CN111378731A - Sex-specific molecular marker primer for Trachinotus ovatus and application of sex-specific molecular marker primer - Google Patents
Sex-specific molecular marker primer for Trachinotus ovatus and application of sex-specific molecular marker primer Download PDFInfo
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- CN111378731A CN111378731A CN202010099453.XA CN202010099453A CN111378731A CN 111378731 A CN111378731 A CN 111378731A CN 202010099453 A CN202010099453 A CN 202010099453A CN 111378731 A CN111378731 A CN 111378731A
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Abstract
The invention discloses a sex-specific molecular marker primer for Trachinotus ovatus, which is a primer P2, wherein the primer P2 comprises a forward primer P2-F and a reverse primer P2-R, and the base sequences are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Also discloses a kit for identifying the sex of trachinotus ovatus and application of the primer or the kit in identifying the sex of trachinotus ovatus. The primer has the advantages of good specificity, high sensitivity, high accuracy, no damage to the living bodies of the trachinotus ovatus, capability of rapidly and accurately identifying the sex of the trachinotus ovatus in batches at one time, and important scientific research value and practical application value.
Description
Technical Field
The invention belongs to the technical field of sex of trachinotus ovatus, and particularly relates to sex-specific molecular marker primers for trachinotus ovatus and application thereof.
Background
Trachinotus ovatus (Trachinotus ovatus) belongs to the order Perciformes, the family trachinodaceae, the genus Trachinotus, commonly known as golden pompanus, yellow wax pompano and yellow wax carangid. After the artificial hastening of production is broken through in 2000, large-scale cultivation is started in China, the marine cultivation variety which is important in China is developed at present, the yield exceeds 10 ten thousand tons, and the marine cultivation variety has the characteristics of rapid growth, strong disease resistance, preference for turbulence and the like, and is the best variety for large-scale cultivation in deep and distant sea in the future. Although the breeding scale is increased, the breeding work is delayed, wherein the important limiting factor is that the sex of the trachinotus ovatus cannot be distinguished from the shape, so that the sex proportion cannot be controlled in artificial breeding. The sex of the trachinotus ovatus does not show morphological difference in early embryonic development and young fish stage, and the sex cannot be identified from the body size and the body surface characteristics of reproductive organs. Although in scientific research it is possible to identify sex by dissection, dissection inevitably leads to death of the individual and is of no consequence for production. The sex identification is the most critical link of genetic breeding, and the failure of in vivo identification of the sex of the trachinotus ovatus seriously hinders the development of breeding work such as genetic breeding and the like.
The problems existing in the prior art are as follows:
(1) in scientific research work, the physiological sex of trachinotus ovatus can be identified through methods such as dissection, but dissection inevitably causes death of dissected individuals, and the method is low in efficiency and cannot realize large-scale identification.
(2) In production practice, the sex of the trachinotus ovatus living body cannot be identified through phenotype.
(3) At present, no technology for identifying the sex of a living body by a biotechnology means exists.
Under the current technical conditions, the sex of the trachinotus ovatus can be accurately identified by developing sex-specific molecular markers. Although there are many types of molecular markers, they represent different manifestations of detection technology, and most fundamentally, the specific sequence that determines sex on the genome varies from species to species. According to the characteristics of the male and female specific sequences, a proper molecular marker can be selected for sex identification.
Disclosure of Invention
The invention aims to provide sex-specific molecular marker primers for Trachinotus ovatus and a kit comprising the primers.
The invention also aims to provide a sex identification method for the trachinotus ovatus.
The last purpose of the invention is to provide a method for identifying the sex of trachinotus ovatus by using the primer or the kit.
The first object of the present invention can be achieved by the following technical solutions: the sex-specific molecular marker primer for the egg-shaped pompano is primer P2, the primer P2 comprises a forward primer P2-F and a reverse primer P2-R, and the base sequence of the forward primer P2-F is shown as SEQ ID NO: 1, the base sequence of the reverse primer P2-R is shown as SEQ ID NO: 2, respectively.
Specifically, the base sequence of the primer P2 is as follows:
forward primer P2-F: 5'-CATGGACAAGAAGGTGGTGC-3', respectively;
reverse primer P2-R: 5'-TACCCAGTGCAAGCTCTCTC-3' are provided.
