CN111118174A - Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology - Google Patents
Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology Download PDFInfo
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- CN111118174A CN111118174A CN202010024588.XA CN202010024588A CN111118174A CN 111118174 A CN111118174 A CN 111118174A CN 202010024588 A CN202010024588 A CN 202010024588A CN 111118174 A CN111118174 A CN 111118174A
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Abstract
The invention discloses a PCR and sequencing technology-based macrobrachium rosenbergii sex identification method, which comprises the following steps: (1) and performing PCR amplification by taking the extracted macrobrachium rosenbergii genome DNA as a template, wherein amplification primers are as follows: MrW-F: 5'-CCCAACAGTTATGTTAGTCTAATCTGC-3', respectively; MrW-R: 5'-CTGCAGCCTTTTTTAGACGGAG-3', respectively; (2) carrying out agarose gel electrophoresis separation on the amplification product, and recovering a main band near 1300 bp; (3) sequencing analysis is carried out on the recovered main band by utilizing an upstream primer MrW-F, wherein the sequencing result of a male individual is successful, and the sequencing result of a female individual is an overlapped peak after 100 bp. The method can realize the early sex identification of the macrobrachium rosenbergii and has the characteristics of rapidness and accuracy.
Description
Technical Field
The invention belongs to the technical field of sex identification of aquatic organisms, and particularly relates to a method for identifying the sex of macrobrachium rosenbergii based on PCR and sequencing technologies.
Background
Macrobrachium rosenbergii (Macrobrachium rosenbergii) is a large freshwater shrimp with fast growth speed and is one of the largest freshwater shrimps in the world. The meat is originally produced in Indian ocean-Pacific tropical region, and can live in various fresh water or saline water areas, and has strong adaptability to environment, high nutritive value, sweet taste and convenient cultivation. Since 1976, after macrobrachium rosenbergii was introduced into mainland china, with the improvement of artificial breeding technology and the development of breeding technology, the breeding area and yield are continuously increased, and the industrial scale is continuously enlarged. In recent years, with the increasing market demand, macrobrachium rosenbergii becomes an important economic freshwater shrimp in China. Although macrobrachium rosenbergii is large in size and is the largest freshwater shrimp in the world, the macrobrachium rosenbergii has a problem of large difference between male and female body types. In China and some natural waters in southeast Asia, the male shrimp body is as long as 40cm and the weight is as high as 600g, while the female shrimp body is only 25cm and the weight is only about one third of the weight of the male shrimp (about 200g) during sexual maturity, so the market value of male individuals is higher. From the economic benefit perspective, the total-male breeding can improve the average yield per mu by about one third on the existing basis, and the total-male breeding can bring about one third increase of the newly increased yield value for the macrobrachium rosenbergii industry by about 27 hundred million yuan according to the calculation of the breeding scale of China. Therefore, the sex identification of the macrobrachium rosenbergii is required regardless of production and scientific research so as to provide a necessary method tool for subsequent grouping culture or parthenocarpic culture research.
In order to breed unisexual whole male or whole female macrobrachium rosenbergii populations, the genetic sex of macrobrachium rosenbergii needs to be clarified, and in particular, in the case where the sex cannot be distinguished by morphological features in the early stage, the sex needs to be analyzed and identified, so that a quick and accurate sex identification method is required.
Disclosure of Invention
The invention aims to provide a PCR and sequencing technology-based macrobrachium rosenbergii sex identification method, aiming at quickly and accurately identifying the genetic sex of macrobrachium rosenbergii, especially under the condition that the sex cannot be distinguished by morphological characteristics in the early stage, for breeding a unisexual full-male or full-female population of the macrobrachium rosenbergii.
The invention is realized in such a way that a macrobrachium rosenbergii sex identification method based on PCR and sequencing technology comprises the following steps:
(1) and performing PCR amplification by taking the extracted macrobrachium rosenbergii genome DNA as a template, wherein amplification primers are as follows:
MrW-F：5'-CCCAACAGTTATGTTAGTCTAATCTGC-3'；
MrW-R：5'-CTGCAGCCTTTTTTAGACGGAG-3'；
(2) carrying out agarose gel electrophoresis separation on the amplification product, and recovering a main band near 1300 bp;
(3) sequencing analysis is carried out on the recovered main band by utilizing an upstream primer MrW-F, wherein the sequencing result of a male individual is successful, and the sequencing result of a female individual is an overlapped peak after 100 bp.
Preferably, in step (1), the PCR amplification system is: dNTP (2.5mmo 1/L) 2. mu.L, 2 XExTaqBuffer (Vazyme) 2.5. mu.L, Taq enzyme (2.5U/. mu.L) 0.5. mu.L, MrW-F and MrW-R (10. mu. mol/L) each 1. mu.L, genomic DNA template (50 ng/. mu.L) 2. mu.L, double distilled water to make up 25. mu.L;
the reaction procedure of the PCR amplification is as follows: pre-denaturation at 95 ℃ for 3min, amplification for 40 cycles (94 ℃, 30sec, 56-60 ℃, 30sec, 72 ℃, 39sec), extension for 10min at 72 ℃.
Preferably, in the step (1), a tissue sample of the macrobrachium rosenbergii individual at any period or part is collected, and its genomic DNA is chemically extracted for sex determination in the early stage of gonadal differentiation.
