrifuging at 12000 r/min for 5min, and collecting supernatant; extracting with chloroform for 5min, centrifuging at 12000 rpm for 5min, and collecting supernatant. Adding 5mol/L NaCl solution with the volume of 1/25, uniformly mixing, and adding absolute ethyl alcohol with the volume twice that of the NaCl solution at the temperature of-20 ℃ to precipitate DNA for 15 min; the DNA thus picked up was washed with 70% ethanol for several tens of minutes, dried, and dissolved sufficiently in 500. mu.L of sterilized water, and then quantitatively diluted to a concentration of 50 ng/. mu.L for use.
2. Designing SNP primers: on the basis that the transcriptome library of the exopalaemon carinicauda contains an EC12 SNP core sequence, the high conservation of sequences at two sides of the EC12 SNP core sequence in the same species relative to the core sequence is utilized, and accordingly, specific primers are designed at two ends of the core sequence and are used for amplifying a DNA fragment at the site. Because the SNP core sequence has base mutation, the restriction enzyme cutting site of the corresponding restriction enzyme is changed, and the change of the DNA sequence length can be obtained by utilizing the change of the restriction enzyme cutting site to carry out restriction enzyme cutting reaction, which is the root of detecting SNP polymorphism. The specific primer sequences at two ends of the SNP core sequence are as follows: the annealing temperature in the case of using the forward primer 5'-CTC GCA CTT TGC CAC TTG-3' and the reverse primer 5'-GGT GGT TCC GAT TTG TCC-3' was 55 ℃.
The EC12 SNP core sequence is: CTCGCACTTTGCCACTTGCATTTGATGTCGTCAATTTCGTTATAGATGAAGTACTTCATATTTCTAATTTCCGCTTCGGTCTTGGCTTCCTTGGCTTGTGCAATGGCCTTTGCTAAGCTTTGTTCAATGGAGTCGAAGGTGTCTTTGATCTTGCCGACTCCACTGACAGCATTTGCGATGGATGGTAAGATGGCGTTGACCTCACCTTCCAGGCTGTGAAGATTTCCGTCGAGCTGACTGAAGTGTTGCAACTCCTGTTCGAGTTCTCGGTTTTTGGCTTCGAGGCTGTTGATCTGTTGCTGATGACGATTGATTTCCTGGTTGCGTGATGCAATATGATTACGGAGATTCTGGATCTGACTATTACCATTAGCAATGAAATTTCTCTTTTCGTTGGCCAGCTGTTCTTCTCTTTGCTTGGCGTCTTTGTAGAACTTATTTCCAATGGTCCATCCGACGACTGGGACAAATCGGAACCACC are provided.
3. And (3) PCR amplification: first, the samples were loaded as follows: exopalaemon carinicauda genomic DNA (50 ng/. mu.L), 2. mu.L; 10 XPCR Buffer, 2.0. mu.L; mg (magnesium)2+(25mmol/L), 2. mu.L; taq enzyme (5U/. mu.L), 0.2. mu.L; dNTPs (2.5 mmol/L each), 2. mu.L; primers of the invention (10mmol/μ L each), forward and reverse primers 2 μ L each; sterile water was added to 20. mu.L. Secondly, carrying out PCR reaction, wherein the program parameters of a PCR amplification instrument are as follows: denaturation at 94 deg.C for 5 min; 94 ℃ 40sec, 55 ℃ 40sec, 72 ℃ 40sec, 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
4. Detection and enzyme digestion of PCR products: after the PCR reaction is finished, carrying out electrophoresis on the PCR product in 1% agarose gel, wherein the voltage of an electrophoresis apparatus is 160V, and the electrophoresis time is 20-30 minutes, so that the reaction can be stopped; then using restriction enzymeHind III, carrying out enzyme digestion reaction, wherein the enzyme digestion reaction system is 20 mu L and comprises 5 mu L of PCR product; 2.0 μ L of 10X Buffer;Hind III, 1 mu L of endonuclease; add ddH2O to 20. mu.L. And (3) keeping the temperature of the enzyme digestion reaction at 37 ℃ for 1 hour, then carrying out electrophoretic separation on the enzyme digestion reaction product in 1% agarose gel electrophoresis, carrying out electrophoresis at 160V for about 25 minutes, and taking a picture by using a full-automatic gel imager to obtain the polymorphism map of the palaemon carinicauda at the EC12 SNP site. As shown in figure 1, the results of enzyme cutting electrophoresis maps show that 1-10, 14-15 and 19-20 are heterozygous individuals, 13 and 18 are homozygous individuals, and 11-12 and 16-17 are homozygous individuals. The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although reference is made to the foregoing implementationThe present invention is described in detail, and it will be apparent to those skilled in the art that the technical solutions described in the foregoing embodiments may be modified or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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tgttcaatgg agtcgaaggt gtctttgatc ttgccgactc cactgacagc atttgcgatg 180
