CN116042856B - Molecular marker, primer group, kit and method for identifying sex of snakehead - Google Patents
Molecular marker, primer group, kit and method for identifying sex of snakehead Download PDFInfo
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Abstract
The application relates to the technical field of sex detection of snakeheads, and discloses a molecular marker, a primer group, a kit and a method for identifying sex of snakeheads. The molecular marker is a DNA sequence of the female and male differences of the channa linezolid, and the DNA sequence is shown as SEQ ID NO.1. The method for identifying the sex of the snakehead comprises the following steps: obtaining genome DNA benzene of the snakehead, carrying out PCR reaction by taking the genome DNA sample as a template, and judging the sex of the snakehead to be detected according to an electrophoresis chart of a PCR reaction product. The identification primer group, the kit and the method provided by the embodiment of the application are not influenced by the development period and the environment of individuals, the sex identification is simple, convenient and quick, the requirement on samples is low, the accuracy of identification results is high, and therefore the defects that other methods are complex in operation, long in time consumption and the like are overcome, and the method is particularly suitable for quick identification of large samples.
Description
Technical Field
The application relates to the technical field of sex detection of channa lineolata, in particular to a molecular marker, a primer group, a kit and a method for identifying sex of channa lineolata.
Background
The Channa (Channa straata) belongs to weever-shaped orders (Perciformes), weever sub-orders (Anabathoidei), snakeheadaceae (Channidae), snakeheads (Channa), commonly called Thailand snakeheads, thailand tiger, tiger maculons, and is distributed in China in south China, southeast Asia and south Asia, and is one of important freshwater economic fishes and one of large aquarium ornamental fishes. The snakehead has strong vitality, high growth speed, and can be marketed in the same year after cultivation, and has more meat, less thorns and delicious taste, and is deeply favored by the cultivation people and consumers. In the artificial breeding and selective breeding process, female and male screening of parent fish is required to obtain high-quality fries, and the sex of the snakehead fish must be accurately identified to establish families. In the breeding process, the first maturation of the channa argus takes two years, the sex of the channa argus cannot be identified through appearance forms before sexual maturation, and sexes of sexes can be accurately identified only through the types of gametes produced by the sexes in breeding seasons, so that the artificial breeding and improved variety breeding processes of the channa argus are greatly limited.
The prior art does not specify the sex determination mechanism of the channa linezolid, and does not determine whether a heterogenic sex chromosome exists, and the genetic sex thereof cannot be identified from a cytological perspective. There is little difference in the morphology between the male and female fish in the on-line snakehead embryo and the young stage, so that the sex cannot be identified by the appearance morphology in the stage before sexual maturity. The existence of these problems has caused great trouble in breeding, farming and related basic research of genetics, especially sex-determining molecular mechanism research. Therefore, the development of the molecular marker for specific sex of the channa argus and the establishment of the molecular method for rapidly identifying the sex of the channa argus are important means for controlling the sex proportion of male and female parents, identifying sex chromosomes, researching sex determination and the evolution mechanism of the sex chromosomes, and have very important significance for developing monosomic breeding and breeding so as to improve the yield of fish and increase the income of aquaculture.
Disclosure of Invention
The inventor screens out a snakehead male specific DNA sequence through the genome sequencing of the snakehead, the re-sequencing of the female and male snakehead and the GWAS analysis, designs a sex specific molecular marker to be applied to the genetic sex identification of the snakehead, can accurately distinguish the snakehead sex by utilizing the molecular marker through a molecular biological method, and can be used as a molecular technology for rapidly identifying the snakehead genetic sex applied to a farm. Therefore, the embodiment of the application at least discloses the following technical scheme:
in a first aspect, the embodiment of the application discloses a molecular marker of a snakehead male-female difference, wherein the molecular marker is a DNA sequence of the snakehead male-female difference, and the DNA sequence is selected from SEQ ID NO.1.
In a second aspect, the present application discloses a primer set for amplifying the molecular marker of the first aspect, wherein the primer set comprises DNA molecules with nucleotide sequences shown as SEQ ID NO. 2-3.
In a third aspect, embodiments of the present application disclose a kit for identifying the sex of a channa lineolata, comprising a primer set according to the second aspect.
In a fourth aspect, embodiments of the present application disclose a method for identifying the gender of a channa lineolata, comprising the steps of:
obtaining a genome DNA sample of the snakehead to be detected;
taking the genome DNA sample as a template, and carrying out PCR reaction by adopting a primer group shown as SEQ ID NO. 2-3;
performing polyacrylamide gel electrophoresis on the obtained PCR reaction product, and judging according to a polyacrylamide gel electrophoresis diagram:
if two target bands with the length of 257bp and 255bp exist in the PCR reaction product, the corresponding snakehead to be detected is male;
if only one strip of the item with the length of 257bp exists in the PCR reaction product, the corresponding snakehead to be detected is female.