The invention also provides a kit for identifying the sex of the trachinotus ovatus, which comprises the sex-specific molecular marker primer for the trachinotus ovatus.
The second object of the present invention can be achieved by the following technical solutions: a sex identification method for egg-shaped pompano comprises the following steps: PCR amplification is adopted to amplify specific fragments in genome DNA of male and female individuals, and the specific fragments specifically comprise: synthesizing the primer, extracting the genome DNA of the trachinotus ovatus to be detected, carrying out PCR amplification by adopting the primer and taking the genome DNA as a template, detecting an amplified product by adopting a gel electrophoresis method, and identifying male and female individuals according to an electrophoresis result, wherein the heterozygous peak type is a female individual, and the homozygous peak type is a male individual.
In the sex determination method for trachinotus ovatus:
preferably, the PCR reaction system used for PCR amplification is 20 ng/. mu.L DNA template 1.0. mu.L, 10 × Taqbuffer (Mg)2+free) 2.5. mu.L, concentration 2.5mM dNTPs2.0. mu.L, concentration 25mM MgCl21.5. mu.L of 10mM forward and reverse primers 0.5. mu.L each, 5U/. mu.L of Taq 0.1. mu.L, ddH2The content of O is 25.0 mu L.
Preferably, the PCR reaction procedure used in PCR amplification is: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 10sec, annealing at 55 ℃ for 40sec, extension at 72 ℃ for 45sec, cycling for 35 times, and extension at 72 ℃ for 10 min.
The third object of the present invention can be achieved by the following technical solutions: the primer or the reagent kit is applied to identifying the sex of the trachinotus ovatus.
Therefore, the successful development of the sex-specific molecular marker primer for the trachinotus ovatus breaks through the technical bottleneck of sex identification of the trachinotus ovatus living body, and provides a tool for sex control of the trachinotus ovatus living body. By applying the technology, sex identification of trachinotus ovatus fries in the early hatching period can be realized, the cultivation efficiency is improved, and the cost is reduced; the sex ratio of the breeding group can be controlled, and inbreeding depression is avoided; can promote the development of research works such as sex determination and sex control of the trachinotus ovatus.
Compared with the prior art, the invention has the following advantages:
(1) the sex-specific molecular marker primer for trachinotus ovatus designed by the invention has the advantages of good specificity, good sensitivity, high accuracy, convenient operation of the identification method, simplicity and feasibility, and the identification success rate can reach 100%;
(2) the method can rapidly and accurately identify the sex of the trachinotus ovatus in batches at one time;
(3) the method disclosed by the invention has the advantages that no substantial damage is caused to the living trachinotus ovatus, the method can be used for identifying the sex of the trachinotus ovatus in living bodies, and the method is applied to identification of the sex of the living bodies, so that the technical bottleneck that the living bodies cannot identify the sex is broken through;
(4) the primer, the kit and the method have important scientific research value and practical application value for sex determination and sex control of the trachinotus ovatus.
Drawings
FIG. 1 is an illustration of capillary electrophoresis peak pattern of trachinotus ovatus individuals in example 1 by using P2 primer, and sequencing by using forward primer P2-F, upper: heterozygous peak type for female individuals; the following: a male individual homozygous peak pattern; the base indicated by the arrow is the target detection site.
Detailed Description
The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are biochemical reagent raw materials which are conventionally commercially available or commercially available, and the laboratory instruments used are laboratory-scale instruments, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.
Example 1
The primer is primer P2, the primer P2 comprises forward primer P2-F and reverse primer P2-R, and the base sequence of the forward primer P2-F is shown as SEQ ID NO: 1, the base sequence of the reverse primer P2-R is shown as SEQ ID NO: 2, respectively.
Specifically, the base sequence of the primer P2 is as follows:
forward primer P2-F: 5'-CATGGACAAGAAGGTGGTGC-3', respectively;
reverse primer P2-R: 5'-TACCCAGTGCAAGCTCTCTC-3' are provided.