gatggtaaga tggcgttgac ctcaccttcc aggctgtgaa gatttccgtc gagctgactg 240
aagtgttgca actcctgttc gagttctcgg tttttggctt cgaggctgtt gatctgttgc 300
tgatgacgat tgatttcctg gttgcgtgat gcaatatgat tacggagatt ctggatctga 360
ctattaccat tagcaatgaa atttctcttt tcgttggcca gctgttcttc tctttgcttg 420
gcgtctttgt agaacttatt tccaatggtc catccgacga ctgggacaaa tcggaaccac 480
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Sequence listing
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ctcgcacttt gccacttgca tttgatgtcg tcaatttcgt tatagatgaa gtacttcata 60
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tgttcaatgg agtcgaaggt gtctttgatc ttgccgactc cactgacagc atttgcgatg 180
gatggtaaga tggcgttgac ctcaccttcc aggctgtgaa gatttccgtc gagctgactg 240
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tgatgacgat tgatttcctg gttgcgtgat gcaatatgat tacggagatt ctggatctga 360
ctattaccat tagcaatgaa atttctcttt tcgttggcca gctgttcttc tctttgcttg 420
gcgtctttgt agaacttatt tccaatggtc catccgacga ctgggacaaa tcggaaccac 480
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Claims (2)
1. The method for detecting the SNP marker at the EC12 locus of the exopalaemon carinicauda is characterized by comprising the following steps of:
firstly, extracting genome DNA of individuals in different geographical groups or the palaemon carinicauda groups for later use;
designing specific primers at two ends of a core sequence containing an EC12 SNP marker in a palaemon carinicauda transcriptome library; the core sequence of the EC12 SNP marker is shown as SEQ ID NO. 3; the specific primer sequences are respectively as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;
the primers are used for carrying out PCR amplification on the genome DNA of individuals in different geographical groups or groups of the palaemon carinicauda, and PCR products are detected; the PCR amplification system is as follows: 100ng of exopalaemon carinicauda genomic DNA; 10 XPCR Buffer, 2.0. mu.L; 25mmol/L Mg2+2 μ L; taq enzyme, 1U; dNTP, 2. mu.L; 2 mu L of each positive primer and negative primer; adding sterilized water to 20 μ L; the PCR reaction program is: denaturation at 94 deg.C for 5 min; 94 ℃ 40sec, 55 ℃ 40sec, 72 ℃ 40sec, 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;
carrying out restriction enzyme digestion reaction by using PCR products, analyzing according to the appeared bands, and determining the base of each individualTyping and obtaining a genetic polymorphism map of the exopalaemon carinicauda; the detection and enzyme digestion of the PCR product are as follows: carrying out electrophoresis on the PCR product in 1% agarose gel, and carrying out 160V constant voltage electrophoresis for 20-30 minutes for detection; after the PCR product is detected to be qualified, restriction endonuclease is utilizedHindIII, carrying out enzyme digestion reaction, wherein the enzyme digestion reaction system is 20 mu L and comprises 5 mu L of PCR product and 2.0 mu L of 10X Buffer,HindIII endonuclease 1. mu.L, add ddH2O to 20 μ L; and (3) preserving the temperature of the enzyme digestion reaction at 37 ℃ for 1 hour, then carrying out agarose gel electrophoresis on the enzyme digestion product, carrying out 160V constant voltage electrophoresis for 25 minutes for detection, and finally taking a picture by using a full-automatic gel imager to obtain the polymorphic map of the palaemon carinicauda at the EC12 SNP site.
2. The construction method of the exopalaemon carinicauda EC12 site SNP marker genetic polymorphism map is characterized by comprising the following steps:
firstly, extracting genome DNA of individuals in different geographical groups or the palaemon carinicauda groups for later use;
designing specific primers at two ends of a core sequence containing an EC12 SNP marker in a palaemon carinicauda transcriptome library;
the primers are used for carrying out PCR amplification on the genome DNA of individuals in different geographical groups or groups of the palaemon carinicauda, and PCR products are detected;
carrying out restriction enzyme digestion reaction by using the PCR product, analyzing according to the appeared bands, determining the genotype of each individual, and obtaining the genetic polymorphism map of the palaemon carinicauda;
the specific primer sequences are respectively as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.
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