Compared with the prior art, the application has at least one of the following beneficial effects:
the identification primer group, the kit and the method provided by the embodiment of the application can be used for sex identification of the snakehead by taking genomic DNA as a template and adopting simple PCR and electrophoresis results, two specific DNA bands (shown as SEQ ID NO. 1) are amplified in a male individual, and only one band (shown as SEQ ID NO. 1) is amplified in a female individual, so that the time for accurately identifying the genetic sex of the snakehead is shortened. The detection method is suitable for identifying the genetic sex of the channa glabra in the simple environment of the farm, and the detection time and the cost are saved. The method for detecting the genetic sex of the snakehead has important significance and application value in promoting sustainable healthy development of snakehead breeding industry by breeding parent snakehead, establishing families and controlling the sex ratio of the female and male of a breeding population.
The identification primer, the kit and the method provided by the embodiment of the application are not influenced by the development period and the environment of individuals, the sex identification is simple, convenient and quick, the requirement on samples is low, the accuracy of identification results is high, and therefore the defects that other methods are complex in operation, long in time consumption and the like are overcome, and the method is particularly suitable for quick identification of large samples. The identification primer, the kit and the method provided by the embodiment of the application are low in cost, simple in operation, rapid and accurate, and suitable for popularization and application.
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FIG. 1 shows the results of a GWAS analysis using the genomic sequencing of snakehead and the resequencing of male and female individuals combined with gender as provided in the examples of the present application.
FIG. 2 is a diagram of polyacrylamide gel electrophoresis of detection of 10 DNA samples of the male snakehead and 10 DNA samples of the female snakehead using the primer set according to the embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In order to establish a method for rapidly identifying the channa maculata, shorten the time for breeding parent channa maculata, establishing families and controlling the proportion of sexes and sexes of cultured groups, and reduce the cost, the inventor utilizes the resequencing to combine with genome sequencing of the channa maculata, and screens out DNA fragments (SEQ ID NO. 1) with the difference of the sexes and the sexes through GWAS analysis, thereby establishing a channa maculata genetic sex identification method, and the method can rapidly and accurately identify the genetics and the sexes of the channa maculata.
In the screening examples of DNA fragments differing from the male and female of the snakehead, a snakehead was subjected to genomic DNA extraction, illumina, nanopore, HI-C sequencing respectively, and then subjected to genome de novo assembly and chromosome mounting using Flye v2.6, racon v1.5, pilon v1.24, juicer v1.6 and 3D-DNA v201008 software to obtain the snakehead male whole genome sequence. Extracting genome DNA of the male and female snakeheads, constructing and sequencing a resequencing database, and sequencing at least 10×; after filtering the sequencing data, the re-sequenced sequences were aligned to the channa genome using BWA mem v0.7.17, repeated sequences were tagged with picard v2.27.4, SNP and InDel variation detection using GATK v4.2.5, GWAS analysis of gender using GEMMA v0.98.5 software analysis, sex-related SNP sites were screened, and DNA sequences upstream and downstream of the sites were extracted, the nucleotide sequences of which are shown in SEQ ID No.1.
To verify the possibility of using a DNA fragment (shown as SEQ ID NO: 1) with the sex differentiation of snakehead as a molecular marker for sex identification, the embodiment of the application also discloses a primer group, comprising an upstream primer and a downstream primer for amplifying a sequence shown as SEQ ID NO.1, and the nucleotide sequences are shown as SEQ ID NO. 2-3.
Furthermore, the embodiment of the application also discloses a kit for sex identification, which comprises the primer set. Preferably, the kit further comprises a DNA polymerase, mg 2+ One or more of dNTPs, PCR Buffer, and sterile water. Wherein the DNA polymerase is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V, such as Taq polymerase or rTap enzyme.
In some embodiments, the kit further comprises a positive control and a negative control, wherein the positive control is a liquid preparation containing the genomic DNA of the male channa maculata, and the negative control is a liquid preparation containing the genomic DNA of the female channa maculata, so that sex differentiation and identification of the sample to be detected are facilitated.
Based on the detection principle, the primer group and/or the kit are used for carrying out PCR amplification on a genomic DNA sample of the snakehead, and the amplification product is identified. Specifically, the embodiment of the application also discloses a method for identifying the sex of the snakehead, which comprises the following steps:
(1) Obtaining a genome DNA sample of the snakehead to be detected;
(2) Taking the genome DNA sample as a template, and carrying out PCR reaction by adopting a primer group shown as SEQ ID NO. 2-3; and
(3) Performing polyacrylamide gel electrophoresis on the obtained PCR reaction product, and judging according to a polyacrylamide gel electrophoresis diagram:
if two target bands with the length of 257bp and 255bp exist in the PCR reaction product, the corresponding snakehead to be detected is male;
if only one strip of the item with the length of 257bp exists in the PCR reaction product, the corresponding snakehead to be detected is female.
In some embodiments, the genomic DNA sample of the channa argus of step (1) is a genomic DNA liquid sample isolated and extracted from the blood of the channa argus. The genome DNA extraction method can be extracted by CTAB (cetyltrimethylammonium bromide) method.
