CN112746111B - Northern snakehead male molecular marker primer and application thereof - Google Patents
Northern snakehead male molecular marker primer and application thereof Download PDFInfo
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Abstract
The invention discloses a male molecular marker primer of channa maculata, which is a primer pair M12 or a primer pair P2, wherein the primer pair M12 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair P2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, and the nucleotide sequences of the primers are respectively shown as SEQID NO:1 to SEQ ID NO:4, the primer can be used for rapidly and accurately identifying the sex of the channa maculata. Meanwhile, the method for identifying the sex of the channa maculata is short in time consumption and high in efficiency. And the application of the primer and the method in the aspect of male breeding of channa maculata.
Description
Technical Field
The invention belongs to a channa maculata sex identification technology, and particularly relates to channa maculata male molecular marker primers and application thereof.
Background
In recent years, sequencing technology has been rapidly developed, and high-throughput sequencing cost has become lower and lower. More and more economical fish markers of sex are found based on high throughput sequencing technologies, such as tilapia nilotica (Oreochromis niloticus), scatophagus parvus (scatophagogorgus), cynoglossus semilaevis (cynoglossus semilaevis), macrospinus dabryanus (mastacember), channa maculata (siniperca chuatsi), and fugu rubripes (takifuguribubripes). The successful development of these sex-specific molecular markers has greatly accelerated the development of parthenocarpy breeding.
Channaeus (the scientific name: channaacula) belongs to snakehead family and snakehead fish, is freshwater bottom-layer fish, and is inhabited in coastal aquatic plants and muddy substrate shallow water areas. The channa maculata is typical carnivorous fish, young fishes mainly feed on copepods, cladocera, larval fishes, aquatic insects and the like, and adult fishes feed on various small trash fishes including crucian carps, loaches and the like. The Channa argus meat has delicious taste, white and tender meat quality, rich nutrition and higher economic value. In addition, the channa maculata grows to have obvious sex dimorphism, and the male of the channa maculata grows faster than the female.
The male fish grows faster than the female fish, so that the realization of the culture of the all-male channa maculata can obviously improve the aquaculture benefit. The existing research shows that the snakehead and the channa maculata belong to XY sex determination type, therefore, XY false female fish is needed to be obtained at first in the cultivation of the all-male channa maculata, and the obtained false female fish is mainly obtained by a method of induced reversion of genetic male fish. The sex of the channa maculata is difficult to distinguish from the external form, and the traditional test cross method is time-consuming and labor-consuming, so that a stable and reliable molecular marker needs to be developed to screen XY false female fish. So far, although there are reports of molecular markers capable of successfully identifying the genetic sex of channa maculata at home and abroad, the molecular markers are mainly single nucleotide polymorphism markers and are difficult to use in practice.
Therefore, the development of a simple molecular marker capable of accurately identifying the genetic sex has great production and application values in the male-and-female breeding of the channa maculata.
Disclosure of Invention
The invention aims to provide a channa maculata male molecular marker primer, which can identify the sex of channa maculata on a molecular level.
The invention also aims to provide a method for carrying out sex determination on the channa maculata by using the primer, which has the advantages of short time consumption and accurate detection result.
The last purpose of the invention is to provide the application of the northern snakehead male molecular marker primer and the northern snakehead sex identification method in the whole male breeding of northern snakehead.
In order to achieve the first purpose, the invention adopts the following technical scheme:
a channa maculata male molecular marker primer is a primer pair M12 or a primer pair P2, the primer pair M12 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair P2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO:1, the nucleotide sequence of the Contig-1 downstream primer is shown as SEQ ID NO:2, the nucleotide sequence of the Contig-2 upstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the Contig-2 downstream primer is shown as SEQ ID NO:4, respectively.
Specifically, the nucleotide sequence of each primer from 5 'to 3' is as follows:
contig-1 upstream primer: 5 'GCAAGCCAGTTTGTATGGAGTA-3';
contig-1 downstream primer: 5 'CCAGAGAGGTGGATGTTCTTTATC-3';
contig-2 upstream primer: 5 'CCTCTGTGTTCCTCCTGTCCTCCTGATAGCTC-3';
contig-2 downstream primer: 5 'AAAGACCATAGCTGAGTACGGCAA-3'.
