CN116042857A - Combined molecular marker and primer for identifying genetic male of yellow river carp and application of combined molecular marker and primer - Google Patents
Combined molecular marker and primer for identifying genetic male of yellow river carp and application of combined molecular marker and primer Download PDFInfo
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Abstract
A group of combined molecular markers and primers for identifying genetic male of yellow river carp and application thereof relate to fish genetic sex identification and sex control. The target sequences are shown as SEQ ID No. 1 and 2, the primer sequences are shown as SEQ ID No. 3-6, when primers SEQ ID No. 3 and 4 are used for amplifying 146bp fragments, namely the to-be-detected yellow river carp has the nucleotide sequence shown as SEQ ID No. 1, or primers SEQ ID No. 5 and 6 are used for amplifying 162bp fragments and 119bp fragments, namely the to-be-detected yellow river carp lacks the nucleotide sequence shown as SEQ ID No. 2, the to-be-detected yellow river carp is a genetic male yellow river carp; when primers SEQ ID No. 3 and 4 were used without amplified fragments and primers SEQ ID No. 5 and 6 were used to amplify a 162bp fragment, it was a genetic female yellow river carp. Proved by verification, the group of marks has high accuracy, small damage to fish bodies and wider application range.
Description
Technical Field
The invention belongs to the technology of fish genetic sex identification and sex control, and particularly relates to a group of combined molecular markers, primers and application thereof for identifying genetic males of yellow river carps, wherein the combined molecular markers are used for identifying the genetic males of the yellow river carps more widely.
Background
Fish have a rich variety of sex-determining patterns, and understanding the sex-determining mechanisms thereof helps to improve sex control and reproductive control techniques. And the sex of fish is often closely related to the reproduction and many important economic traits. Sex control is therefore a great concern in aquaculture.
With the development of molecular biology techniques, various types of molecular marker techniques have been developed, including mainly restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP), random amplified polymorphic DNA (randomly amplified polymorphic DNA, RAPD), amplified fragment length polymorphism (Amplified Fragment Length Polymorphism, AFLP), microsatellite marker (microsatellite), single nucleotide polymorphism (single nucleotide polymorphism, SNP). In recent years, with the rapid development of Next Generation Sequencing (NGS) technology and the accumulation of genomic resources, methods for screening sex-specific markers are also continuously innovating. There are many species for which the development of sex molecular markers has been carried out using the molecular marker technique described above. Because of the genomic sequence structure of different species, molecular markers of one species are often not useful for genetic sex identification of other species, and therefore, specific molecular markers for the sex of a particular species often require specialized development.
Cyprinus carpio belongs to Cyprinus carpio belonging to Cyprinidae (Cypriniformes) of Cyprinidae, is currently an important economic fish which is widely cultivated internationally, is widely cultivated in more than 100 countries worldwide, has 400 ten thousand tons of 2018 annual yield and occupies 10 percent of the global freshwater fish yield, and has important industrial value (FAO 2020). Under the artificial culture environment, females have remarkable growth advantages, so that the development of sex control technology of culture populations has commercial application potential. The development of sex molecular markers is an important tool for developing sex control technology. The existing sex molecular marker of the yellow river carp is only suitable for partial males.
Disclosure of Invention
The first object of the invention is to provide a set of combined molecular markers for identifying genetic males of yellow river carps.
The second object of the present invention is to provide a set of primers for detecting male specific sequences and male specific deletions of yellow river carp.
The third object of the invention is to provide the combined molecular marker or the application of the primer in identifying the genetic sex of the yellow river carp, which is used for identifying whether the yellow river carp carries a male linkage site.
A group of combined molecular markers for identifying the genetic male of the yellow river carp are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the genotype of the molecular marker fragment with SEQ ID NO. 1 or the deletion fragment with SEQ ID NO. 2 is the genetic male genotype.
The method for obtaining the combined molecular marker comprises the following steps: based on the existing old genome sequence of the carp, resequencing 40 female yellow river carps and 40 male yellow river carps, and identifying regions which are obviously related to the sex of the yellow river carps through whole genome association analysis, wherein the regions are respectively positioned on chromosome 6 and chromosome 50 of the carp; further carrying out female-male depth comparison analysis on genome resequencing data, and finding a section of male specific sequence and a section of male specific deletion on chromosome 6 and chromosome 50 respectively, wherein the two sections of sequences are mutually exclusive in a genetic male individual; primers are respectively designed at the side sequences of the two sequences, and whether the yellow river carp carries a male linkage site is identified through the difference of amplified fragments; the individual with the nucleotide sequence SEQ ID NO. 1 or the sequence shown in the deletion SEQ ID NO. 2 is genetic male carp.
