CN110184361B - Molecular marker for identifying genetic sex of hucho taimen and identification method - Google Patents
Molecular marker for identifying genetic sex of hucho taimen and identification method Download PDFInfo
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- CN110184361B CN110184361B CN201910512054.9A CN201910512054A CN110184361B CN 110184361 B CN110184361 B CN 110184361B CN 201910512054 A CN201910512054 A CN 201910512054A CN 110184361 B CN110184361 B CN 110184361B
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Abstract
A molecular marker for identifying the genetic sex of hucho taimen and an identification method thereof relate to a molecular marker for identifying the genetic sex of fish and an identification method thereof. The nucleotide sequence of the peculiar band of the male hucho taimen in the molecular marker for identifying the genetic sex of hucho taimen is shown as SEQ ID NO:1, the nucleotide sequence of the common band of the hucho taimen male and female fish is shown as SEQ ID NO:2, respectively. The identification method comprises the following steps: 1. extracting genome DNA; 2. PCR amplification; 3. and (4) performing gel electrophoresis, and displaying that the hucho taimen female fish has one strip and the hucho taimen male fish has two strips. The method can rapidly and efficiently identify the sex of the hucho taimen juvenile fish, only one pair of primers is needed for amplification, and the sex of the hucho taimen can be identified according to the number of bands of an agarose gel electrophoresis amplification product without sequencing.
Description
Technical Field
The invention relates to a molecular marker for identifying genetic sex of fish and an identification method.
Background
Hucho taimen (pallas) belongs to Salmoniformes, salmonidae and Hucho, is rare and rare cold water fishes in China, has tender meat, delicious taste, large individuals, late sexual maturity, sexual maturity of 5-6 years for female fishes and sexual maturity of 4-5 years for male fishes, and has no secondary characteristics. The sex mature hucho taimen only has marital color in the breeding season, the tail of the fish body turns red, and the marital color of part of the parent fish is not obvious, so that the sex is difficult to be identified through the marital color. Although the growth rates of male and female hucho taimen are not greatly different, the roe of the hucho taimen is rich in nutrition and can be used as caviar, and the commodity price far exceeds that of male fish; and the sperm of one male fish can be matched with the 3-5 female hucho taimen during artificial insemination, so that the breeding cost can be saved by pertinently adjusting the proportion of the male and female fish according to different breeding purposes.
The hucho taimen in 1998 is listed in Chinese endangered animal red book, and the endangered grade is easy to endanger. The resource of hucho salmon has been reduced and is difficult to see in recent years, and the hucho salmon is listed in the red list of Chinese species in 2004. Hucho taimen belongs to rare and rare fishes, so the sex ratio of wild resources is always needed to be known in the field investigation process, but the fishes with the age of less than 3 can not identify the sex, usually the fishes with the age of more than 3 need to kill the fishes, the gonads are taken, and the male and female can be identified by slicing, which leads to the situation that the wild resources are barren at present, and therefore, a method for identifying the male and female without killing the fishes is urgently needed.
Disclosure of Invention
The invention provides a molecular marker and an identification method for genetic sex identification of hucho taimen, which can accurately identify the sex of hucho taimen without killing the hucho taimen and have positive significance for artificial breeding and field resource investigation of hucho taimen.
The nucleotide sequence of the specific band of the male hucho taimen in the molecular marker for identifying the genetic sex of hucho taimen is shown as SEQ ID NO:1, the nucleotide sequence of the common band of the hucho taimen male and female fish is shown as SEQ ID NO:2, respectively.
The invention relates to a molecular marker primer for identifying the genetic sex of hucho taimen, wherein the upstream primer of the molecular marker is 5-.
The invention discloses a method for identifying the genetic sex of hucho salmon, which comprises the following steps:
1. extracting the genomic DNA of a sample to be identified;
2. performing PCR amplification by taking the genomic DNA in the step one as a template, wherein the molecular marker upstream primer in a PCR amplification system is 5-;
3. and performing gel electrophoresis on the PCR product, and displaying that the female hucho taimen has one strip and the male hucho taimen has two strips.
The sex identification method for hucho taimen by adopting the molecular marker PCR amplification method is convenient to operate, efficient and rapid and can be completed within 4.5 h. The body length of hucho taimen eggs is generally 2-3cm when the hucho taimen eggs are hatched out of membranes, the method needs a small amount of samples, hucho taimen which are just hatched out of membranes can be identified, the survival of seedlings can still be guaranteed after sampling, the method is not limited by the age, and the problem that the male and female hucho taimen cannot be morphologically identified is solved.
