CN108411002B - PCR primer and method for identifying hybrid of echinus purpureus and echinus intermedius - Google Patents
PCR primer and method for identifying hybrid of echinus purpureus and echinus intermedius Download PDFInfo
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Abstract
The invention discloses a PCR primer for identifying hybrid of echinus purpureus and echinus intermedia, which has the following nucleotide sequence: an upstream primer: 5 'ttttatccctcttttatyttct 3'; a downstream primer: 5 'cctcwaagttaagattgggac 3'; the identification method is sequentially carried out according to the following steps: extracting DNA of an individual sea urchin to be identified, which has the shape of purple sea urchin; diluting the extracted DNA to 10 ng/mu l as a template, and carrying out PCR amplification by using an upstream primer and a downstream primer under the following PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃ and 2min at 72 ℃ for 35 cycles; 5min at 72 ℃; and (4) carrying out electrophoresis on the PCR amplification product to obtain a band which is a purple sea urchin-Strongylocentrotus intermedius hybrid.
Description
Technical Field
The invention relates to the field of molecular markers, in particular to a PCR primer and an identification method for identifying hybrid species of echinus purpureus and echinus intermedia.
Background
Sea urchin ball (A)Strongylocentrotus intermedius) Also known as strongylocentrotus nudus, it is native to northern Japan and the coastal region of the far east of Russia. Compared with other edible sea urchins, the sea urchins in the middle ball have the characteristics of good gonad color, high unsaturated fatty acid content, sweet taste and the like, are well loved by consumers in southeast Asia, have increased market demand year by year, and have formed a culture scale in Liaoning, Shandong and the like at present. Research shows that the optimal growth water temperature of the intermediate sea urchins is 15-20 ℃, and when the water temperature is increased to 23 ℃, a great amount of death of the intermediate sea urchins can be caused. Purple sea urchin (A)Anthocidaris crassispina) Is a sea urchin variety naturally distributed in the sea area in south China, and has higher tolerance to high-temperature environment. Experiments prove that the hybrid of the purple sea urchin and the Strongylocentrotus intermedius which are generated by hybridization by taking the purple sea urchin (female parent) and the Strongylocentrotus intermedius (male parent) as parents has better performance than that of the female parentCompared with the sea urchin with the shape of a ball, the sea urchin with the gonad quality has obvious high temperature resistance potential, and is a new sea urchin variety with rapid growth, good high temperature resistance and excellent gonad quality. However, since the hybrid of echinus-echinus intermedia has a similar appearance to echinus, it is difficult to distinguish them with the naked eye, and a problem of confusion in cultivation is likely to occur. Therefore, the effective identification of purple-middle hybrid sea urchins and purple sea urchins is an important problem to be solved urgently in sea urchin culture and market operation.
At present, there are two main methods for identifying the type of sea urchin: one is according to the external morphological characteristics of sea urchin, such as shell shape, body type symmetry, mouth position, width ratio and radian of interstep belt and interstep belt, tube foot hole number and arrangement mode, etc., the method belongs to the traditional classification identification method, not only needs to kill sea urchin and has low identification efficiency, but also is not suitable for the identification of purple sea urchin and purple sea urchin hybrids with similar appearance; the other method is to screen specific molecular markers on a large scale by a molecular biology method, generally by utilizing genome sequencing and a comparative genomics method, and the method has the advantages of high identification accuracy, huge cost, long time consumption and high requirements on the professional performance of operating and analyzing personnel and the precision of instruments and equipment.
The mitochondrial genome is a genetic system other than the nuclear genome of eukaryotic cells and is an important subject for the study of molecular evolution and phylogeny of species. Compared with a complex nuclear genome, the mitochondrial genome contains fewer genes, is easy to obtain and is relatively simple to operate. However, there has been no report on the identification of the hybrid of echinus violaceus-echinus intermedia using mitochondrial DNA sequences.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a PCR primer and an identification method for identifying hybrid of echinacea purpurea and echinacea intermedia.
The technical solution of the invention is as follows: a PCR primer for identifying hybrid of echinus purpureus and echinus intermedia is characterized in that the nucleotide sequence is as follows:
an upstream primer: 5 'ttttattatccctcttttatyttct 3'
A downstream primer: 5 'ccccwaagttaagattgggac 3'.
The method for identifying the PCR primers for identifying the hybrid of the echinacea purpurea and the echinacea purpurea-Strongylocentrotus intermedius is characterized by sequentially carrying out the following steps:
a. extracting DNA of an individual sea urchin to be identified, which has the shape of purple sea urchin;
b. diluting the extracted DNA to 10 ng/mu l as a template, and carrying out PCR amplification by using an upstream primer and a downstream primer under the following PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃ and 2min at 72 ℃ for 35 cycles; 5min at 72 ℃;
c. and (4) carrying out electrophoresis on the PCR amplification product to obtain a band which is a purple sea urchin-Strongylocentrotus intermedius hybrid.
