CN114717302B - Molecular marker, kit and method for distinguishing genders of triangular breams - Google Patents
Molecular marker, kit and method for distinguishing genders of triangular breams Download PDFInfo
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Abstract
The invention relates to a molecular marker for distinguishing the sex of triangular breams, a kit and a distinguishing method, and a primer pair of the molecular marker for distinguishing the sex of the triangular breams is HLJFLf3 and HLJFLr3. The method for distinguishing the sex of triangular breams comprises the following steps: 1. extracting genome DNA of a triangular bream sample to be detected; 2. performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf3 and HLJFLr3 as primers to obtain a PCR product; 3. and (3) carrying out electrophoresis detection on the PCR product, wherein a corresponding sample with a 582bp band is male and a sample without an amplification band is female in the electrophoresis result. The invention discloses a sex-specific molecular marker and a sex identification method for triangular breams, which can determine the individual sex of the triangular breams in the early development stage, further control the sex ratio of a reserve parent fish population and reduce the culture and maintenance cost. The sex determination method can determine the individual sex of the triangular bream in the early development stage, and lays a foundation for researching the association between the sex and the growth of the triangular bream and other commercial traits.
Description
Technical Field
The invention relates to a molecular marker, a kit and a distinguishing method for distinguishing the sex of triangular bream.
Background
The bream fish is an important member of freshwater fish families in China, and mainly comprises widely known megalobrama amblycephala (megalobrama amblycephala) distributed in large lakes at the middle and lower reaches of the Yangtze river, megalobrama amblycephala in the water systems of the Yangtze river and the Hainan river, and megalobrama amblycephala widely distributed in a plurality of water systems.
The triangular bream is large in size and good in meat quality, is suitable for cooking and eating habits of consumers in different regions, is easy to raise and catch, has strong disease resistance and stress resistance, and is suitable for various culture modes such as ponds, net cages and the like. Although scholars indicate that the triangular breams are expected to become the most potential megalobrama fishes after the megalobrama amblycephala, the triangular breams are undeniably inferior to the megalobrama amblycephala in the aspects of cultivation quantity, market acceptance, variety breeding and the like. At present, triangular breams only store 3 naturally distributed populations of reservoirs of Heilongjiang, hangzhou Qiantanjiang and Hongan gold sand river in Hubei due to over fishing, environmental damage and the like. The germplasm characteristics and new species cultivation research of the triangular breams are imperatively developed by utilizing modern molecular genetics means.
According to the investigation in recent years, the wild Heilongjiang triangular bream needs 7-year old formula to reach sexual maturity. Under the culture condition, the sexual maturity age of the triangular breams in Heilongjiang requires 5 years, and the southern population requires 3 years. Triangular bream has no obvious sex characteristics in the early ontogenesis stage before sexual maturity. For the cultured fishes with late sexual maturity and relatively large individuals during maturation, the parent fish culture period is long, and the colony culture and maintenance cost is high. If breeding enterprises can master the sex ratio of a backup parent fish population at any time through early sex identification, the number of parent breeding can be properly reduced, so that the breeding and maintenance cost is effectively reduced, and the overall economic benefit is improved.
Disclosure of Invention
The invention provides a molecular marker, a kit and a distinguishing method for distinguishing the sex of triangular bream.
The primer pair of the molecular marker for distinguishing the sex of the triangular bream is HLJFLf3 and HLJFLr3, and the specific sequences are as follows:
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
further, the sex molecular marker nucleotide sequence of the triangular bream in Heilongjiang is shown as SEQ ID NO:1 is shown in the specification; the sex molecular marker nucleotide sequences of the triangular breams in the Qiantang river and the triangular breams in the Jinsha river reservoir are shown as SEQ ID NO:2, respectively.
The sex molecular markers of the triangular breams in Heilongjiang river and the sex molecular markers of the triangular breams in Qiantangjiang river and the triangular breams in Jinsha river reservoir are 582bp.
The kit for distinguishing the sex of the triangular breams comprises a molecular marker primer pair HLJFLf3 and HLJFLr3.HLJFLf3:5 'GATGATGTCAAATGAACTAG-3'; HLJFLr3:5 'ATGGGATGGAGCAATGCAATG-3'.
