CN114592076A - Siniperca scherzeri male molecular marker primer and application thereof - Google Patents
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Abstract
The invention discloses a siniperca scherzeri male molecular marker primer, which is a primer pair N1 or a primer pair N2, wherein the primer pair N1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair N2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, and the nucleotide sequences of the Contig-1 upstream primer, the Contig-1 downstream primer, the Contig-2 upstream primer and the Contig-2 downstream primer are respectively shown as SEQ ID NO: 1-SEQ ID NO: 4, respectively. The primer can be used for rapidly and accurately identifying the sex of siniperca scherzeri. Also discloses a method for identifying sex of siniperca scherzeri, which has short time consumption and high efficiency. The invention further discloses application of the siniperca scherzeri male molecular marker primer and the method in siniperca scherzeri total female breeding and total female hybrid siniperca chuatsi breeding.
Description
Technical Field
The invention belongs to the technical field of fish sex identification, and particularly relates to a siniperca scherzeri male molecular marker primer and application thereof.
Background
In recent years, high-throughput sequencing technology is rapidly developed, a new efficient means is provided for developing molecular markers, and more high-throughput sequencing methods are successful in developing sex-specific molecular markers in various fish species. For example, the sex marker was successfully found in Western mosquito-eating fish using RNA-seq, the sex marker was found in Nile tilapia (Oreochromyniloticus), European bass (Dicentra percha), halibut (Alaskan halibut) and Hypophthal chub (Hypophthalmichix) using simplified genome technology, and the sex marker was developed in species such as Atlantic cod (Gadusmorhua), Thunnus albus (Thunnus), Pseudosciaenopus (Larihyscocea) and Channa argus (Channaargus) using whole genome sequencing and resequencing technology.
Siniperca scherzeri (sinipercachlerzeri), belonging to the order Perciformes (Perciformes), mandarinaceae (Sinipercidae), mandarin (Siniperca), also known as Siniperca scherzeri. The mandarin fish is a unique and rare fish in fresh water in China, has delicious meat, rich nutrition and no muscle thorns, and is deeply loved by consumers at home and abroad. Compared with siniperca scherzeri, the siniperca scherzeri is easier to domesticate and eat icy fresh feed, and easier to realize feed-based culture, and hybrid species of the siniperca scherzeri and the siniperca scherzeri show excellent characteristics of two mandarin fishes, so that the culture prospect is wide.
Various researches show that the siniperca chuatsi is determined by XY chromosome sex, so that the male and female genomes are subjected to sequencing analysis, a plurality of specific DNA fragments are found in the male genomes, and the sex determination of the siniperca chuatsi is determined as male heterozygosis through PCR verification. In the parthenocarpic mandarin fish breeding, pseudo-male mandarin fish is firstly obtained, and XX pseudo-male siniperca chuatsi is also obtained in the all-female hybrid mandarin fish. If no molecular marker exists, the sex chromosome composition of the male parent, namely XX pseudo-male fish or normal XY male fish, can be judged only through a test cross experiment. The sex of the mandarin fish is difficult to distinguish from the external form, and the independent cultivation of the test cross parents needs to occupy a large amount of cultivation resources and takes a long time, so the test cross method takes time, labor and resources. At present, no molecular marker capable of successfully identifying the genetic sex of siniperca scherzeri 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 parthenocarpic breeding of siniperca scherzeri and hybrid siniperca chuatsi.
Disclosure of Invention
The invention aims to provide a siniperca scherzeri male molecular marker primer, which can identify male siniperca scherzeri at a molecular level.
The invention also aims to provide the siniperca scherzeri gender identification method which is short in time consumption and accurate in detection result.
The last purpose of the invention is to provide the primer or the method for the breeding of siniperca scherzeri and the hybridization of siniperca scherzeri (siniperca scherzeri)♂Siniperca chuatsi with X-shaped tilting tip♀) Application in breeding.