The invention provides a group of two pairs of primers for detecting male specific sequences and male specific deletions of yellow river carps, wherein one pair of primers is positioned at two sides of the male specific sequences on chromosome 6, the other pair of primers is positioned at two sides of the male specific deletions on chromosome 50, and forward and reverse primer sequences of the primers are respectively shown as SEQ ID NO. 3-6:
the invention provides a kit for detecting the genetic sex of yellow river carps, which comprises the molecular marker or the primer.
The invention provides application of the molecular marker or the primer in identifying the genetic sex of yellow river carp.
The specific steps of the application can be as follows:
1) Collecting and storing individual fin living tissues of individual yellow river carp to be identified;
2) Extracting genome DNA of a yellow river carp sample to be identified;
3) Designing and synthesizing a specific primer by using the molecular marker;
4) Performing PCR amplification by using the primer;
and taking the genomic DNA of the yellow river carp to be detected as a template, and carrying out PCR amplification reaction by using the primers SEQ ID NO. 3-4 and the primers SEQ ID NO. 5-6. The labeled primer purification method was PAGE purification using a concentration of 10. Mu.M. PCR reaction System was prepared using a PCR premix kit (brand: TAKARA, cat# RR 903A): 2 XPromix Taq 12.5. Mu.L, 10. Mu.M forward and reverse primers each 0.8. Mu.L, template DNA 2. Mu.L, and ddH2O to a total volume of 25. Mu.L. The reaction conditions of the primers SEQ ID NO. 3-4 are as follows: denaturation at 98℃for 10sec, annealing at 59.4℃for 30sec, elongation at 72℃for 20sec, cycle number 30; extending at 72℃for 5min. The reaction conditions of the primer of SEQ ID NO. 5-6 are as follows: denaturation at 98℃for 10sec, annealing at 55℃for 30sec, extension at 72℃for 20sec with cycle number 30; extending at 72℃for 5min.
5) Typing a PCR amplification product by agarose gel electrophoresis, and judging whether the yellow river carp to be identified carries a male specific site or not according to the amplification condition of a target band and the position difference of the target band, wherein the specific steps are as follows:
preparing 2.5% agarose gel, adding nucleic acid dye (product number: 345D 0101), taking 6ul of PCR product for sample application, marking with 100bp DNA Ladder, electrophoresis for 35min under 120v voltage, shooting the strips by using a gel shooting instrument, recording the genotype of each sample according to the conditions of the strips amplified by the two pairs of primers, and judging whether the yellow river carp to be identified carries a male specific site according to the amplification conditions and the position difference of the target strips; if the target fragment can be amplified to be 146bp (A) by using the primer mark 1 or a strip of 119bp (B) can be amplified by using the primer mark 2, judging that the tail yellow river carp carries a male specific site and is a genetic male yellow river carp; if the primer mark 1 is used for amplifying the fragments, and only the 162bp (b) band is amplified by the primer mark 2, judging that the tail yellow river carp does not carry a male specific site, and the tail yellow river carp is the inherited female yellow river carp.
The invention also provides application of the combined molecular marker or the primer in breeding of yellow river carp. Can judge the sex of the yellow river carp during breeding.
The invention has the advantages of simple operation, small damage to fish bodies, applicability to the whole life history, wider applicable material range and the like, and is specifically expressed as follows:
1. can more widely identify the genetic male of the yellow river carp: the existing sex molecular marker of the yellow river carp is only suitable for partial males (the identification effect is the same as that of the single marker 1), but the invention discovers that the yellow river carp has two unlinked sex obvious association areas which are respectively positioned on No. 6 chromosome and No. 50 chromosome in the resequencing analysis, and develops molecular markers in the two areas respectively, and the two markers are combined to be used for widely identifying the genetic males of the yellow river carp, so that the limitation that the sex molecular marker of the yellow river carp can only identify partial males before is overcome.
2. The PCR product obtained by the amplification of the combined molecular marker detection primer can be used for detecting the polymorphism of the strip by using 2.5% agarose gel without additional restriction enzyme digestion or complicated polyacrylamide gel electrophoresis operation.