The method for distinguishing the sex of triangular breams comprises the following steps:
1. extracting genome DNA of a triangular bream sample to be detected;
2. performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf3 and HLJFLr3 as primers to obtain a PCR product;
3. and (3) carrying out electrophoresis detection on the PCR product, wherein a corresponding sample with a 582bp band is male and a sample without an amplified band is female in the electrophoresis result, and thus the sex identification of the triangular bream is completed.
The invention discloses a sex-specific molecular marker and a sex identification method of triangular breams, which can determine the individual sex at the early development stage of the triangular breams, further control the sex ratio of a reserve parent fish population and reduce the breeding and maintenance cost.
The sex of individuals can be determined in the early development stage of the triangular bream, and a foundation is laid for researching the correlation between the sex and the growth of the triangular bream and other commercial traits.
The invention can realize the high-efficiency identification of the sex of the existing 3 triangular breams (black dragon river, qian tang river and Jinsha river). The accuracy of sex identification reaches 100%, and the method has the advantages of simple operation process, short time consumption and the like.
Drawings
FIG. 1 is a diagram showing the results of gel electrophoresis detection of a part of samples in example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive efforts based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.
The first embodiment is as follows: the primer pair of the molecular marker for distinguishing the sex of the triangular bream in the embodiment is HLJFLf3 and HLJFLr3, and the specific sequence is as follows:
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
the sex molecular markers of the triangular breams of the black dragon river, the triangular breams of the qiantangjiang river and the triangular breams of the Jinsha river reservoir are 582bp.
The second embodiment is as follows: the kit for distinguishing the genders of the triangular breams in the embodiment comprises a molecular marker primer pair HLJFLf3 and HLJFLr3.HLJFLf3:5 'GATGATGTCAAATGAACTAAG-3'; HLJFLr3:5 'ATGGGATGGAGCAAATGCAATG-3'.
The third concrete implementation mode: the difference between this embodiment and the second embodiment is that the kit further comprises deionized water, 10 XTaq Buffer, mgCl 2 dNTPs and Taq DNA polymerase. The rest is the same as the second embodiment.
The fourth concrete implementation mode: the method for distinguishing the genders of triangular breams in the embodiment comprises the following steps:
1. extracting genome DNA of a triangular bream sample to be detected;
2. performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf3 and HLJFLr3 as primers to obtain a PCR product;
3. and (3) carrying out electrophoresis detection on the PCR product, wherein a corresponding sample with a 582bp band is male and a sample without an amplification band is female in an electrophoresis result, and thus the sex identification of the triangular bream is completed.
Wherein, the PCR amplification system in the second step is shown in Table 1:
TABLE 1
| Composition (I) | Dosage of |
| Deionized water | 13.8 |
| 10×Taq Buffer | 2μL |
| MgCl 2 | 1.6μL |
| 10mM dNTPs | 0.4 |
| 10 μ M primer HLJFLf3 | 0.6 |
| 10 μ M primer HLJFLr3 | 0.6μL |
| 5U/. Mu.L Taq DNA polymerase | 0.08μL |
| 100 ng/. Mu.L genomic DNA | 1.0μL |
Wherein, the PCR amplification conditions in the step two are as follows: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃; then extending for 5min at 72 ℃, and preserving heat at 4 ℃.
Example 1
In the embodiment, 50 black longjiang triangular breams, qiantanjiang triangular breams and golden sha river reservoir triangular breams are selected randomly, and the sampling time is in the breeding period after the individuals reach sexual maturity.
1. About 60mg of each tail of the fin-ray tissue is sampled, numbered and recorded for sex, and fin-ray genomic DNA is extracted. The extracted fin-ray genomic DNA was stored at-20 ℃ in a 2ml EP tube containing 75% ethanol for further use.
In this example, a DNA extraction kit was used to extract genomic DNA from fin rays.
2. Performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf3 and HLJFLr3 as primers to obtain a PCR product;
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
the PCR amplification system is shown in Table 1. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃; then extending for 5min at 72 ℃, and preserving heat at 4 ℃.