In some embodiments, the reaction system of the PCR reaction described in step (2) comprises, in 25. Mu.L, 10 XPCR Buffer, 50 ng/. Mu.L of genomic DNA, 1. Mu.L, 0.2 to 0.4. Mu.M upstream primer, 0.2 to 0.4. Mu.M downstream primer, 0.2 to 0.4mM dNTP 0.8. Mu.L, 0.025 to 0.05U/. Mu.L of DNA polymerase, and the balance double distilled water.
In some embodiments, the reaction system of the PCR reaction comprises, in 25. Mu.L, 10 XPCR Buffer, 50 ng/. Mu.L of genomic DNA 1. Mu.L, 0.4. Mu.M upstream primer, 0.4. Mu.M downstream primer, 0.4mM dNTPs 0.8. Mu.L, 0.05U/. Mu.L rTase 0.75. Mu.L, and the balance double distilled water.
In some embodiments, the reaction procedure of the PCR reaction in step (2) comprises the following sub-steps: (1) 94-95 ℃, 3-5 min, (2) 94-95 ℃, 28-35 s, (3) 53-63 ℃, 28-35 s, (4) 72 ℃, 28-40 s, (5) 72 ℃ 5-15min, wherein the substeps (2) to (4) are 30-40 cycles. In some embodiments, the temperature of step (3) in the reaction sequence is 50 to 54 ℃. In some embodiments, the temperature of step (3) in the PCR reaction is 52℃in the PCR reaction system if the target band shown in SEQ ID NO.1 is amplified.
In some embodiments, in step (3), if there are two target bands of 257bp and 255bp in length in the PCR reaction product, the corresponding snakehead to be detected is male. Wherein the nucleotide sequences of the bands of both items are identical to the sequence of SEQ ID NO.1.
As shown in FIG. 1, in one embodiment, the target bands are recovered and sent to Bio-company for sequencing, and the sequence is consistent with the sequence shown in SEQ ID NO.1, by using a polyacrylamide gel electrophoresis of PCR amplification products obtained by using the primer set CsS-1 primer respectively. Moreover, the corresponding samples of the strips carrying two items are all male snakeheads, and the samples of the strips carrying one item are all female snakeheads, so that the primer group, the kit and the sex identification method provided by the embodiment of the application can accurately identify the sex of the snakeheads.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (10)
1. And identifying molecular markers of the sex of the snakehead, wherein the molecular markers are DNA sequences of the sex difference of the snakehead, and the DNA sequences are shown as SEQ ID NO.1.
2. The primer pair for amplifying the molecular marker of claim 1, wherein the primer pair comprises a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 2-3.
3. A kit for identifying the sex of channa argus, comprising the primer pair of claim 2.
4. The kit of claim 3, further comprising a DNA polymerase, mg 2+ dNTP, PCRBuffer and sterile water.
5. The kit according to claim 4, further comprising a positive control and a negative control, wherein the positive control is a liquid preparation containing genomic DNA of a male channa maculata, and the negative control is a liquid preparation containing genomic DNA of a female channa maculata.
6. The kit of claim 4, wherein the DNA polymerase is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV, and DNA polymerase V.
7. A method for identifying the sex of snakehead, which is characterized by comprising the following steps:
obtaining a genome DNA sample of the snakehead to be detected;
taking the genome DNA sample as a template, and carrying out PCR reaction by adopting primer pairs shown as SEQ ID NO. 2-3;
performing polyacrylamide gel electrophoresis on the obtained PCR reaction product, and judging according to a polyacrylamide gel electrophoresis diagram:
if two target bands with the length of 257bp and 255bp exist in the PCR reaction product, the corresponding snakehead to be detected is male;
if only one strip of the item with the length of 257bp exists in the PCR reaction product, the corresponding snakehead to be detected is female.
8. The method according to claim 7, wherein the reaction system of the PCR reaction comprises, in terms of 25. Mu.L, 10 XPCR Buffer, 50 ng/. Mu.L of genomic DNA 1. Mu.L, 0.2 to 0.4. Mu.M of upstream primer, 0.2 to 0.4. Mu.M of downstream primer, 0.2 to 0.4mM of dNTPs 0.8. Mu.L and 0.025 to 0.05U/. Mu.L of DNA polymerase 0.75. Mu.L;
the reaction program of the PCR reaction comprises the following substeps: (1) 94-95 ℃, 3-5 min, (2) 94-95 ℃, 28-35 s, (3) 53-63 ℃, 28-35 s, (4) 72 ℃, 28-40 s, (5) 72 ℃ 5-15min, wherein the substeps (2) to (4) are 30-40 cycles.
9. The method according to claim 8, wherein the reaction system of the PCR reaction comprises, in 25. Mu.L, 10 XPCR Buffer, 50 ng/. Mu.L of genomic DNA, 1. Mu.L, 0.4. Mu.M upstream primer, 0.4. Mu.M downstream primer, 0.4mM dNTP, 0.8. Mu.L, 0.05U/. Mu.L rTase, 0.75. Mu.L and the balance double distilled water.
10. The method of claim 8, wherein the temperature of step (3) in the PCR reaction procedure is 52 ℃.
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| WO2012156563A1 (en) * | 2011-05-13 | 2012-11-22 | Universidad Del País Vasco | Method for identifying the sex of fish |
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