The first object of the present invention can be achieved by the following technical solutions: the siniperca scherzeri male molecular marker primer is a primer pair N1 or a primer pair N2, the primer pair N1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair N2 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-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'-TCCCAGACACAAACCCCTAC-3', respectively;
contig-1 downstream primer: 5'-CCCATTTTCCACGTCAGTCT-3', respectively;
contig-2 upstream primer: 5'-GTTGGCCCATCTGAAGTGTT-3', respectively;
contig-2 downstream primer: 5'-GCGCTCTCAGCAGAAGAAAT-3' are provided.
The second object of the present invention can be achieved by the following technical solutions: a siniperca scherzeri gender identification method comprises the following steps:
(1) designing the primer pair N1 or the primer pair N2;
(2) extracting a siniperca scherzeri DNA sample;
(3) and (3) carrying out PCR amplification by using the DNA sample in the step (2) as a template and adopting a primer pair N1 or a primer pair N2, carrying out electrophoresis detection after the amplification reaction is finished, wherein if an electrophoresis result shows that a specific DNA strip exists, the detected siniperca scherzeri DNA sample is male siniperca scherzeri, and if the electrophoresis result shows that the specific DNA strip does not exist, the detected siniperca scherzeri DNA sample is female siniperca scherzeri.
The sex identification method of the siniperca scherzeri comprises the following steps:
preferably, the siniperca scherzeri DNA sample in step (2) is extracted by column centrifugation.
Preferably, when PCR amplification is performed using primer pair N1 or primer pair N2 in step (3), the 20 μ LPCR reaction system includes 50ng DNA, 0.8 μ L of each of the upstream and downstream primers of primer pair N1 or 0.8 μ L of each of the upstream and downstream primers of primer pair N2, 2 XTaqMasterMix 10 μ L, and ddH is used for less than 20 μ L2And (4) supplementing and finishing.
Preferably, when the primer pair N1 or N2 is used for PCR amplification in 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.
Preferably, when the electrophoresis detection is performed in the step (3), the length of the amplified fragment is detected by agarose gel electrophoresis with the mass percentage of 1%.
Preferably, when the primer pair N1 or the primer pair N2 is used for PCR amplification in the step (3), if the electrophoresis result shows that a specific DNA strip exists at the 537bp position or the 613bp position, the detected siniperca scherzeri DNA sample is male siniperca scherzeri, and if the electrophoresis result shows that the specific DNA strip does not exist at the 537bp position or the 613bp position, the detected siniperca scherzeri DNA sample is female siniperca scherzeri.
When the primers are adopted to carry out PCR amplification on N1 in the step (3), if the electrophoresis result shows that a specific DNA strip is contained at the 537bp position, the detected siniperca scherzeri DNA sample is male siniperca scherzeri, and if the electrophoresis result shows that the specific DNA strip is not contained at the 537bp position, the detected siniperca scherzeri DNA sample is female siniperca scherzeri.
When the primer pair N2 is adopted to carry out PCR amplification in the step (3), if the electrophoresis result shows that a specific DNA strip is present at the 613bp position, the detected siniperca scherzeri DNA sample is male siniperca scherzeri, and if the electrophoresis result shows that the specific DNA strip is absent at the 613bp position, the detected siniperca scherzeri DNA sample is female siniperca scherzeri.
The last objective of the present invention can be achieved by the following technical solutions: the siniperca scherzeri male molecular marker primer or the siniperca scherzeri male molecular marker method is applied to siniperca scherzeri total female breeding and total female hybrid mandarin fish breeding.
The invention has the following beneficial effects:
(1) the method is based on genome high-throughput sequencing data of siniperca scherzeri, utilizes analysis methods such as genome assembly and comparison, deep analysis and the like to quickly and accurately screen the siniperca scherzeri male specific molecular marker, designs a male specific molecular marker primer for identifying the sex of siniperca scherzeri for the first time, and can quickly and accurately identify the sex of the siniperca scherzeri.