The invention overcomes the defects of the prior art and provides a macrobrachium rosenbergii sex identification method based on PCR and sequencing technology. The invention utilizes DNA extracted from any tissues of macrobrachium rosenbergii and by means of amplification primer of sex-specific fragment to obtain male and female sex-specific fragments, and the specific fragment is single in male individual, but not single in female individual.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
(1) compared with the traditional method for identifying the sex of the macrobrachium rosenbergii through the external morphological characteristics of the macrobrachium rosenbergii, such as the difference of a male foramen and a second gastropod, the method has higher accuracy in identifying the sex of the macrobrachium rosenbergii, and the condition of inaccurate identification caused by external damage can be avoided;
(2) the invention can realize the early sex identification of the macrobrachium rosenbergii: in the process of breeding parent macrobrachium rosenbergii, early sex determination is very critical to realize early character recording and breeding, but since the early macrobrachium rosenbergii individuals are small, the sex characteristics are not obvious or difficult to distinguish and identify, but the sex determination is carried out through genetic materials, and the sex determination of the macrobrachium rosenbergii can be realized by using limited genetic materials at any time;
(3) the method for identifying the sex of the macrobrachium rosenbergii is quick and accurate, the mass sex identification of the individual macrobrachium rosenbergii can be realized by utilizing the genetic material difference characteristic of the macrobrachium rosenbergii and combining the popular and mature PCR and sequencing technology of biology, and generally hundreds of pairs of parent shrimp individuals can be accurately identified one day.
Drawings
FIG. 1 is a schematic diagram of PCR product detection and tapping recovery bands for sex determination of Macrobrachium rosenbergii;
FIG. 2 is a graph showing the sequencing results of the tapping recovery bands of male and female individuals.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A macrobrachium rosenbergii sex identification method based on PCR and sequencing technology comprises the following steps:
(1) DNA extraction: collecting 10 muscle tissue samples of female and male macrobrachium rosenbergii individuals in a culture base of the macrobrachium rosenbergii in Deqing, and extracting genome DNA by a chemical method;
(2) preparing a PCR amplification mixed solution: the PCR amplification system is as follows: dNTP (2.5mmo 1/L) 2. mu.L, 2 XExTaqBuffer (Vazyme, USA) 2.5. mu.L, Taq enzyme (2.5U/. mu.L) 0.5. mu.L, MrW-F and MrW-R (10. mu. mol/L) each 1. mu.L, genomic DNA template (50 ng/. mu.L) 2. mu.L, double distilled water to make up 25. mu.L;
(3) PCR amplification reaction: the PCR reaction program is: first at 95 ℃ pre-denaturation for 3min, then amplification for 40 cycles (94 ℃, 30sec, 56-60 ℃, 30sec, 72 ℃, 39sec), and finally extension for 10min at 72 ℃;
(4) tapping and recovering PCR products: PCR product detection was performed using 1% agarose Gel to which nucleic staining solution Gel Red was added, and as shown in FIG. 1, a main band of about 1300bp in size was recovered;
(5) sequencing analysis: sequencing analysis was performed on the recovered sample using the forward primer MrW-F, and the sequencing results for the male individuals were successful and the female individuals were overlapping peaks (from 100bp later), as shown in FIG. 2.
All individual sequencing results are shown in table 1:
TABLE 1 sequencing sex determination of Macrobrachium rosenbergii
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.
Claims (3)
1. A macrobrachium rosenbergii sex identification method based on PCR and sequencing technology is characterized by comprising the following steps:
(1) and performing PCR amplification by taking the extracted macrobrachium rosenbergii genome DNA as a template, wherein amplification primers are as follows:
MrW-F：5'-CCCAACAGTTATGTTAGTCTAATCTGC-3'；
MrW-R：5'-CTGCAGCCTTTTTTAGACGGAG-3'；
(2) carrying out agarose gel electrophoresis separation on the amplification product, and recovering a main band near 1300 bp;
(3) sequencing analysis is carried out on the recovered main band by utilizing an upstream primer MrW-F, wherein the sequencing result of a male individual is successful, and the sequencing result of a female individual is an overlapped peak after 100 bp.
2. The method for sex determination of macrobrachium rosenbergii based on PCR and sequencing technology according to claim 1, wherein in step (1), the PCR amplification system is: dNTP (2.5mmo 1/L) 2. mu.L, 2 XExTaq Buffer (Vazyme) 2.5. mu.L, Taq enzyme (2.5U/. mu.L) 0.5. mu.L, MrW-F and MrW-R (10. mu. mol/. mu.L) each 1. mu.L, genomic DNA template (50 ng/. mu.L) 2. mu.L, double distilled water to make up 25. mu.L;
the reaction procedure of the PCR amplification is as follows: pre-denaturation at 95 ℃ for 3min, amplification for 40 cycles (94 ℃, 30sec, 56-60 ℃, 30sec, 72 ℃, 39sec), extension for 10min at 72 ℃.
3. The method for sex determination of macrobrachium rosenbergii based on PCR and sequencing technology as claimed in claim 1, wherein in step (1), tissue samples of macrobrachium rosenbergii individuals at any time or position are collected, and their genomic DNA is extracted by chemical method to perform sex determination at the early stage of gonadal differentiation.
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Cited By (2)
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| CN111996261A (en) * | 2020-07-31 | 2020-11-27 | 湖南银鱼农业科技有限公司 | Macrobrachium rosenbergii sex molecular marker primer and application thereof |
| CN113234755A (en) * | 2021-03-31 | 2021-08-10 | 浙江省农业科学院 | Expression plasmid of exogenous gene of macrobrachium rosenbergii and high-efficiency transcription expression method thereof |
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Cited By (3)
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| CN113234755A (en) * | 2021-03-31 | 2021-08-10 | 浙江省农业科学院 | Expression plasmid of exogenous gene of macrobrachium rosenbergii and high-efficiency transcription expression method thereof |
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