The molecular marker for identifying the genetic sex of hucho taimen provides a scientific basis for identifying the sex of hucho taimen.
The method can rapidly and efficiently identify the sex of the hucho taimen juvenile fish, only one pair of primers is needed for amplification, and the sex of the hucho taimen can be identified according to the number of bands of an agarose gel electrophoresis amplification product without sequencing.
Drawings
FIG. 1 is a gel electrophoresis identification chart of 1 female fish and 1 male fish selected in example 1.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the method for identifying the genetic sex of hucho taimen in the embodiment comprises the following steps:
1. extracting the genomic DNA of a sample to be identified;
2. performing PCR amplification by using the genomic DNA in the step I as a template, wherein the molecular marker upstream primer in the PCR amplification system is 5-;
3. and performing gel electrophoresis on the PCR product to display the female hucho taimen with one strip and the male hucho taimen with two strips.
The second embodiment is as follows: the present embodiment differs from the first embodiment in that: the PCR amplification reaction system in the second step is 20 μ l:2 XTaq PCR mix 10 mul, 2 mul of genomic DNA of a sample to be identified, the concentration of a molecular marker primer is 10 mul, the upstream and downstream primers of the molecular marker are respectively 1 mul, and the rest is complemented by ultrapure water without enzyme; the PCR reaction is as follows: pre-denaturation at 95 ℃ for 3min, followed by 30 cycles of denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, and extension at 72 ℃ for 30sec; finally, extension is carried out for 5min at 72 ℃. The rest is the same as the first embodiment.
Under the PCR amplification reaction condition of the embodiment, the target sequence is thoroughly denatured, the activity of Taq enzyme is not influenced, the primer is completely extended, and the effective amplification amount can be achieved
The third concrete implementation mode: the difference between this embodiment and the first or second embodiment is:
the method comprises the following steps: 0.4cm of hucho taimen to be identified is taken 2 Cracking the fin ray sample by using 100 mu l of cracking solution, and then digesting the sample by using a PCR instrument, wherein the digestion process is that digestion is carried out for 2 hours at 55 ℃ and digestion is carried out for 10 minutes at 98 ℃; then evenly mixing, centrifuging at 1000-2000 rpm for 1-2min, and taking supernatant fluid to obtain the sample genome DNA. The other embodiments are the same as the first or second embodiment.
The fourth concrete implementation mode is as follows: this embodiment is different from the first to third embodiments in that the lysate has a proteinase K concentration of 0.5mg/ml, a Tris concentration of 10mM at pH8.0, a KCl concentration of 50mM, a Tween20 concentration of 0.3% (w/v), and an NP-40 concentration of 0.3% (w/v). The other is the same as one of the first to third embodiments.
Example 1
1. Group acquisition of known sex
And (3) identifying male and female fish when the cold water fish experiment station at Bohai sea of the institute of aquatic science and research, heilongjiang aquatic research, china, lays 30 tails of male and female fish samples respectively, shearing fin strip samples, and pasting the fin strip samples on filter paper for drying in the shade.
2. Extracting the genomic DNA of a sample to be identified:
0.4cm of hucho taimen to be identified is taken 2 Cracking the fin ray sample by using 100 mu l of cracking solution, and then digesting the sample by using a PCR instrument, wherein the digestion process is that the digestion is carried out for 2 hours at the temperature of 55 ℃ and for 10 minutes at the temperature of 98 ℃; then evenly mixing the mixture by Vortex, centrifuging the mixture for 1 to 2min at 1000 to 2000rpm, and taking supernatant fluid to obtain the sample genome DNA. The lysate had a proteinase K concentration of 0.5mg/ml, a Tris concentration of 10mM, a KCl concentration of 50mM, a Tween20 concentration of 0.3% (w/v), and an NP-40 concentration of 0.3% (w/v).
3. And (3) PCR amplification:
performing PCR amplification by using the genome DNA of the step as a template, wherein the molecular marker upstream primer in a PCR amplification system is 5-; the PCR amplification reaction system is 20 ul: 2 xTaq PCR mix 10 mul, 2 mul of genomic DNA of a sample to be identified, the concentration of a molecular marker primer is 10 mul, the upstream primer and the downstream primer of the molecular marker are respectively 1 mul, and the rest is complemented by ultrapure water without enzyme; the PCR reaction is as follows: pre-denaturation at 95 ℃ for 3min, then performing 30 cycles of denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, and extension at 72 ℃ for 30sec; finally, extension is carried out for 5min at 72 ℃.