(2) The method establishes a method for identifying the sex of siniperca scherzeri by using male molecular marker primers of siniperca scherzeri, and can identify the sex of the siniperca scherzeri by completing a PCR reaction by using specific primer pairs N1 or N2 respectively.
Drawings
FIG. 1 is a schematic diagram showing the distribution of lengths of male fish-specific candidate fragments in example 1;
FIG. 2 is a schematic diagram showing the acquisition of candidate fragments of the Y chromosome in example 1;
FIG. 3 is a schematic diagram showing the distribution of the lengths of the Y chromosome-specific fragments in example 1;
FIG. 4 is an electrophoretogram of PCR products of 2 pairs of primers in example 1, in which amplified fragments were detected only in a male sample and not in a female sample;
FIG. 5 is the electrophoresis chart of the PCR products of the primer pair N1 and N2 in the detection of male and female individuals (24 tails) in Guangdong Shaoyuan population in example 2;
FIG. 6 is an electrophoresis chart of PCR products detected by the primer pair N1 and N2 in example 2 in hermaphrodite population (24 tails) in Hunan province.
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 siniperca scherzeri male molecular marker primer provided by the embodiment is a primer pair N1 or a primer pair N2, the primer pair N1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair N2 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-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'-TCCCAGACACAAACCCCTAC-3';
contig-1 downstream primer: 5'-CCCATTTTCCACGTCAGTCT-3', respectively;
contig-2 upstream primer: 5'-GTTGGCCCATCTGAAGTGTT-3', respectively;
contig-2 downstream primer: 5'-GCGCTCTCAGCAGAAGAAAT-3' are provided.
The siniperca scherzeri male molecular marker primer is obtained by the following steps:
one) sequencing library construction and re-sequencing
1. Preparing a siniperca scherzeri DNA sample:
cutting 30 female tail and 3 male individual tail fins of siniperca scherzeri during propagation, sequentially numbering female fish F1-F30 and male fish M1-M3, preparing DNA samples by a conventional column type centrifugation method (a general column type genome extraction kit, Beijing kang is century Biotechnology Co., Ltd.) according to the operation steps of a specification, and detecting the quality and concentration by using 1% agarose gel electrophoresis and a micro spectrophotometer (NanoDrop 2000) 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: 95,411,700,900bp and 93,279,312,300 bp.
Taking an equal amount of DNA samples from 27 parts of F4-F30, uniformly mixing to form a female fish DNA mixed pool, constructing a 350-plus 500bp 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: 34,267,784,100 bp.
Library construction procedures reference was made specifically to the Novogen eNGS DNA Library Prep Kit instructions and sequencing was performed by Guangzhou Ruike Gene technology, Inc.
II) enrichment of Y chromosome-specific DNA fragments
1. The method comprises the steps of male fish genome assembly:
the genome assembly of the male fish sequencing reads is carried out by using an all method in SOAPdenovo 2.04-r240 (https:// github. com/aquaskyline/SOAPdenovo2), so that 1056209 scaffolds of male fish with the total size of 825034054bp are obtained, wherein N50=27347 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 the method of bwav0.7.17-r1188(http:// bio-bwa. sourceform. net /) aln software, the reads which cannot be aligned by the male fish sequencing reads are filtered out, and the regions which can be aligned by the male fish sequencing reads but are not aligned by the female fish sequencing reads are selected, and the regions are presumed to be male-specific DNA fragments (FIG. 1 and FIG. 2).
3. Enrichment of male-specific DNA fragments:
taking a male genome as reference, taking female DNA mixed pool sequencing reads as query, and adopting aln method of bwav0.7.17-r1188(http:// bio-bw. sourceform. net /) software to carry out sequence comparison to find out male specific DNA fragments which are not covered by any reads. 59 DNA fragments and no read of any female fish DNA mixed pool can be compared, and the lengths of the male specific DNA fragments are mainly distributed within 1000bp and account for about 64.4 percent of the total number; contig with a length ≧ 1000bp accounted for 35.9% of the total (FIG. 3).
