CN110777210B - Siniperca chuatsi male molecular marker primer and application thereof - Google Patents
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Abstract
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.
Description
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 (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 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.
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 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.
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 primer pair 1 or the primer pair 2 is adopted for PCR amplification in the step (3), 20 mul of PCR reaction system contains 50ng of DNA, 0.8 mul of each of the Contig-1 upstream primer and the Contig-1 downstream primer or 0.8 mul of each of the Contig-2 upstream primer and the Contig-2 downstream primer, 10 mul of 2 xTaq MasterMix, and ddH is used for the rest2And (4) supplementing by using oxygen.
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 primer pair 1; the molecular size of the specific band obtained by using primer set 2 was 489 bp.
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.
Example 1
The embodiment provides mandarin fish male molecular marker primers, which comprise a primer pair 1 or 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 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'-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.
The siniperca chuatsi male molecular marker primer is obtained by the following steps:
one) sequencing library construction and re-sequencing
1. Preparing a mandarin fish DNA sample:
shearing female 24-tail and male 4-tail individual tail fins of the mandarin fish during reproduction, sequentially numbering female fish F1-F24 and male fish M1-M4, preparing DNA samples by adopting a conventional column type centrifugation method (a general column type genome extraction kit, the background health is century biotechnology limited) according to the operation steps of the instruction, and detecting the quality and the concentration by using 1% agarose gel electrophoresis and a micro spectrophotometer (NanoDrop2000) 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-one 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: 94,431,090,900bp and 94,469,916,300 bp.
Taking the same amount of DNA samples from 21 parts of F4-F24, uniformly mixing to form a female fish DNA mixed pool, constructing a 350-and 500-bp fragment sequencing library, and performing double-end PE150 sequencing by using an Illumina Hiseq X Ten platform to obtain female fish DNA mixed pool sequencing clean data: 36,249,884,100 bp.
The Library construction procedure was specifically described in the Novogene NGS DNA Library Prep Kit, and sequencing was performed by Beijing Nuo Po-derived science and technology, Inc.
II) enrichment of Y chromosome-specific DNA fragments
1. The method comprises the steps of male fish genome assembly:
genome assembly is carried out on male fish M1 sequencing reads by using an all method in SOAPdenovo v2.04-r240(https:// github. com/aquaskyline/SOAPdenovo2), so as to obtain 1037309scaffolds with the total size of 819033064bp, wherein N50 is 28567 bp.
2. Obtaining male fish candidate DNA fragments:
female fish F1-F3 sequencing reads and male fish M1-M3 sequencing reads are aligned to male fish genomes by a method of bwa v0.7.17-r1188(http:// bio-bw. sourceform. net /) aln software, the 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 (figure 1 and figure 2).
3. Enrichment of male-specific DNA fragments:
taking a male genome as reference, taking female fish DNA mixed pool sequencing reads as query, and adopting a method of bwa v0.7.17-r1188(http:// bio-bw. sourceform. net /) software aln to carry out sequence comparison to find out male fish specific DNA fragments which are not covered by any reads. The results of comparison of 27 DNA fragments without any read of a female fish DNA mixed pool can be obtained, and the lengths of the male fish DNA fragments are mainly distributed in 300-600bp and account for 66.7 percent of the total number; contigs with a length ≧ 300bp account for 100% of the total. These 27 contigs contained specific DNA fragments from the Y chromosome (fig. 3).
Three) screening of molecular markers of the Y chromosome
1. Designing and synthesizing a primer:
based on the above 27 male fish Contig sequences, 6 Contig sequences were randomly selected, and PCR primers were designed using Primer5 software (the software used is referred to Primer PREMIER version5.0 for Windows and Power Macintosh) according to Contig-xxF/R (xx stands for Contig number), for example, Contig-1 Primer 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 6 pairs of primers are selected to carry out PCR detection on each 4 male and female individuals, the total PCR amplification reaction system is 20 mul, and comprises 10 mul of 2 XTaq MasterMix (Beijing kang is century Biotechnology Co., Ltd.), 0.8 mul of each of upstream and downstream primers (10 mul/L) and 50ng of template DNA. The PCR reaction program is: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; final extension at 72 ℃ for 5 min. Agarose gel electrophoresis results show that 2 pairs of primers only obtain amplified bands in male individuals, no amplified fragment is detected in female individuals (FIG. 4), and the two pairs of primers are respectively Contig-1F/R and Contig-2F/R, which represents that the corresponding Contig is Contig-1 and Contig-2; the other 4 pairs of primers have non-specific hybrid bands and diffuse bands which cannot be accurately judged in the amplification products of the male and female individuals (FIG. 5), and the male and female sex is difficult to be strictly distinguished.
2 of these cases, Contig: contig-1 and Contig-2 are Y chromosome specific fragments and are male molecular markers of mandarin fish.