3. The damage to the fish body is small: compared with the currently used anatomic inspection of gonads, the method does not need to kill an experimental object, and only needs to take 15 mg tail fins; is particularly suitable for objects needing keep-alive.
4. Is suitable for the whole life history: compared with the current method for anatomically checking the gonads, the method can be used not only in the life stage with clear gonad differentiation, but also in the early stage of gonad undeveloped or differentiated.
Drawings
FIG. 1 is a diagram showing the result of electrophoresis for verifying the sex of yellow river carp.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
In order to achieve the aim of the invention, the invention provides a group of combined markers for identifying whether the yellow river carp carries a male linkage site, the detected target sequence is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the individual with the nucleotide sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 is the genetic male carp. The molecular marker is obtained by the following steps: based on the existing carp genome sequence, by resequencing 40 female yellow river carps and 40 male yellow river carps, the whole genome association analysis identifies the regions which are obviously associated with the sex of the yellow river carps and are respectively positioned on the No. 6 chromosome and the No. 50 chromosome of the carp. Further carrying out female-male depth comparison analysis on genome resequencing data, and finding a section of male specific sequence and a section of male specific deletion on chromosome 6 and chromosome 50 respectively, wherein the two sections of sequences are mutually exclusive in genetic male individuals. Primers were designed to flank the two sequences, respectively. And (5) identifying whether the yellow river carp carries a male linkage site or not through the difference of the amplified fragments. The individual with the nucleotide sequence SEQ ID NO. 1 or the sequence shown in the deletion SEQ ID NO. 2 is genetic male carp.
The invention provides a group of two pairs of primers for detecting the specific sequence and the specific deletion of the male, wherein one pair of primers is positioned at two sides of the specific sequence of the male on chromosome 6, the other pair of primers is positioned at two sides of the specific deletion of the male on chromosome 50, and the forward and reverse primer sequences of the primers are respectively shown as SEQ ID NO. 3-6.
The invention also provides application of the two pairs of primers in identifying whether the yellow river carp carries a male linkage site. The method comprises the following steps:
1) Extracting genomic DNA of yellow river carp;
and (3) recording the gender after the physiological gender is judged by dissecting the yellow river of two ages, cutting fin strips, and putting the fin strips into absolute ethyl alcohol for preservation.
Extracting genome DNA with a marine animal extraction kit (product number DP 324), diluting the extracted DNA 5 times, and storing in a refrigerator at-20deg.C.
2) Identification of yellow river carp genotype using molecular markers
And (3) performing PCR amplification by using the primers, and performing PCR amplification reaction by using the genomic DNA of the yellow river carp to be detected as a template and using the primers SEQ ID NO. 3-4 and the primers SEQ ID NO. 5-6. The labeled primer purification method was PAGE purification using a concentration of 10. Mu.M. PCR reaction System was prepared using a PCR premix kit (brand: TAKARA, cat# RR 903A): 2 XPromix Taq 12.5. Mu.L, 10. Mu.M forward and reverse primers each 0.8. Mu.L, template DNA 2. Mu.L, and ddH2O to a total volume of 25. Mu.L. The reaction conditions of the primers SEQ ID NO. 3-4 are as follows: denaturation at 98℃for 10sec, annealing at 59.4℃for 30sec, elongation at 72℃for 20sec, cycle number 30; extending at 72℃for 5min. The reaction conditions of the primer of SEQ ID NO. 5-6 are as follows: denaturation at 98℃for 10sec, annealing at 55℃for 30sec, extension at 72℃for 20sec with cycle number 30; extending at 72℃for 5min.
Two pairs of PCR primers were screened based on the flanking sequence design of the two sites, and the detailed information of the two pairs of specific primers was finally determined as shown in Table 1.
TABLE 1 specific primer information
3) Agarose gel electrophoresis detection and judgment according to PCR amplified products
Preparing 2.5% agarose gel, adding nucleic acid dye (product number: 345D 0101), taking 3-6 uL of PCR product for sample application, marking with 100bp DNA Ladder, electrophoresis for 35min under 120v voltage, shooting the strips by using a gel shooting instrument, recording the genotype of each sample according to the conditions of the strips amplified by two pairs of primers, wherein in FIG. 1, A is 146bp strip amplified by part of male of mark 1, B is 119bp strip amplified by other part of male of mark 2, and B is 162bp strip amplified by all fish of mark 2. If A or B can be amplified in one individual, the individual is genetic male yellow river carp; if an individual can amplify neither A nor B, it is a genetic female yellow river carp (FIG. 1). Judging whether the yellow river carp to be identified carries a male specific site or not according to the amplification condition and the position difference of the target band. If the target fragment can be amplified to be 146bp (A) by using the primer mark 1 or a strip of 119bp (B) can be amplified by using the primer mark 2, judging that the tail yellow river carp carries a male specific site; if the primer mark 1 is used for amplifying no fragment and only the 162bp (b) band is amplified by the primer mark 2, judging that the tail yellow river carp does not carry a male specific site.
