CN114164262B - Molecular technology-based sex identification method for northern pikes - Google Patents
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Abstract
The invention discloses a molecular technology-based sex identification method for northern pikes, which is characterized in that a specific molecular marker for genetic sex of northern pikes is claimed, and the difference exists between an X chromosome and a Y chromosome of northern pikes, so that the difference can be used for identifying the genetic sex of northern pikes. The invention overcomes the defects of the prior art, provides a specific molecular marker based on sex linkage, can simply and quickly realize genetic sex, and greatly saves manpower and material resources.
Description
Technical Field
The invention relates to the technical field of fish genetic sex identification and sex control in the technical field of aquatic organisms, in particular to a molecular technology-based sex identification method for northern pikes.
Background
In fish culture with female growth speed obviously faster than that of male, the realization of total-female culture by sex control technology is one of the key technologies for improving the culture yield, and the northern pike is the fish with economic value in this way. Therefore, the sex-related gene studies of northern pikes are increasing. The scholars at home and abroad also develop a series of experimental researches on sex control of the northern pikes. The Luczynski et al induced the northern pike to develop gynogenesis, obtained gynogenesis fries of all-female northern pikes, and determined the chromosome sex determination class of male gametophyte allotype (XY) of northern pikes. Demska-Zake et al have found that feeding 11 beta-hydroxyandrogendione to northern pike fries can convert all of them to male or intermittent. Recent studies on the determining mechanism of fish sex and gene have achieved remarkable results, and Zhang Junjie et al have also made many related studies.
The research of the genetic sex identification technology of the northern pikes is carried out, so that the method has important scientific significance and wide application prospect. Traditional fish sex control breeding needs to judge whether the parent genotype is XX pseudo-male fish through test cross, which is time-consuming and labor-consuming. Based on the sex identification technology, sex identification can be simply and rapidly realized, and manpower and material resources are greatly saved. Therefore, a person skilled in the art provides a molecular technology-based sex identification method for northern pikes, so as to solve the problems in the background art.
Disclosure of Invention
In order to solve the technical problems, the invention provides a molecular technology-based sex identification method for northern pikes, which extracts DNA of northern pikes fin, adopts a 4# primer (reseqSNP 381),
the 4# primer (reseqSNP 381) was synthesized and dissolved, and identified by PCR amplification reaction,
the sequences of the 4# primers are respectively:
GGACTTTTTCCTACATACCTCAC and TCAGCCACTATATCTATCTTACCG.
Preferably: the extraction steps of the DNA of the northern pike fin are as follows:
(a) 30mg of fin sample tissue preserved by absolute ethyl alcohol is sheared as much as possible, and air-dried for 1h;
(b) Adding 600 μl of lysate, 10 μl proteinase K (20 mg/ml), mixing well in water bath at 56 ℃ and lysing cells for 2h;
(c) Centrifugation at 8000rpm for 5min, and taking 550. Mu.L of supernatant;
(d) Adding 550 mu L of phenol-chloroform isoamyl alcohol, manually and gently mixing for 10min, centrifuging for 10min at 13000rpm and 4 ℃;
(e) Carefully aspirate 400 μl of supernatant (without protein to be aspirated into the middle) into a new EP tube;
(f) Adding 800 μL of absolute ethanol pre-cooled at-20deg.C, standing at-20deg.C for 30min, and centrifuging at 13000rpm for 10min at 4deg.C;
(g) The supernatant was discarded and the DNA was a white precipitate on the walls and bottom of the EP. Adding 70% ethanol, and centrifuging at 13000rpm for 5min after gently blowing;
(h) Repeating the operation step g;
(i) 70 μL ddH was added 2 O is dissolved;
(j) 1.5% agarose gel electrophoresis, the concentration and purity of the DNA extraction effect can be measured by a full-wavelength enzyme-labeled instrument, and the concentration of each DNA sample is diluted and adjusted to 50-100 ng/. Mu.L according to the DNA concentration measured by the enzyme-labeled instrument.