In order to achieve the second object, the invention adopts the following technical scheme:
a method for sex determination of channa maculata comprises the following steps:
(1) Designing the primer pair M12 or the primer pair P2;
(2) Preparing a channa maculata DNA sample;
(3) Taking the channa maculata DNA sample in the step (2) as a template, adopting a primer pair M12 to carry out PCR amplification, carrying out electrophoresis detection after the amplification reaction is finished, and if the electrophoresis result shows that a specific DNA strip is provided, wherein the channa maculata DNA sample detected at the moment is male channa maculata;
or carrying out PCR amplification on the P2 by using a primer, carrying out electrophoresis detection after the amplification reaction is finished, wherein the detected channa maculata DNA sample is male channa if the electrophoresis result shows that the specific channa DNA strip and two non-specific male and female shared NDA strips exist, and the detected channa maculata DNA sample is female channa maculata if the electrophoresis result shows that the non-specific channa DNA strip exists.
Preferably, the DNA sample preparation in step (2) adopts a column type centrifugation method to extract the DNA of the channa maculata.
Preferably, in the PCR amplification using the primer pair M12 or the primer pair P2 in step (3), 50ng of DNA, C is contained in the 20. Mu.LPCR reaction system0.8. Mu.L of the ontig-1 forward primer and the Contig-1 backward primer respectively or 0.8. Mu.L of the Contig-2 forward primer and the Contig-2 backward primer respectively, 10. Mu.L of 2 XTAQA Plus Mastermix II, and ddH for the rest 2 And (4) supplementing by using oxygen.
Preferably, when the primer pair is used for PCR amplification in step (3), the PCR reaction procedure is as follows: firstly, pre-denaturation is carried out for 3min at 94 ℃; then carrying out denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extending at 72 ℃ for 2min for 35 cycles; final extension at 72 ℃ for 5min.
Preferably, when the electrophoresis detection is performed in the step (3), agarose gel electrophoresis with the mass percentage of 1% is adopted to detect the length of the amplified fragment.
Preferably, the electrophoresis result in the step (3) shows that the male has a specific band, and the molecular size of the male specific DNA band amplified by the primer pair M12 is 902bp; the molecular size of the male specific DNA band amplified by the primer pair P2 is 1915bp, and the other two non-specific male and female common DNA bands are 679bp and 1010bp respectively.
In order to achieve the third object, the invention adopts the following technical scheme:
the northern snakehead male molecular marker primer and the northern snakehead sex identification method are applied to the northern snakehead all-male breeding.
The invention has the following beneficial effects:
(1) The invention is based on the genome sequencing data of male and female channa maculata, utilizes the methods of genome assembly, comparison, deep analysis and the like to accurately screen the male molecular sequence of channa maculata, designs a male molecular marker primer for identifying the sex of channa maculata for the first time, and the primer can quickly and accurately identify the sex of channa maculata;
(2) The method establishes a method for identifying the sex of the channa maculata by using the channa maculata male molecular marker primer, completes a PCR reaction by using one of two pairs of specific primers, can identify the genetic sex of the channa maculata, and has the advantages of short time consumption, high efficiency and resource saving.
Drawings
FIG. 1 is a schematic diagram showing the distribution of the lengths of the unique fragments of the Y chromosome in example 1;
FIG. 2 is an electrophoretogram of PCR products of two pairs of primers in which amplified fragments were detected only in a male sample and corresponding amplified fragments were not detected in a female sample in example 1;
FIG. 3 is an electrophoretogram of male and female individual (48 tails) detection PCR products of the primer pair M12 in example 2;
FIG. 4 is an electrophoresis diagram of the PCR products for male and female individual detection (48-tailed) of the primer pair P2 in example 2.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to specific examples so that those skilled in the art can better understand and implement the technical solutions of the present invention. Reagents or materials used in the examples were commercially available, unless otherwise specified.
Example 1
The embodiment provides a snakehead fish male molecular marker primer, which is a primer pair M12 or a primer pair P2, wherein the primer pair M12 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair P2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO:1, the nucleotide sequence of the Contig-1 downstream primer is shown as SEQ ID NO:2, the nucleotide sequence of the Contig-2 upstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the Contig-1 downstream primer is shown as SEQ ID NO:4, respectively.