Example 2
The embodiment provides a method for identifying the sex of siniperca chuatsi by using a mandarin fish male molecular marker primer, which comprises the following steps:
(1) designing the siniperca chuatsi male molecular marker primer, and referring to the embodiment 1 in the specific process;
(2) preparing a siniperca chuatsi DNA sample:
visual inspection of the mandarin fish with mature nature is obtained from the Siniperca Chuatsi Miao GmbH of Fushan city, Guangdong province, and 24 fish of male and female mandarin fish are obtained by dissection. The tail fins were cut out, and DNA samples were prepared by the same conventional column centrifugation method (universal column genome extraction kit, beijing kang, century biotechnology limited) with reference to the instruction manual.
(3) And (3) PCR amplification verification:
PCR verification is carried out on 24 mandarin fish DNA samples of the male and female by adopting primers Contig-1F/R and Contig-2F/R. The total PCR amplification reaction system is 20 μ L, including 10 μ L of 2 × Taq MasterMix (Beijing kang is century Biotechnology Co., Ltd.), 0.8 μ L of each of the upstream and downstream primers (10 μmol/L), 50ng of template DNA, and ddH for the rest2And (4) supplementing by using oxygen.
The PCR reaction program is: 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.
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 specific band amplified by the primers Contig-1F/R is 374 bp; the molecular size of the specific band amplified by Contig-2F/R was 489 bp. No band could be amplified in the 24 female DNA samples (FIG. 6: Contig-1F/R amplification result graph and FIG. 7: Contig-2F/R amplification result graph). The molecular markers Contig-1F/R and Contig-2F/R can be used for identifying the sex of the mandarin fish.
Example 3
The embodiment provides an application of the siniperca chuatsi male molecular marker primer in full-female breeding of siniperca chuatsi, which comprises the following steps:
(1) after the mandarin fish fries are cultured to 10 days old, bait fish is fed with eel feed containing 100mg/kg of androgen 17 alpha-methyl testosterone, and the bait fish fed with hormone feed is fed with mandarin fish. Continuously feeding for two months, adopting natural illumination during the culture period, keeping the temperature of the culture water at 26-28 ℃, and feeding after being fed with satiety.
(2) Screening the mandarin fish fed with androgen by the method in example 2 to obtain the male fish with genotype XX, namely pseudo male fish, and the specific process refers to example 2.
(3) And (4) continuing to breed the XX pseudo-male fish identified by the mark until the XX pseudo-male fish is sexually mature, and mating the XX pseudo-male fish with the normal XX female fish in a breeding season to obtain offspring which are the all female 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
<110> Zhongshan university
Guangdong Liangshi Aquatic Seed Industry Co.,Ltd.
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Claims (8)
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) carrying out PCR amplification by using the DNA in the step (2) as a template and adopting a primer pair 1 or a primer pair 2, carrying out electrophoresis detection after the amplification reaction is finished, wherein if an electrophoresis result shows that a specific strip exists, the detected Siniperca Chuatsi DNA sample is a male Siniperca Chuatsi, and if the electrophoresis result shows that the specific strip does not exist, 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) of PCR amplification using primer pair 1 or primer pair 2, the 20 μ l PCR reaction system contains 50ng of DNA, 0.8 μ l of each of the Contig-1 forward primer and the Contig-1 downstream primer or 0.8 μ l of each of the Contig-2 forward primer and the Contig-2 downstream primer, and 10 μ l of 2 XTAQQ Mastermix.
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 amplified by primer pair 1 in the specific band shown by electrophoresis in step (3) is 374 bp; the molecular size of the specific band amplified by primer pair 2 was 489 bp.
8. Use of the siniperca chuatsi male molecular marker primer of claim 1 for breeding mandarin fish in all females.
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| CN111593055B (en) * | 2020-04-15 | 2023-03-10 | 广东梁氏水产种业有限公司 | Siniperca chuatsi male specific anti-mullerian hormone AMHY gene and application thereof |
| CN112106704A (en) * | 2020-10-28 | 2020-12-22 | 广东梁氏水产种业有限公司 | Application of bdellovibrio bacteriovorus in improvement of domestication rate of siniperca chuatsi artificial feed |
| CN112359104A (en) * | 2020-12-10 | 2021-02-12 | 中国科学院水生生物研究所 | Development method and application of sex specific marker of mandarin fish |
| CN113462787B (en) * | 2021-07-29 | 2023-08-08 | 广州大学 | Male molecular marker of optical barb cheilus and application thereof |
| CN114592076B (en) * | 2022-05-10 | 2022-08-16 | 中山大学 | Siniperca scherzeri male molecular marker primer and application thereof |
| CN116904474B (en) * | 2023-06-30 | 2024-04-02 | 中国长江三峡集团有限公司中华鲟研究所 | Male sex-specific molecular marker of siniperca scherzeri and genetic sex identification method |
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