Collecting 1 holomorphic families of the trachinotus ovatus, including 2 parents and 100 offspring individuals, and identifying physiological sex after dissection. And (3) carrying out marker development on parents and filial generations by using a high-throughput sequencing and taking the genome sequence of the trachinotus ovatus as a reference sequence, and positioning sex quantitative character loci to a single interval by using linkage analysis. Indels within the interval that are significantly linked to gender were developed as gender-specific markers.
Primers are designed from partial sequences (trachinotus ovatus sex-specific molecular markers) in the sex determination interval in the trachinotus ovatus genome sequence by using Primer3, wherein the trachinotus ovatus sex-specific molecular marker sequences are as follows:
wherein the box represents the forward primer, the underline represents the reverse primer (the forward sequence, the base sequence of P2-R after reverse complementation, and the square bracket represents the detected target variation, which is shown in SEQ ID NO: 3 of the sequence table.
To verify the accuracy of the primers, this example is further illustrated by the following verification test in example 2, which is specifically described below.
Example 2
The method for identifying the sex of the trachinotus ovatus provided by the embodiment comprises the following steps of: amplifying specific fragments in genome DNA of male and female individuals by adopting PCR amplification, and specifically comprises the following steps: the primers in the embodiment 1 are designed, the genomic DNA of the trachinotus ovatus to be detected is extracted according to the method in the embodiment 1, the primers in the embodiment 1 are adopted, the extracted genomic DNA is used as a template to perform PCR amplification, an amplification product is detected by a gel electrophoresis method (specifically, an automatic sequencer ABI 3730XL can be adopted), and male and female individuals are identified according to the electrophoresis result.
The implementation object of the embodiment is trachinotus ovatus, 47 females and 47 males are taken, the sex is identified by dissection, and then the feasibility of the primer is verified.
(1) Extraction of DNA
Cutting about 1 square centimeter of fin from the living body of the trachinotus ovatus (the process has no substantial damage to the living body of the trachinotus ovatus, the sampled trachinotus ovatus is put back to be observed to grow healthily, the process is the same as the process), preserving in 95% alcohol, and putting back the sampled trachinotus ovatus.
Tissue DNA is extracted using a commercially available DNA extraction kit or a conventional phenol-mimetic method or the like.
(2) Amplification of specific marker of trachinotus ovatus
The forward primer P2-F (5'-CATGGACAAGAAGGTGGTGC-3') and the reverse primer P2-R (5'-TACCCAGTGCAAGCTCTCTC-3') of the primer P2 are used as amplification primers, the extracted DNA of the trachinotus ovatus is used as a template, and common Taq enzyme is used for PCR amplification.
The PCR reaction system is 20 ng/. mu.L DNA template 1.0. mu.L, 10 × Taq buffer (Mg)2+free) 2.5. mu.L, concentration 2.5mM dNTPs2.0. mu.L, concentration 25mM MgCl21.5 μ L, 10mM forward and reverse primers 0.5 μ L each, 5U/. mu.LTaq0.1 μ L, ddH2Supplementing 25.0 mu L of O;
the PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 10sec, annealing at 55 ℃ for 40sec, extension at 72 ℃ for 45sec, cycling for 35 times, and extension at 72 ℃ for 10 min.
(3) Gender specific fragment detection
And (3) carrying out base detection on the amplification product in the step (2) on an automatic sequencer ABI 3730XL by using a forward primer P2-F.
FIG. 1 is an illustration of peak pattern detection of trachinotus ovatus by capillary electrophoresis using primer P2-F, sequencing using forward primer P2-F, top: heterozygous peak type for female individuals; the following: the male individual is homozygous peak type, i.e. the heterozygous peak type is female individual, and the homozygous peak type is male individual.
As a result, 47 cases were consistent with the heterozygous type in FIG. 1 and were female, and 47 peak types were consistent with the homozygous type in FIG. 1 and were male, that is, 47 males and 47 females were present in the result, and the result was completely consistent with the result of the dissection experiment, so that the accuracy of male and female identification was 100% using the primers and method of the present invention.