Specifically, the nucleotide sequence of each primer from 5 'to 3' is as follows:
contig-1 upstream primer: 5 'GCAAGCCAGTTTGTATGGAGTA-3';
contig-1 downstream primer: 5 'CCAGAGAGGTGGATGTTCTTTATC-3';
contig-2 upstream primer: 5;
contig-2 downstream primer: 5 'AAAGACCATAGCTGAGTACGGCAA-3'.
The snakehead fish male molecular marker primer is obtained in the following mode and comprises the following steps:
(I) sequencing library construction and re-sequencing
1. Preparation of a Channa maculata DNA sample:
in the breeding period, female 31-tailed and male 3-tailed individual tail fins of the channa maculata are cut, a DNA sample is prepared by adopting a conventional column type centrifugation method (a marine animal tissue genome extraction kit, tiangen Biochemical technology limited company) and referring to the operation steps of the instruction, and the quality and concentration detection is carried out by using 1% agarose gel electrophoresis and a micro-spectrophotometer (NanoDrop 2000), so as to meet the requirement of high-throughput sequencing.
2. Sequencing library construction and high throughput sequencing:
taking female fish F1-F3 and male fish M1-M3 DNA samples, respectively constructing a 350-500bp fragment sequencing library, and performing double-End (Pair-End) PE150 sequencing by using an Illumina Hiseq X Ten platform to respectively obtain female and male genome sequencing clean data:60,506,228,100bp and 65,442,598,200bp.
And (2) uniformly mixing equivalent DNA from the rest 28 female fish DNA samples to form a female fish DNA mixing pool, constructing a 350-500bp fragment sequencing library, and performing PE150 double-end sequencing by using an Illumina Hiseq X Ten platform to obtain female fish DNA mixing pool sequencing clean data:30,925,893,000bp.
Library construction procedures were specifically described in the Novogene NGS DNA Library Prep Kit, and sequencing was performed by the Somatode science and technology, inc., beijing Novo.
(II) enrichment of Y chromosome-specific DNA fragments
1. The method comprises the steps of male fish genome assembly:
male fish M3 sequencing reads were subjected to genome assembly using the all method in SOAPdenovo v2.04-r240 (https:// github. Com/aquaskyline/SOAPdenovo 2) to obtain male fish strips 541,932scaffolds, total size 667,573,559bp, where N50=57,259bp.
2. Obtaining male fish candidate DNA fragments:
female fish F1-F3 sequencing reads and male fish M1-M3 sequencing reads are compared to male fish genome by means of the aln method of bwa v0.7.17-r1188 (http:// bio-bw. Sourceform. Net /) software, reads which cannot be aligned at both ends of the male fish sequencing reads are filtered out, and regions which can be aligned with the male fish sequencing reads but are not aligned with the female fish sequencing reads are selected, and the regions are male fish specific DNA fragments (Table 1).
TABLE 1 is the information table of the specific candidate segments of the male fish
2. Enrichment of male-specific DNA fragments:
taking a male genome as reference, taking female fish DNA mixed pool sequencing reads as query, and adopting an aln method of bwa v0.7.17-r1188 (http:// bio-bwa. Source. Net /) software to carry out sequence comparison so as to find out the male fish specific DNA fragment which is not covered by any reads. 23 DNA fragments with the length being larger than or equal to 300bp can be compared without reading of any female fish DNA mixing pool, and the lengths of the male fish DNA fragments are mainly distributed in 300-1000bp and account for 78.26 percent of the total length. These 23 contigs contained specific DNA fragments from the Y chromosome (fig. 1).
Screening of molecular markers of (III) Y chromosome
1. Designing and synthesizing a primer:
based on the 23 male fish Contig sequences, 11 Contig sequences were randomly selected, and Primer5 software (software using reference Primer PREMIER version5.0 for Windows and Power Macintosh) was used to design PCR primers with the Primer numbers of Contig-xxF/R (xx stands for Contig number), e.g., the Primer number of Contig-1 is Contig-1F/R (F stands for upstream Primer, R stands for downstream Primer, the same applies below). The primers were synthesized by Enwei fundi (Shanghai) trade Co., ltd.