Preferably: the PCR amplification reaction: the operation steps are as follows:
(a1) PCR reaction system: PCR reaction premix (mix) was used, 12.5. Mu.L of each reaction;
(b1) The PCR reaction procedure was as follows:
firstly, carrying out denaturation at 94 ℃ for 10min, then carrying out 35 cyclic reactions, specifically comprising denaturation at 94 ℃ for 30s, then carrying out annealing at 57 ℃ for 30s, extending at 72 ℃ for 1min after annealing, extending at 72 ℃ for 10min after the reaction is finished, and finally storing at 4 ℃.
Preferably: the PCR amplification reaction is identified by agarose gel electrophoresis detection and sex identification.
Preferably: the agarose gel electrophoresis detection and sex identification steps are as follows:
(a2) Agarose gel electrophoresis:
detecting PCR amplified products by agarose gel electrophoresis of 2% under 150V voltage by using an agarose level electrophoresis apparatus, and adopting DL2000 as electrophoresis maker (the band of the electrophoresis maker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom);
(b2) Sex identification:
the 4# primer (reseqSNP 381) amplified a different band between the male and female fish. Electrophoresis of the amplified product of the female fish DNA to obtain a single band, wherein the size of the amplified product is 606bp. The DNA amplified product of the male fish is electrophoresed to obtain two very obvious bands, one band is 606bp in size, and the other band is about 320bp in size.
Preferably: and determining the sequencing quality of the sequencing result by adopting Chromas, and performing sequence splicing and comparison analysis by using DNAMAN.
Preferably: and the sequence comparison is carried out on the sequencing result of the double ends of the male specific band and the sequencing result of the single end of the male and female common band of the 4# primer (reseqSNP 381).
The invention has the technical effects and advantages that:
the invention claims a specific molecular marker of the genetic sex of the northern pike, which has a difference between the X chromosome and the Y chromosome of the northern pike, and can be used for identifying the genetic sex of the northern pike. The invention overcomes the defects of the prior art, provides a specific molecular marker based on sex linkage, can simply and quickly realize genetic sex, and greatly saves manpower and material resources.
Drawings
FIG. 1 is a diagram showing the result of amplification of 24 pairs of primers with bands on the DNA pairs of 4 female fish and 4 male fish in the sex identification method of northern pike provided in the example of the present application;
fig. 2 is a diagram showing the results of sequencing and splicing the two ends of a male specific band of a primer # 4 (reseqSNP 381) in the sex identification method of northern pike provided in the example of the present application;
FIG. 3 is a diagram showing the sequencing result of a male-female common band obtained by PCR amplification of a 4# primer (reseqSNP 381) in the method for sex identification of northern pike provided in the embodiment of the present application;
FIG. 4 is a diagram showing PCR amplification results of 45 female fish and 66 male fish with the 4# primer (reseqSNP 381) in the sex identification method of northern pike provided in the embodiment of the present application;
Detailed Description
The invention will be described in further detail with reference to the drawings and the detailed description. The embodiments of the invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
In this example there is provided a method of sex identification of northern pike,
(1) Extracting the DNA of the fin of the northern pike, adopting a 4# primer (reseqSNP 381),
the 4# primer (reseqSNP 381) was synthesized and dissolved, and identified by PCR amplification reaction,
the sequences of the 4# primers are respectively:
GGACTTTTTCCTACATACCTCAC and TCAGCCACTATATCTATCTTACCG.
