Siniperca chuatsi male molecular marker primer and application thereof
阅读说明：本技术 翘嘴鳜雄性分子标记引物及其应用 (Siniperca chuatsi male molecular marker primer and application thereof ) 是由 张勇 韩崇 李桂峰 李水生 韩林强 梁建辉 林浩然 于 2019-11-12 设计创作，主要内容包括：本发明提供一种翘嘴鳜雄性分子标记引物,所述引物至少包括引物对1、引物对2中一种,所述引物对1包括Contig-1上游引物和Contig-1下游引物,所述引物对2包括Contig-2上游引物和Contig-2下游引物,其中Contig-1上游引物的核苷酸序列如SEQ ID NO：1所示,Contig-1下游引物的核苷酸序列如SEQ ID NO：2所示,Contig-2上游引物的核苷酸序列如SEQ ID NO：3所示,Contig-1下游引物的核苷酸序列如SEQ ID NO：4所示。该引物可对翘嘴鳜的性别进行快速准确地鉴定。同时还提供一种鉴别翘嘴鳜鱼性别的方法,该方法耗时短,效率高。(The invention provides a siniperca chuatsi male molecular marker primer, which at least comprises one of a primer pair 1 and a primer pair 2, wherein the primer pair 1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair 2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown as 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. The primer can be used for rapidly and accurately identifying the sex of siniperca chuatsi. Meanwhile, the method for identifying the sex of the siniperca chuatsi is short in time consumption and high in efficiency.)
1. The siniperca chuatsi male molecular marker primer is characterized by comprising at least one of a primer pair 1 and a primer pair 2, wherein the primer pair 1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair 2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown as 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.
2. A method for sex identification of siniperca chuatsi by using the siniperca chuatsi male molecular marker primer as claimed in claim 1, which comprises the following steps:
(1) designing a primer pair 1 and a primer pair 2;
(2) preparing a siniperca chuatsi DNA sample;
(3) and (3) performing PCR amplification by using the DNA in the step (2) as a template and adopting a primer pair 1 or a primer pair 2, after the amplification reaction is finished, performing electrophoresis detection, wherein if an electrophoresis result shows that the DNA has a specific DNA band, the detected Siniperca Chuatsi DNA sample is a male Siniperca Chuatsi, and if the electrophoresis result shows that the DNA does not have the specific band, the detected Siniperca Chuatsi DNA sample is a female Siniperca Chuatsi.
3. The method for sex determination of siniperca chuatsi as claimed in claim 2, wherein the DNA sample preparation in step (2) is performed by column centrifugation to extract siniperca chuatsi DNA.
4. The method for sex determination of siniperca chuatsi as claimed in claim 2, wherein in the step (3) using primer pair 1 or primer pair 2 for PCR amplification, 20 μ l of PCR reaction system contains DNA50ng, 0.8 μ l of each of Contig-1 upstream primer and Contig-1 downstream primer or 0.8 μ l of each of Contig-2 upstream primer and Contig-2 downstream primer, and 10 μ l of 2 XTAQAQASTAMASTERMix.
5. The method for sex determination of siniperca chuatsi as claimed in claim 4, wherein in the step (3) of PCR amplification with primer pair, the PCR reaction procedure is as follows: firstly, pre-denaturation is carried out for 2min at 94 ℃; then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; and final extension at 72 ℃ for 5 min.
6. The method for sex determination of siniperca chuatsi as claimed in claim 4 or 5, wherein the step (3) comprises detecting the length of the amplified fragment by electrophoresis using agarose gel with a mass percentage of 1%.
7. The method for sex determination of siniperca chuatsi as claimed in claim 6, wherein the molecular size of the specific band obtained by using primer pair 1 as the specific band shown in step (3) is 374 bp; the molecular size of the specific band obtained by using primer set 2 was 489 bp.
8. Use of the siniperca chuatsi male molecular marker primer of claim 1 for breeding mandarin fish in all females.
Technical Field
The invention belongs to the fish sex identification technology in the technical field of aquatic organisms, and particularly relates to a siniperca chuatsi male molecular marker primer and application thereof.
Background
In recent years, with the rapid development of molecular biology technology, sex-specific markers of various fishes have been developed, such as nile tilapia (oreochromis niloticus), rainbow trout (oncorhynchus mykiss), pelteobagrus fulvidraco (pelteobagrus fulvidraco), cynoglossus semilaevis (cynoglossus semilaevis), and takifugu rubripes (takifugurus), but these markers are mainly obtained by molecular marker development technologies such as microsatellite (SSR), restriction site associated DNA (RAD), and Amplified Fragment Length Polymorphism (AFLP), and these molecular marker development methods are long in time consumption, large in workload, and low in success rate. The appearance of high-throughput sequencing technology provides a new high-efficiency means for developing molecular markers, and the sex-specific molecular markers of the snakeheads and the large yellow croakers are successfully developed by the high-throughput sequencing method at present.