Screening and confirmation of molecular markers of the Y chromosome:
the 11 pairs of primers were selected for PCR detection of 4 individuals each of male and female.
The total PCR amplification system was 20. Mu.L, including 10. Mu.L of 2 XTaq Plus Mastermix II (Biotech Co., ltd., nanjing NuoZan), 0.8. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), and 50ng of template DNA.
The PCR reaction program is: pre-denaturation at 94 ℃ for 2min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 35 cycles; final extension at 72 ℃ for 5min.
Agarose gel electrophoresis results show that 2 pairs of primers have specific amplification bands in male individuals, no specific amplification fragment is detected in female individuals (figure 2), and the two pairs of primers are respectively Contig-1F/R and Contig-2F/R, which means that the corresponding Contig is Contig-1 and Contig-2.
From the PCR amplification of 11 pairs of primers in 4 individuals each of males and females, 2 of them: contig-1 and Contig-2 are specific fragments of Y chromosome and are male molecular markers of channa maculata.
Example 2
The method for identifying the sex of the channa maculata by utilizing the channa maculata male molecular marker primer provided by the embodiment comprises the following steps:
(1) Designing the male molecular marker primer of the channa maculata, and referring to the embodiment 1 in the concrete process;
(2) Preparation of a Channa maculata DNA sample:
the visually mature channa maculata is obtained from a Baijin three-hydrate water-borne sprout limited company of Fushan City in Guangdong province, and 24 fish of each female channa maculata and each male channa maculata are obtained through dissection. The tail fins were cut out, and DNA samples were prepared by the conventional column centrifugation method (Marine animal tissue genome extraction kit, tiangen Biochemical technology Co., ltd.) according to the procedure of the instruction.
(3) And (3) PCR amplification verification:
PCR verification is carried out on the 24 male and female channa maculata DNA samples by primers of Contig-1F/R and Contig-2F/R.
The total PCR amplification system is 20. Mu.L, including 10. Mu.L of well-known century 2 XTaq Plus Mastermix II (Biotechnology Co., ltd., nanjing Novozam), 0.8. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), and 50ng of template DNA.
The PCR reaction program is: firstly, performing pre-denaturation at 94 ℃ for 2min; then carrying out denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extending at 72 ℃ for 2min for 35 cycles; final extension at 72 ℃ for 5min.
After the PCR reaction is finished, the product is subjected to agarose gel electrophoresis with the mass percentage of 1%, and the result is that: primers Contig-1F/R and Contig-2F/R can amplify bands in the DNA sample of the male fish of 24 tails, and at the moment, the molecular size of the male specific DNA band amplified by the primers Contig-1F/R is 902bp; the molecular size of the male specific DNA band amplified by using the primer Contig-2F/R is 1915bp, and the other two male and female common non-specific DNA bands are 679bp and 1010bp respectively. No male-specific DNA band could be amplified in the 24-tailed female DNA samples (FIG. 3. The molecular markers Contig-1F/R and Contig-2F/R can be used for identifying the sex of the channa maculata.
Example 3
The embodiment provides application of the northern snakehead male molecular marker primer to northern snakehead all-male breeding, which comprises the following steps:
(1) When the fry of the channa maculata is cultured to 10 days old, the channa maculata is fed by feed containing 100mg/kg of estradiol. Continuously feeding for two months, adopting natural illumination during the culture period, culturing at the water temperature of 26-28 deg.C, and feeding after full feeding.
(2) The method in example 2 is adopted to screen the female snakeheads fed with estrogen, so as to obtain the female fish with genetic type XY, namely pseudo-female fish (namely male snakeheads described in the text), and the specific process refers to example 2.
(3) And (4) continuously culturing the XY false female fish identified by the mark until the XY false female fish reaches sexual maturity, mating with normal XY male fish in a breeding season, and obtaining offspring containing XY and YY male fish.
(4) Further screening YY super-male fish in offspring by adopting a male specific molecular marker through a fluorescent quantitative PCR or test cross means, breeding the obtained YY super-male fish to sexual maturity, and mating the obtained YY super-male fish with XX female fish to obtain the offspring of the full-male fish.