(2) The extraction steps of the DNA of the northern pike fin are as follows:
(a) About 30mg of fin sample tissue preserved by absolute ethyl alcohol is sheared as much as possible, and air-dried for 1h;
(b) Adding 600 μl of lysate, 10 μl proteinase K (20 mg/ml), mixing well in water bath at 56 ℃ and lysing cells for 2h;
(c) Centrifugation (8000 rpm for 5 min) gave about 550. Mu.L of supernatant;
(d) Adding 550 mu L of phenol-chloroform isoamyl alcohol, manually and gently mixing for 10min, centrifuging for 10min at 13000rpm and 4 ℃;
(e) Carefully aspirate 400 μl of supernatant (without protein to be aspirated into the middle) into a new EP tube;
(f) Adding 800 μL of absolute ethanol pre-cooled at-20deg.C, standing at-20deg.C for 30min, and centrifuging at 13000rpm for 10min at 4deg.C;
(g) The supernatant was discarded and the DNA was a white precipitate on the walls and bottom of the EP. Adding 70% ethanol, and centrifuging at 13000rpm for 5min after gently blowing;
(h) Repeating the operation step g;
(i) Adding 70 mu L of ddH2O for dissolution;
(j) 1.5% agarose gel electrophoresis, the concentration and purity of the DNA extraction effect can be measured by a full-wavelength enzyme-labeled instrument, and the concentration of each DNA sample is diluted and adjusted to 50-100 ng/. Mu.L according to the DNA concentration measured by the enzyme-labeled instrument.
Example 1
The PCR reaction test procedure was as follows:
(a1) The PCR reaction was performed using PCR reaction premix (mix) from Beijing Co., ltd. Of Tiangen Biochemical technology, each of which was 12.5. Mu.L, and the specific reaction system was shown in Table 1.
(b1) The PCR reaction procedure was as follows: firstly, carrying out denaturation at 94 ℃ for 10min, then carrying out 35 cyclic reactions, specifically comprising denaturation at 94 ℃ for 30s, then carrying out annealing at 57 ℃ for 30s, extending at 72 ℃ for 1min after annealing, extending at 72 ℃ for 10min after the reaction is finished, and finally storing at 4 ℃.
Agarose gel electrophoresis detection and sex identification:
(a2) Agarose gel electrophoresis:
the PCR amplification products were detected by agarose gel electrophoresis at a voltage of 150V using an agarose level electrophoresis apparatus (DYCP-31 DN), and DL2000 was used as an electrophoresis maker (the band was 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom).
(b2) Sex identification:
the 4# primer (reseqSNP 381) amplified a different band between the male and female fish. Electrophoresis of the amplified product of the female fish DNA to obtain a single band, wherein the size of the amplified product is 606bp. The DNA amplified product of the male fish is electrophoresed to obtain two very obvious bands, one band is 606bp in size, and the other band is about 320bp in size.
Sequencing and analysis of PCR amplified products
Sequencing the PCR amplified product, wherein single-end sequencing is carried out on the common female and male bands amplified by the primer, and double-end sequencing is carried out on the sex-specific bands amplified by the primer. And determining the sequencing quality of the sequencing result by using Chromas, and performing sequence splicing and alignment analysis by using DNAMAN.
The results of the sequencing splice of the two ends of the male specific strip of the primer # 4 (reseqSNP 381) are shown in FIG. 2. The male specific band length is 338bp, one end of the sequence is matched with the reference genome (except 2 bases), and the other end is matched with the reference genome (except 12 bases) by 284 bases (shown in figure 3).
The sequence was aligned with the single-end sequencing result of the male and female consensus bands, and the result is shown in FIG. 3. In the figure, the fragment amplified at 734304bp-735909bp of chromosome 24 (NC_ 025991.4) is a sheared version of a primer design sequence, and the fragment is deleted from 51bp-324bp, and 273bp is deleted.
FIG. 4 shows the result of PCR amplification of 45-tail female fish and 66-tail male fish DNA with the 4# primer (reseqSNP 381) flanking one specific SNP site at the 734587bp position of chromosome 24 (NC_ 025991.4). As can be seen from the figure, the amplification results are very different between the female fish and the male fish, 42 female fish are amplified to obtain a single band, and the size of the amplified product is basically consistent with the size (606 bp) of the predetermined product when the primer is designed. Of the 66 males, 64 amplified two very distinct bands, one band of size consistent with the predetermined product size (606 bp) and the other band of about 320bp. In addition, the amplification result of 3 out of 45 females was identical to that of the males, two bands were present, and the amplification result of 2 out of 66 males was identical to that of the females, with only one band consistent with the predetermined product (606 bp). Wherein: the upper two rows are 45 females and the lower three rows are 66 males. The center of each plot is DL2000maker, the bands of which are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art and which are included in the embodiments of the present invention without the inventive step, are intended to be within the scope of the present invention. Structures, devices and methods of operation not specifically described and illustrated herein, unless otherwise indicated and limited, are implemented according to conventional means in the art.