3. And (3) carrying out electrophoresis detection on the PCR product by using 1.5% agarose gel, wherein a sample with a 582bp band is identified as a male, and a sample without an amplified band is identified as a female.
The accuracy of the identification result of the embodiment reaches 100%.
The results of gel electrophoresis of a part of the samples are shown in FIG. 1. In the lane samples numbered 1-8 in the figure 1, the samples are black longjiang triangular breams, wherein the samples in the lanes 1, 3, 6 and 7 are male, and a 582bp band is amplified; the samples in lanes 2, 4, 5 and 8 are female and no band was amplified. The Lane samples numbered 9-15 in FIG. 1 are Qiantanjiang triangular breams, wherein the Lane samples 9, 11, 12 and 15 are male and all amplify 582bp bands; the samples in lanes 10, 13, and 14 are female and no band was amplified. 16-23 in the figure 1 are triangular breams in a Jinsha river reservoir, wherein the samples in lanes 17, 18, 20 and 23 are male and are all amplified to form a 582bp band; lanes 19, 21, and 22 are female and no band was amplified. Lane NC of figure 1 is a negative control.
The method disclosed by the invention has the sex identification accuracy of 100% for the triangular breams in the Heilongjiang river, the triangular breams in the Qiantangjiang river and the triangular breams in the Jinsha river reservoir.
Sequencing the amplified band, wherein the sex molecular marker nucleotide sequence of the triangular bream in Heilongjiang is shown in SEQ ID NO:1 is shown in the specification; the sex molecular marker nucleotide sequences of the triangular breams in the Qiantang river and the triangular breams in the Jinsha river reservoir are shown as SEQ ID NO:2, respectively.
Example 2
In the embodiment, about 50g of each of about 50g of Heilongjiang triangular breams and Qiantangjiang triangular breams are randomly selected as experimental fishes.
1. And (3) respectively carrying out PIT marking and numbering recording on the experimental fish individuals, independently culturing, shearing about 50mg of fin ray samples from each tail, and extracting the fin ray tissue genome DNA. The extracted fin-ray genomic DNA was stored at-20 ℃ in a 2ml EP tube containing 75% ethanol for further use.
In this example, a DNA extraction kit was used to extract genomic DNA from fin-rays.
2. Performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf3 and HLJFLr3 as primers to obtain a PCR product;
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
the PCR amplification system is shown in Table 1. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃; then extending for 5min at 72 ℃, and preserving heat at 4 ℃.
3. And (3) carrying out electrophoresis detection on the PCR product by using 1.5% agarose gel, wherein a sample with a 582bp band is identified as a male, and a sample without an amplified band is identified as a female.
When the experimental fish individual reaches about 500g (the survival individual bred by the triangular breams in Heilongjiang river is 45 tails, and the survival individual bred by the triangular breams in Qiantangjiang river is 41 tails), the experimental fish is marked, identified, anesthetized and dissected, and the sex is identified through the sex gland morphology. The sex result of the experimental fish after anatomical identification is completely consistent with the sex result identified by the molecular marker in the embodiment, and the accuracy is 100%.