Three) screening of molecular markers of the Y chromosome
1. Designing and synthesizing a primer:
based on the 59 male fish Contig sequences, 48 PCR primers were designed using Primer5 software (the software used is referred to Primer PREMIER version5.0 for Windows and Power Macintosh) with the primers numbered Contig-xxF/R (xx stands for Contig number), e.g., Contig-1F/R for Contig-1 (F stands for upstream Primer and 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 primers are selected to carry out PCR detection on each 3 male and female individuals, the total PCR amplification reaction system is 20 mu L, including 10 mu L of 2 XTaqMasterMix (Beijing Kangji is century Biotechnology Co., Ltd.), each of the upstream primer and the downstream primer (10 mu mol/L) is 0.8 mu L, the template DNA is 50ng, and ddH is used for part less than 20 mu L2And (4) supplementing oxygen.
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 two pairs of primers N1 and N2 only obtain amplified bands in male individuals, no amplified fragment is detected in female individuals (FIG. 4), and the two pairs of primers are Contig-1F/R and Contig-2F/R respectively, which represents that the corresponding Contig is Contig-1 and Contig-2, and can distinguish sex.
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 method for sex identification of siniperca scherzeri by using the male molecular marker primer of siniperca scherzeri provided by the embodiment comprises the following steps:
(1) designing the siniperca scherzeri male molecular marker primer, wherein the specific process refers to example 1;
(2) preparing a siniperca scherzeri DNA sample:
obtaining the mature mandarin fish from Guangdong Shaoshaoguan and Hunan kingdom, and obtaining 24 fish of each male and female mandarin fish through dissection. Respectively cutting tail fins, and preparing DNA samples by a conventional column type centrifugation method (a general column type genome extraction kit, Beijing kang is century Biotechnology Co., Ltd.) according to the operation steps of the instruction;
(3) and (3) PCR amplification verification:
performing PCR verification on the 12 mandarin fish DNA samples of the male and female by using an upstream primer Contig-1F/R of a primer pair N1 or a downstream primer Contig-2F/R of a primer pair N2;
the total system of PCR amplification reaction is 20 μ L, including 10 μ L of 2 × TaqMasterMix (Beijing kang is century Biotechnology Co., Ltd.), 0.8 μ L of each of the upstream and downstream primers (10 μmol/L) of primer pair N1 or primer pair N2, 50ng of template DNA, and ddH for part less than 20 μ L2And (4) supplementing and finishing.
The PCR reaction program is: firstly, performing pre-denaturation at 94 ℃ for 2 min; 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: the primers Contig-1F/R of the primer pair N1 and Contig-2F/R of the primer pair N2 can amplify bands in the DNA samples of 12 male fishes in each of two populations, wherein the molecular size of the specific band amplified by the upstream and downstream primers Contig-1F/R of the primer pair N1 is 537 bp; the molecular size of the specific band amplified using the upstream and downstream primers Contig-2F/R of the primer pair N2 was 613 bp. No bands could be amplified in the DNA samples of 12 female fish in each of the two populations (FIG. 5: the amplification results of N1 and N2 in Guangdong Shaokou population males and females and FIG. 6: the amplification results of N1 and N2 in Hunan kingdom females). The molecular marker primer pair N1: Contig-1F/R and the primer pair N2: Contig-2F/R can be used for identifying the sex of the mandarin fish.