Claims (5)
1. A molecular technology-based sex identification method for northern pike is characterized in that DNA of northern pike fin is extracted, 4# primer is synthesized and dissolved, PCR amplification reaction is used for identification,
the sequences of the 4# primers are respectively:
primer 1: GGACTTTTTCCTACATACCTCAC and primer 2: TCAGCCACTATATCTATCTTACCG; the 4# primer is used for amplifying different bands between the female fish and the male fish, single bands are obtained by electrophoresis of a female fish DNA amplification product, the size of the amplification product is 606bp, and two bands are obtained by electrophoresis of a male fish DNA amplification product, wherein one band is 606bp, and the other band is 320bp.
2. The molecular technology-based sex identification method for northern pikes, as claimed in claim 1, wherein the step of extracting the DNA of the northern pikes fin is as follows:
(a) 30mg of fin sample tissue preserved by absolute ethyl alcohol is air-dried for 1h;
(b) Adding 600 mu L of lysate, 10 mu L of proteinase K (20 mg/ml), mixing uniformly, and carrying out water bath at 56 ℃ to lyse cells for 2h;
(c) Centrifugation at 8000rpm for 5min, and taking 550. Mu.L of supernatant;
(d) Adding 550 mu L of phenol-chloroform isoamyl alcohol, manually and gently mixing for 10min, centrifuging for 10min at 13000rpm and 4 ℃;
(e) Carefully aspirate 400 μl of supernatant into a new EP tube;
(f) Adding 800 μL of absolute ethanol pre-cooled at-20deg.C, standing at-20deg.C for 30min, and centrifuging at 13000rpm for 10min at 4deg.C;
(g) Removing supernatant, wherein DNA is white precipitate at the wall and bottom of EP, adding 70% ethanol, lightly blowing, and centrifuging at 13000rpm for 5min;
(h) Repeating step (g);
(i) 70 μL ddH was added 2 O is dissolved;
(j) 1.5% agarose gel electrophoresis, the concentration and purity of the DNA extraction effect can be measured by a full-wavelength enzyme-labeled instrument, and the concentration of each DNA sample is diluted and adjusted to 50-100 ng/. Mu.L according to the DNA concentration measured by the enzyme-labeled instrument.
3. The molecular technology-based sex identification method of northern pike as claimed in claim 1, wherein the PCR amplification reaction: the operation steps are as follows:
(a1) PCR reaction system: the PCR reaction premix mix was used, 12.5. Mu.L of each reaction;
(b1) The PCR reaction procedure was as follows:
firstly, carrying out denaturation at 94 ℃ for 10min, then carrying out 35 cyclic reactions, specifically comprising denaturation at 94 ℃ for 30s, then carrying out annealing at 57 ℃ for 30s, extending at 72 ℃ for 1min after annealing, extending at 72 ℃ for 10min after the reaction is finished, and finally storing at 4 ℃.
4. The molecular technology-based sex identification method of northern pike as claimed in claim 1, wherein the identification of the PCR amplification reaction comprises agarose gel electrophoresis detection and sex identification.
5. The molecular technology-based sex identification method of northern pike as claimed in claim 4, wherein the steps of agarose gel electrophoresis detection and sex identification are as follows:
(a2) Agarose gel electrophoresis:
performing 2% agarose gel electrophoresis at 150V by using an agarose level electrophoresis apparatus, detecting PCR amplification products, and using DL2000 as an electrophoresis maker;
(b2) Sex identification:
the 4# primer is used for amplifying different bands between the female fish and the male fish, single bands are obtained by electrophoresis of a female fish DNA amplification product, the size of the amplification product is 606bp, and two bands are obtained by electrophoresis of a male fish DNA amplification product, wherein one band is 606bp, and the other band is 320bp.
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