The invention provides a universal primer for amplifying a mitochondrial DNA sequence of sea urchin and a method for identifying a hybrid of purple sea urchin and Strongylocentrotus intermedius by using the universal primer, which can carry out living body sampling (tube foot and the like) at any time and quickly and accurately identify the hybrid of purple sea urchin and Strongylocentrotus intermedius and a female parent of the hybrid of purple sea urchin.
Drawings
FIG. 1 is an electrophoresis diagram of PCR products of primer sets (i) to (v) using DNA of Strongylocentrotus intermedius as a template in example 1 of the present invention.
FIG. 2 is an electrophoresis diagram of PCR products of primer sets (i) to (v) using DNA of echinacea as a template in example 1 of the present invention.
FIG. 3 is an electrophoresis diagram of PCR products of primer sets (first to fifth) using DNA of hybrid species of echinacea purpurea and echinacea intermedia as templates in example 1 of the present invention.
FIG. 4 is an electrophoresis test chart for identifying hybrid species of Echinacea purpurea and Echinacea purpurea-Strongylocentrotus intermedius in example 2 of the present invention.
Detailed Description
Example 1:
1. the Nagassum intermedius (GenBank Accession number KC 490912.1) and the Marsh sea urchin (horse dung sea urchin) are obtained respectively by logging in an NCBI database (https:// www.ncbi.nlm.nih.gov /)Hemicentrotus pulcherrimus) (GenBank Accession number KC 490911.1) full length mitochondrial genome sequence, passage orderComparing the sequences, and designing 5 groups of PCR primer pairs with higher universality, wherein the nucleotide sequences are as follows:
an upstream primer: 5 'taacggattaaagcacagcactgaa 3';
a downstream primer: 5 'cgcatagagcttgaagggaatttaa 3';
② an upstream primer: 5 'tcttgttttcttgtttttgtgagtt 3';
a downstream primer: 5 'ctcgtgtatcaacatccattcc 3';
third, upstream primer: 5 'tgccatgattgcaataggagt 3';
a downstream primer: 5 'atctacaaagtgtcagtatcaggca 3';
fourthly, upstream primer: 5 'ccacttctcaacccatcaccacttt 3';
a downstream primer: 5 'ctattccttgggggcctatttcttc 3';
the upstream primer: 5 'ttttattctcccttttttatytct 3';
a downstream primer: 5 'ccccwaagttaagattgggac 3'.
2. Respectively taking DNA of the intermediate strongylocentrotus intermedius, the purple sea urchin and purple sea urchin-intermediate strongylocentrotus intermedius hybrid species as templates, and respectively carrying out PCR reaction by using each group of PCR primers;
2.1 extraction of DNA
3 individuals of each of the echinacea purpurea, the echinacea purpurea and the echinacea intermedia were taken, 30mg of tubular leg tissues were taken, liquid nitrogen was added into a mortar, the mixture was ground into powder, and DNA was extracted by a kit method. The kit is purchased from TianGen company, and the model is a marine animal tissue genome DNA extraction kit (centrifugal column type) (catalog number: DP 324).
2.2 diluting the extracted DNA to 10 ng/mu l as a template, and carrying out PCR reaction by using each group of PCR primers;
the PCR reaction system is a 10. mu.l system:
ddH2O 6.2μl
TAKARA La Taq(5U/μl) 0.2μl
10×LA PCR bufferⅡ(Mg2+Plus) 1.0μl
dNTP Mixture (2.5mM each) 0.8μl
0.4. mu.l of upstream primer
Downstream primer 0.4ul
Template DNA 1.0. mu.l
Wherein the content of the first and second substances,TAKARA La Taq(5U/μl)、10×LA PCR bufferⅡ(Mg2+plus) and dNTP mix (2.5mM each) were purchased from Takara Bio Inc., and primers were synthesized by Biotech (Shanghai) Inc.
The PCR reaction conditions of the primer sets are respectively as follows:
firstly, 5min at 94 ℃; 30s at 94 ℃, 30s at 61 ℃, 3min at 72 ℃ and 30s for 35 cycles; 5min at 72 ℃.
② 94 ℃ for 5 min; 30s at 94 ℃, 30s at 54 ℃ and 4min at 72 ℃ for 35 cycles; 5min at 72 ℃.
③ 94 ℃ for 5 min; 30s at 94 ℃, 30s at 57 ℃ and 4min at 72 ℃ for 35 cycles; 5min at 72 ℃.
Fourthly, 5min at 94 ℃; 30s at 94 ℃, 30s at 53 ℃ and 4min at 72 ℃ for 35 cycles; 5min at 72 ℃.
Fifthly, 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃ and 2min at 72 ℃ for 35 cycles; 5min at 72 ℃.