Example 3
The embodiment provides an application of the siniperca scherzeri male molecular marker primer in siniperca scherzeri total female and total female hybrid mandarin fish breeding, which comprises the following steps:
(1) after the Siniperca scherzeri fries are cultured to 10 days old, bait fishes are fed by eel feed containing 200mg/kg of androgen 17 alpha-methyl testosterone, and the bait fishes which eat hormone feed are immediately fed to the Siniperca chuatsi. Continuously feeding for two months, adopting natural illumination during the culture period, keeping the water temperature at 23-30 ℃, keeping the feeding amount not too much, keeping the fish in an unsupplemented state, and continuously feeding for two months.
(2) Screening the siniperca scherzeri fed with androgen by the method in example 2 to obtain the fish with genotype XX, namely the pseudo-male fish, and the specific process refers to example 2.
(3) And (3) continuously culturing the XX pseudo male fishes identified by the marks until the XX pseudo male fishes are sexually mature, and mating the XX pseudo male fishes with the normal XX spot mandarin fish female fishes in the breeding season to obtain spot mandarin fish offspring which are all female fishes.
(4) And (3) continuously culturing the XX pseudo male fishes identified by the marks until the male fishes are sexually mature, mating the XX pseudo male fishes with the normal XX siniperca chuatsi female fishes in the breeding season, and obtaining filial mandarin fish offspring which is the all-female mandarin fish filial generation.
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 modifications and variations based on the concept of the present invention are included in the scope of the present invention, and the specific scope of the protection is subject to the claims.
Sequence listing
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Guangdong Liang aquatic species Co Ltd
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Claims (8)
1. A siniperca scherzeri male molecular marker primer is characterized in that: the primer is a primer pair N1 or a primer pair N2, the primer pair N1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair N2 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.
2. A siniperca scherzeri gender identification method comprises the following steps:
(1) designing primer pair N1 or primer pair N2 of claim 1;
(2) extracting a siniperca scherzeri DNA sample;
(3) and (3) carrying out PCR amplification by using the DNA sample in the step (2) as a template and adopting a primer pair N1 or a primer pair N2, carrying out electrophoresis detection after the amplification reaction is finished, wherein if an electrophoresis result shows that a specific DNA strip exists, the detected siniperca scherzeri DNA sample is male siniperca scherzeri, and if the electrophoresis result shows that the specific DNA strip does not exist, the detected siniperca scherzeri DNA sample is female siniperca scherzeri.
3. The method of sex identification of siniperca scherzeri as claimed in claim 2, wherein: and (3) extracting the siniperca scherzeri DNA sample in the step (2) by adopting a column type centrifugation method.
4. The method of sex identification of siniperca scherzeri as claimed in claim 2, wherein: when the primer pair N1 or the primer pair N2 is adopted to carry out PCR amplification in the step (3), a 20-mu-L PCR reaction system comprises DNA50ng, 0.8-mu L of each of the upstream primer and the downstream primer of the primer pair N1 or 0.8-mu L of each of the upstream primer and the downstream primer of the primer pair N2, 2 XTaq MasterMix 10-mu L, and ddH is used for the part which is less than 20-mu L2And (4) supplementing and finishing.
5. The method of sex identification of siniperca scherzeri as claimed in claim 2, wherein: when the primer pair N1 or the primer pair N2 is adopted for PCR amplification in the step (3), the PCR reaction program 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 of sex identification of siniperca scherzeri as claimed in claim 2, wherein: 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%.
7. The method of sex identification of siniperca scherzeri as claimed in claim 2, wherein: when the primer pair N1 or the primer pair N2 is adopted for PCR amplification in the step (3), if the electrophoresis result shows that a specific DNA strip is present at the 537bp position or the 613bp position, the detected siniperca scherzeri DNA sample is male siniperca scherzeri, and if the electrophoresis result shows that no specific DNA strip is present at the 537bp position or the 613bp position, the detected siniperca scherzeri DNA sample is female siniperca scherzeri.
8. The siniperca scherzeri male molecular marker primer as claimed in claim 1, and the method as claimed in claim 2, can be used for breeding siniperca scherzeri all-female or hybrid siniperca scherzeri.
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