Sequence listing
<110> research institute for aquatic products of Heilongjiang of China institute for aquatic science
<120> molecular marker, kit and method for distinguishing genders of triangular breams
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 582
<212> DNA
<213> triangular bream (Megalobrama terminalis)
<400> 1
gatgatggtc aaatgaacta gggaaaatca ggccttaaat acatgcagag ggtactcaac 60
tgaacagaaa catgagacaa aacagactac acgttacagt ttctgaatga tgctctatat 120
tcttctgctc atgaacagtg tcagtaaaag tattgcattt catacttgtg gacatttcac 180
aatgtatttc agcttttgca gaaatgtatg taaaagcatg ttgataatga gaccacaatg 240
caaccaatag tacatttgat atttttaatg ctcatttggt taaatttgta aattactgca 300
tttattaact cataatactt actacattca tgtcttcata ttgatccaaa tcaatggcca 360
aagaaaaaaa tgaatttatt tcctcaatgt gttcatggtg gctgtgcaga cagatagttg 420
agtgtttctt ctttccctgg aatatctgta aaatattaag aatattgtga ataatttgtc 480
taaagttgaa ttaatactct cagtagtatt aacaatacag tacatttata tgtgcctttt 540
taactaaaac aattaaaata acattgcatt gctccatccc at 582
<210> 2
<211> 582
<212> DNA
<213> triangular bream (Megalobrama terminalis)
<400> 2
gatgatggtc aaatgaacta gggaaaatca ggccttaaat acatgcagag ggtactcaac 60
tgaacagaaa catgagacaa aacagactac acgttacagt ttctgaatga tgctctatat 120
tcttctgctc atgaacagtg tcagtaaaag tattgcattt catacttgtg gacatttcac 180
aatgtatttc agcttttgca gaaatgtatg taaaagcatg ttgataatga gaccacaatg 240
caaccaatag tacatttgat atttttaatg ctaatttggt taaatttgta aattattgca 300
tttattaact cataatactt actacattca tgtcttcata ttgatccaaa tcaatggcca 360
aagaaaaaaa tgaatttatt tcctcaatgt gttcatggtg gctgtgcaga cagatagttg 420
agtgtttctt ctttccctgg aatatctgta aaatattaag aatattgtga ataatttgtc 480
taaagttgaa ttaatactct cagtagtatt aacaatacag tacatttata tgtgcctttt 540
taactaaaac aattaaaata acattgcatt gctccatccc at 582
Claims (6)
1. A molecular marker for distinguishing the sex of triangular breams is characterized in that primer pairs of the molecular marker are HLJFLf3 and HLJFLr3, and the specific sequence is as follows:
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
2. a kit for distinguishing the sex of triangular bream is characterized in that the kit comprises a molecular marker primer pair HLJFLf3 and HLJFLr3;
wherein, the specific sequences of HLJFLf3 and HLJFLr3 are as follows:
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
3. the kit for distinguishing the sex of triangular bream according to claim 2, wherein the kit further comprises deionized water, 10 XTaq Buffer, mgCl 2 dNTPs and Taq DNA polymerase.
4. The method for distinguishing the sex of the triangular bream is characterized by comprising the following steps of:
1. extracting genome DNA of a triangular bream sample to be detected;
2. performing PCR amplification by taking the extracted genome DNA as a template and HLJFLf3 and HLJFLr3 as primers to obtain a PCR product;
3. performing electrophoresis detection on the PCR product, wherein a corresponding sample with a 582bp band is male and a sample without an amplification band is female in an electrophoresis result, and thus the sex identification of the triangular bream is completed;
wherein, the specific sequences of the HLJFLf3 and the HLJFLr3 are as follows:
HLJFLf3：5'-GATGATGGTCAAATGAACTAG-3'；
HLJFLr3：5'-ATGGGATGGAGCAATGCAATG-3'。
5. the method for distinguishing the sex of triangular bream according to claim 4, wherein the PCR amplification system in step two is as follows:
the amount of the components
13.8 muL deionized water
10×Taq Buffer 2µL
MgCl 2 1.6µL
10mM dNTPs 0.4µL
10 mu M primer HLJFLf3 0.6 mu L
10 mu M primer HLJFLr3 0.6 mu L
5U/muL Taq DNA polymerase 0.08 muL
100 ng/muL genome DNA 1.0 muL
6. The method for distinguishing the sex of triangular bream according to claim 4, wherein the PCR amplification conditions in step two are: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃; then extending for 5min at 72 ℃, and preserving heat at 4 ℃.
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| CN108277287B (en) * | 2018-04-18 | 2021-06-22 | 中国水产科学研究院黑龙江水产研究所 | Molecular marker, kit and distinguishing method for distinguishing black-dragon-river triangular bream from megalobrama fish |
| CN111850138B (en) * | 2020-07-31 | 2021-11-19 | 中国水产科学研究院黑龙江水产研究所 | Molecular marker, kit and method for distinguishing triangular bream and megalobrama amblycephala in Heilongjiang and hybrid thereof |
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