The invention provides a group of combined markers and primers capable of identifying genetic males of yellow river carps and application thereof. The target sequence is shown as SEQ ID No. 1 and SEQ ID No. 2, and the individual with the target sequence SEQ ID No. 1 or the target sequence SEQ ID No. 2 deleted is male. The primer sequences are shown in SEQ ID No. 3 to SEQ ID No. 6 and are used for PCR amplification. When primers SEQ ID No. 3 and SEQ ID No. 4 are used for amplifying a 146bp fragment, namely the to-be-detected yellow river carp has a nucleotide sequence shown as SEQ ID No. 1, or primers SEQ ID No. 5 and SEQ ID No. 6 are used for amplifying a 162bp fragment and a 119bp fragment, namely the to-be-detected yellow river carp lacks the nucleotide sequence shown as SEQ ID No. 2, the to-be-detected yellow river carp is a genetic male yellow river carp; when primers SEQ ID No. 3 and SEQ ID No. 4 are used and have No amplified fragments, namely the to-be-detected yellow river carp does not have the nucleotide sequence shown as SEQ ID No. 1, and primers SEQ ID No. 5 and SEQ ID No. 6 are used for amplifying a 162bp fragment, namely the to-be-detected yellow river carp does not lack the nucleotide sequence shown as SEQ ID No. 2, the to-be-detected yellow river carp is a genetic female yellow river carp. Proved by verification, the group of marks has the characteristics of high accuracy, small damage to fish bodies and wider application range.
Claims (7)
1. A group of combined molecular markers for identifying genetic males of yellow river carps is characterized in that the nucleotide sequences of the combined molecular markers are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and genotypes of the molecular marker fragments with the SEQ ID NO. 1 or the deletion fragments with the SEQ ID NO. 2 are genetic male genotypes.
2. A group of primers for detecting genetic male molecular markers of yellow river carps is characterized in that the nucleotide sequence of the primers is shown in SEQ ID No. 3 to SEQ ID No. 6.
3. A kit for detecting the genetic sex of yellow river carp, characterized in that it comprises a combination molecular marker according to claim 1 or a primer according to claim 2.
4. Use of a combination molecular marker as defined in claim 1 or a primer as defined in claim 2 for identifying the genetic sex of yellow river carp.
5. The use according to claim 4, characterized by the specific steps of:
1) Collecting and storing individual fin living tissues of individual yellow river carp to be identified;
2) Extracting genome DNA of a yellow river carp sample to be identified;
3) Designing a synthetic specific primer using the combination molecular marker of claim 1;
4) PCR amplification using the primer of claim 2;
5) And (3) typing the PCR amplification product by agarose gel electrophoresis, and judging whether the yellow river carp to be identified carries a male specific site or not according to the amplification condition of the target band and the position difference thereof.
6. The method of claim 5, wherein in step 3), the combined molecular markers are used to design and synthesize specific primers by designing and screening two pairs of PCR primers based on the flanking sequences of the two sites, and the detailed information of the two pairs of specific primers is finally determined as follows:
7. the application of claim 5, wherein in the step 5), whether the yellow river carp to be identified carries a male specific site is judged according to the amplification condition of the target band and the position difference thereof, specifically:
if the target fragment can be amplified to 146bp by using the primer mark 1 or the strip of 119bp can be amplified by using the primer mark 2, judging that the tail yellow river carp carries a male specific site and is a genetic male yellow river carp; if the primer mark 1 is used for amplifying the fragments, and the primer mark 2 is used for amplifying the 162bp band only, judging that the tail yellow river carp does not carry a male specific site, and the tail yellow river carp is the inherited female yellow river carp.
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| CN116891853A (en) * | 2023-06-30 | 2023-10-17 | 中国长江三峡集团有限公司中华鲟研究所 | Carp genetic sex identification method and sex marker |
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