Therefore, the primer can be further prepared into a kit or a biological preparation for identifying the trachinotus ovatus males and females.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> research institute for aquatic products in south China sea
Sex-specific molecular marker primer for Trachinotus ovatus and application of sex-specific molecular marker primer
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Trachinotus ovatus (Trachinotus ovatus)
<400>1
catggacaag aaggtggtgc 20
<210>2
<211>20
<212>DNA
<213> Trachinotus ovatus (Trachinotus ovatus)
<400>2
tacccagtgc aagctctctc 20
<210>3
<211>166
<212>DNA
<213> Trachinotus ovatus (Trachinotus ovatus)
<400>3
catggacaag aaggtggtgc tgatcacagg ctgctcctcg ggaatcggtc tcagcctggc 60
tgtccggtta gcatctgacc ccgacaaaac attcaaagga taacagccca catctacaca 120
caatgatgga cctttgttgt atttccgaga gagcttgcac tgggta 166
Claims (6)
1. A sex-specific molecular marker primer for Trachinotus ovatus is characterized in that: the primer is a primer P2, the primer P2 comprises a forward primer P2-F and a reverse primer P2-R, and the base sequence of the forward primer P2-F is shown as SEQ ID NO: 1, the base sequence of the reverse primer P2-R is shown as SEQ ID NO: 2, respectively.
2. A kit for sex identification of trachinotus ovatus is characterized in that: the kit comprises the primer of claim 1.
3. A sex identification method for egg-shaped pompano is characterized in that: amplifying specific fragments in genome DNA of male and female individuals by adopting PCR amplification, and specifically comprises the following steps: synthesizing the primer in claim 1, extracting the genomic DNA of the trachinotus ovatus to be detected, carrying out PCR amplification by using the primer in claim 1 and the genomic DNA as a template, detecting an amplification product by using a gel electrophoresis method, and identifying male and female individuals according to an electrophoresis result, wherein the heterozygous peak type is a female individual, and the homozygous peak type is a male individual.
4. The sex identification method for trachinotus ovatus as claimed in claim 3, wherein the PCR amplification system includes 20ng/μ L DNA template 1.0 μ L, 10 × Taq buffer (Mg)2+free) 2.5. mu.L, 2.0. mu.L of 2.5mM dNTP, 25mM MgCl21.5. mu.L of 10mM forward and reverse primers 0.5. mu.L each, 5U/. mu.L of Taq 0.1. mu.L, ddH2The content of O is 25.0 mu L.
5. The sex identification method for trachinotus ovatus according to claim 3, wherein the sex identification method comprises the following steps: the PCR reaction procedure adopted during PCR amplification is as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 10sec, annealing at 55 ℃ for 40sec, extension at 72 ℃ for 45sec, cycling for 35 times, and extension at 72 ℃ for 10 min.
6. Use of the primer according to claim 1 or the kit according to claim 2 for identifying the sex of trachinotus ovatus.
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Cited By (3)
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| CN112239779A (en) * | 2020-10-21 | 2021-01-19 | 中国水产科学研究院南海水产研究所 | Primer and kit for quickly identifying sex of egg-shaped pompano and application of primer and kit |
| CN113151492A (en) * | 2021-03-24 | 2021-07-23 | 中国水产科学研究院南海水产研究所 | SNP molecular marker related to trachinotus ovatus hypoxia-resistant character and application thereof |
| CN113854202A (en) * | 2021-07-14 | 2021-12-31 | 中国水产科学研究院南海水产研究所 | Molecular marker assisted breeding method for rapid-growth new variety of egg-shaped pompano |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112239779A (en) * | 2020-10-21 | 2021-01-19 | 中国水产科学研究院南海水产研究所 | Primer and kit for quickly identifying sex of egg-shaped pompano and application of primer and kit |
| CN112239779B (en) * | 2020-10-21 | 2023-08-18 | 中国水产科学研究院南海水产研究所 | Primer and kit for rapidly identifying sex of trachinotus ovatus and application of primer and kit |
| CN113151492A (en) * | 2021-03-24 | 2021-07-23 | 中国水产科学研究院南海水产研究所 | SNP molecular marker related to trachinotus ovatus hypoxia-resistant character and application thereof |
| CN113151492B (en) * | 2021-03-24 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | SNP molecular marker related to trachinotus ovatus hypoxia-resistant character and application thereof |
| CN113854202A (en) * | 2021-07-14 | 2021-12-31 | 中国水产科学研究院南海水产研究所 | Molecular marker assisted breeding method for rapid-growth new variety of egg-shaped pompano |
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