3. The obtained PCR products were subjected to electrophoresis, and the results are shown in fig. 1, fig. 2, and fig. 3, respectively.
FIG. 1 is an electrophoresis diagram of PCR products using the DNA of Strongylocentrotus intermedius as a template. As can be seen from FIG. 1, bands appear in all of primer pairs (i) to (v).
FIG. 2 is an electrophoresis diagram of PCR products of primer sets (first to fifth) using DNA of echinacea as a template. As can be seen from FIG. 2, bands appear in the primer pair Nos. (i) and (iv).
FIG. 3 is an electrophoresis diagram of PCR products of primer sets including first to fifth primers using DNA of hybrid species of echinus purpureus and echinus intermedia as templates. As can be seen from FIG. 3, bands appear in the primer pairs (i), (ii), (iv) and (v).
Meanwhile, as can be seen from fig. 2 to 3, the PCR primers for amplifying only the DNA of the hybrid species of echinacea purpurea and echinacea intermedia, which have similar shapes, are the primer groups (i.e., the primer groups (ii), (iii), and (v), which are specific, but the primer group (iii) has stronger identification specificity than the primer group (ii), and shorter amplification time, so the primer group (iv) can be used as the PCR primers for identifying the hybrid species of echinacea purpurea and echinacea intermedia.
The nucleotide sequences of the primer pairs are as follows:
an upstream primer: 5 'ttttattctcccttttttatytct 3';
a downstream primer: 5 'ccccwaagttaagattgggac 3'.
Example 2:
the method for identifying the PCR primers for identifying the hybrid of the echinacea purpurea and the echinacea purpurea-Strongylocentrotus intermedius sequentially comprises the following steps:
a. taking 3 purple-middle hybrid sea urchins with the shape of purple sea urchins and 3 purple sea urchins individuals, randomly disordering the sequence until the sequence cannot be distinguished, taking the mixture as sea urchins to be identified, and extracting DNA of each individual;
b. diluting the extracted DNA to 10 ng/. mu.l as a template, and respectively carrying out PCR amplification by using an upstream primer and a downstream primer, wherein the nucleotides of the primers are as follows:
an upstream primer: 5 'ttttattctcccttttttatytct 3';
a downstream primer: 5 'ccccwaagttaagattgggac 3'.
The PCR reaction system is a 10. mu.l system:
ddH2O 6.2μl
TAKARA La Taq(5U/μl) 0.2μl
10×LA PCR bufferⅡ(Mg2+Plus) 1.0μl
dNTP Mixture (2.5mM each) 0.8μl
0.4. mu.l of upstream primer
Downstream primer 0.4ul
Template DNA 1.0. mu.l
Wherein the content of the first and second substances,TAKARA La Taq(5U/μl)、10×LA PCR bufferⅡ(Mg2+plus) and dNTP mix (2.5mM each) were purchased from Takara Bio Inc., and primers were synthesized by Biotech (Shanghai) Inc.
The PCR reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃ and 2min at 72 ℃ for 35 cycles; 5min at 72 ℃.
The PCR amplification products were subjected to electrophoresis, and the results of the electrophoresis are shown in FIG. 4.
In FIG. 4, samples II, III, and V failed amplification and were purple sea urchins; the samples I, IV and VI are successfully amplified, and a strip appears, which is a hybrid of the purple sea urchin and the Strongylocentrotus intermedius.
Sequence listing
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Claims (2)
1. A PCR primer for distinguishing the hybridized species of purple sea urchin and purple sea urchin-Strongylocentrotus intermedius is characterized by the following nucleotide sequences:
an upstream primer: 5 'ttttattatccctcttttatyttct 3'
A downstream primer: 5 'ccccwaagttaagattgggac 3'.
2. A method for distinguishing hybrid species of echinacea purpurea and echinacea purpurea-echinacea purpurea by using the PCR primer of claim 1 is characterized by comprising the following steps in sequence:
a. extracting DNA of individual sea urchin to be distinguished with purple sea urchin shape;
b. diluting the extracted DNA to 10 ng/mu l as a template, and carrying out PCR amplification by using an upstream primer and a downstream primer under the following PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃ and 2min at 72 ℃ for 35 cycles; 5min at 72 ℃;
c. and (3) carrying out electrophoresis on the PCR amplification product, wherein the obtained band is the purple sea urchin-Strongylocentrotus intermedius hybrid, and the obtained band is the purple sea urchin.
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| CN109182540B (en) * | 2018-10-10 | 2021-06-15 | 大连海洋大学 | Primer and method for screening high-temperature-resistant strongylocentrotus intermedius |
| CN113025728B (en) * | 2021-04-29 | 2023-05-23 | 大连海洋大学 | DNA molecular marker for living identification of sex of echinococci and identification method |
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