Siniperca chuatsi (Sinipercachuatsi) belongs to Perciformes, Sinipercidae and Sinipercica, and is commonly called Sinipercica and Mandarin Fish, and the English names thereof mean Chinese bass (Chinese perch) and Mandarin Fish (Mandarin Fish). The siniperca chuatsi is a unique and rare fish in fresh water in China, has delicious meat, rich nutrition and no muscle thorns, and is deeply favored by consumers at home and abroad. The growth process of the siniperca chuatsi is sex-two-state, and the weight of the female fish is about 10-20% of that of the commercial fish of the same age, so that the full-female parthenocarpic breeding of the siniperca chuatsi has higher economic benefit.
Although no obvious sex chromosome is found by the conventional chromosome karyotype analysis, the male genome has a plurality of specific DNA fragments by sequencing analysis of male and female genomes, so that the sex of the mandarin fish is presumed to be male heterozygosis. The parthenocarpy breeding of the all-female mandarin fish needs to obtain pseudo-male fish firstly, and the pseudo-male fish is obtained mainly through a method of inducing reversion of genetic female fish. In the traditional method, the sex chromosome composition of the male parent, namely XX pseudo-male fish or normal XY male fish, is judged through a test cross experiment. The sex of the mandarin fish is difficult to distinguish from the external form, the sex gland identification through dissection also needs at least three months, and the independent culture of the test cross parent needs to occupy more culture resources, so the test cross method is time-consuming, labor-consuming and resource-consuming. So far, no molecular marker capable of successfully identifying the genetic sex of siniperca chuatsi is reported at home and abroad, so that the development of the molecular marker for accurately identifying the genetic male has great production and application values in the hologynic breeding of the siniperca chuatsi.
Disclosure of Invention
The first purpose of the invention is to provide a siniperca chuatsi male molecular marker primer, which can identify male siniperca chuatsi at a molecular level.
The second purpose of the invention is to provide a method for sex determination of siniperca chuatsi, which is short in time consumption and accurate in detection result.
The third purpose of the invention is to provide application of the siniperca chuatsi male molecular marker primer in full-female mandarin fish breeding.
In order to achieve the first purpose, the invention adopts the following technical scheme:
a siniperca chuatsi male molecular marker primer comprises at least one of a
Specifically, the nucleotide sequence of each primer from 5 'to 3' is as follows:
contig-1 upstream primer: 5'-GCTGCTTTGAAGTCTGATACATG-3', respectively;
contig-1 downstream primer: 5'-TGGTTTGTCAGATTGCACCTGTA-3', respectively;
contig-2 upstream primer: 5'-CATCTCCTCTTAACAGGGACCAT-3', respectively;
contig-2 downstream primer: 5'-TCCGGTTATCCAGAGGAGGTGAT-3' are provided.
In order to achieve the second object, the invention adopts the following technical scheme:
a method for carrying out sex identification on siniperca chuatsi by using the siniperca chuatsi male molecular marker primer comprises the following steps:
(1) designing a
(2) preparing a siniperca chuatsi DNA sample;
(3) and (3) performing PCR amplification by using the DNA in the step (2) as a template and adopting a
And (3) extracting the DNA of the siniperca chuatsi by adopting a column type centrifugation method in the DNA sample preparation in the step (2).
When the
When the primer pair is adopted for PCR amplification in the step (3), the PCR reaction procedure is as follows: firstly, pre-denaturation is carried out for 2min at 94 ℃; then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; and final extension at 72 ℃ for 5 min.
And (3) when the electrophoresis detection is carried out in the step (3), detecting the length of the amplified fragment by adopting agarose gel electrophoresis with the mass percentage of 1%.
The molecular size of the specific band displayed by the electrophoresis result in the step (3) is 374bp by adopting the
In order to achieve the third object, the invention adopts the following technical scheme:
an application of the siniperca chuatsi male molecular marker primer in the full female breeding of siniperca chuatsi.
The invention has the following beneficial effects:
(1) the method is based on genome high-throughput sequencing data of male and female siniperca chuatsi, utilizes an analysis method such as genome assembly and comparison, quickly and accurately screens the male molecular marker of the siniperca chuatsi, designs a male molecular marker primer for identifying the sex of the siniperca chuatsi for the first time, and can quickly and accurately identify the sex of the siniperca chuatsi.
(2) The method establishes a method for identifying the sex of the siniperca chuatsi by using the siniperca chuatsi male molecular marker primer, can identify the sex of the siniperca chuatsi by using two pairs of specific primers and completing a PCR reaction, and has the advantages of short time consumption, high efficiency and resource saving compared with the traditional method for identifying the sex of a male parent through a test cross experiment.
Drawings
FIG. 1 is a schematic diagram showing the length distribution of male fish specific candidate fragments;
FIG. 2 is a schematic diagram of the acquisition of candidate fragments of the Y chromosome;
FIG. 3 is a diagram showing the distribution of the lengths of the individual fragments of the Y chromosome;
FIG. 4 is an electrophoretogram of PCR products of 2 pairs of primers in which amplified fragments were detected only in male samples and not in female samples
FIG. 5 is an electrophoresis diagram of PCR products of 4 pairs of primers for detecting non-specific amplified fragments in male and female samples;
FIG. 6 is an electrophoretogram of male and female individual (48 pieces) detection PCR product of primer Contig-1F/R;
FIG. 7 is an electrophoretogram of male and female individual (48 pieces) detection PCR product of primer Contig-2F/R.
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.