The above embodiments are only used for illustrating the present invention, and the scope of the present invention is not limited to the above embodiments. The object of the present invention can be achieved by those skilled in the art based on the above disclosure, and any improvements and modifications based on the concept of the present invention fall within the protection scope of the present invention, which is defined by the claims.
Sequence listing
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Claims (7)
1. A northern snakehead male molecular marker primer is characterized in that: the primer is a primer pair M12 or a primer pair P2, the primer pair M12 is a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair P2 is a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO:1, the nucleotide sequence of the Contig-1 downstream primer is shown as SEQ ID NO:2, the nucleotide sequence of the Contig-2 upstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the Contig-2 downstream primer is shown as SEQ ID NO:4, respectively.
2. A method for sex identification of channa maculata is characterized by comprising the following steps:
(1) Preparing a channa maculata DNA sample;
(2) Taking the channa maculata DNA sample in the step (1) as a template, adopting the primer in the claim 1 to carry out PCR amplification on M12, carrying out electrophoresis detection after the amplification reaction is finished, and if the electrophoresis result shows that a specific DNA band exists, the channa maculata DNA sample detected at the moment is male channa maculata;
or carrying out PCR amplification on the P2 by using the primer in the claim 1, carrying out electrophoresis detection after the amplification reaction is finished, wherein if the electrophoresis result shows that the specific DNA strip and the two non-specific male and female common DNA strips exist, the detected channa maculata DNA sample is male channa, and if the electrophoresis result shows that the two non-specific male and female common DNA strips exist, the detected channa maculata DNA sample is female channa maculata;
the electrophoresis result in the step (2) shows that the male has a specific band, and the molecular size of the male specific DNA band amplified by adopting a primer pair M12 is 902bp; the molecular size of the male specific DNA band amplified by the primer pair P2 is 1915bp, and the other two non-specific male and female common DNA bands are 679bp and 1010bp respectively.
3. The method for sex determination of channa maculata according to claim 2, characterized in that: in the step (1), a column type centrifugal method is adopted to extract a channa maculata DNA sample.
4. The method for sex identification of channa maculata according to claim 2, characterized in that: when the primer pair M12 or the primer pair P2 is adopted for PCR amplification in the step (2), a 20 mu L PCR reaction system contains 50ng of DNA, 0.8 mu L of each of the Contig-1 upstream primer and the Contig-1 downstream primer or 0.8 mu L of each of the Contig-2 upstream primer and the Contig-2 downstream primer, 10 mu L of 2 xTaq Plus MasterMix II and the balance ddH 2 O。
5. The method for sex determination of channa maculata according to claim 2, characterized in that, when the primer pair is used for PCR amplification in step (2), the PCR reaction program is: firstly, pre-denaturation is carried out for 2min at 94 ℃; then carrying out denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 2min for 35 cycles; final extension at 72 ℃ for 5min.
6. The method for sex determination of channa maculata according to claim 2, characterized in that, when the electrophoresis detection is carried out in step (2), the agarose gel electrophoresis with the mass percentage content of 1% is adopted to detect the length of the amplified fragment.
7. The application of the northern snakehead male molecular marker primer in the claim 1 and the method in the claim 2 in the whole male breeding of northern snakehead.
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| CN114592076B (en) * | 2022-05-10 | 2022-08-16 | 中山大学 | Siniperca scherzeri male molecular marker primer and application thereof |
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| CN103798173B (en) * | 2014-03-12 | 2015-12-02 | 佛山市南海百容水产良种有限公司 | For the progeny testing method of yellow cartfish and snakehead supermale fish and full milter exploitation |
| CN106119378B (en) * | 2016-07-06 | 2019-01-01 | 中国水产科学研究院珠江水产研究所 | SNP site and its detection method for snakehead sex identification |
| CN107094671A (en) * | 2017-05-19 | 2017-08-29 | 佛山市南海百容水产良种有限公司 | A kind of all-male hybridizes the breeding method of Pelteobagrus fulvidraco |
| CN107217099B (en) * | 2017-06-28 | 2018-11-27 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker and its application can be used for snakehead genetic sex and the identification of supermale fish |
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