4. Performing gel electrophoresis on the PCR products, taking 8 mu l of the PCR products, analyzing by using 1.0-1.5% agarose gel electrophoresis, amplifying 1 strip for each 30 female fishes (the common strip of the female fishes and the male fishes is 306bp, shown as SEQ ID NO: 2), and amplifying 2 strips for each 30 male fishes (the common strip of the female fishes and the male fishes also comprises the unique strip of the male fishes, which is 255bp, shown as SEQ ID NO: 1).
The results of gel electrophoresis identification of 1 female fish (lane 1) and 1 male fish (lane 2) are shown in FIG. 1. The identification conclusion of the method is completely consistent with the actual situation, and the method is proved to be accurate.
Sequence listing
<110> institute of aquatic products of Heilongjiang, china institute of aquatic science
<120> molecular marker for identifying genetic sex of hucho taimen and identification method
<130> 2 young
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 255
<212> DNA
<213> genus Hucho
<400> 1
atcaatcatg gctggaggtg tgatgaggaa tcagactgca gaagaactgc agtaactatt 60
tgcttatttg tgtgtgttgt gtgtatgtgt gtttctgtgt gttcatatat atatatacgt 120
agattgtgtg tgcatgtcag ttcctttgtg catcggaaag ctgtgtcagg ttgtcgtgga 180
gatgtgacgg cgaggatgac tgctctgacg gcagcgatga agatgactgt gagtagactg 240
gtacgtgtgt gggtg 255
<210> 2
<211> 306
<212> DNA
<213> genus Hucho
<400> 2
atcaatcatg gctggaggtg tgacggggag tttgactgtg atgaccaatc agatgagaag 60
aactgcagta agtatctgct tatgtgtgtg tatctgtgtt tgtgtacata atgtatgtag 120
attgtgcatg tgtgtgtgtc taactgacct gtcctctccc tccagccacc tccatgtgta 180
ctgcggatca gttccgctgt gcatcgggac gctgtgtcag gctgtcatgg agatgtgacg 240
gcgaggatga ctgctctgac ggcagcgatg aagatgactg tgagtagact ggtacgtgtg 300
tgggtg 306
Claims (5)
1. A method for identifying the genetic sex of hucho taimen is characterized by comprising a molecular marker and a molecular marker primer for identifying the genetic sex of hucho taimen;
the nucleotide sequence of the specific band of the male hucho taimen in the molecular marker for identifying the genetic sex of hucho taimen is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the common strip of the male and female hucho taimen is shown as SEQ ID NO:2 is shown in the specification;
the molecular marker upstream primer is 5-.
2. A method for identifying the genetic sex of hucho taimen is characterized in that the method is used for identifying the genetic sex of hucho taimen according to the following steps:
1. extracting genome DNA of a sample to be identified;
2. performing PCR amplification by using the genomic DNA in the step one as a template, wherein the molecular marker upstream primer in a PCR amplification system is 5'
-CACCCACACACGTACCAGTC-3’；
3. And performing gel electrophoresis on the PCR product to display the female hucho taimen with one strip and the male hucho taimen with two strips.
3. The method for identifying the genetic sex of hucho taimen according to claim 2, wherein the PCR amplification reaction system in the second step is 20 μ l:2 xTaq PCR mix 10 mul, 2 mul of genomic DNA of a sample to be identified, the concentration of a molecular marker primer is 10 mul, the upstream primer and the downstream primer of the molecular marker are respectively 1 mul, and the rest is complemented by ultrapure water without enzyme; the PCR reaction is as follows: pre-denaturation at 95 ℃ for 3min, then performing 30 cycles of denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, and extension at 72 ℃ for 30sec; finally, extension is carried out for 5min at 72 ℃.
4. The method for identifying the genetic sex of hucho taimen according to claim 2 or 3, wherein the first step comprises: taking a fin ray sample of 0.4cm2 of hucho salmon to be identified, cracking the fin ray sample by using 100 microliter of lysate, and then digesting the sample by using a PCR (polymerase chain reaction) instrument, wherein the digestion process is digestion at 55 ℃ for 2 hours and digestion at 98 ℃ for 10 minutes; then mixing evenly, centrifuging for 1-2min at 1000-2000 rpm, and taking supernatant fluid to obtain the sample genome DNA.
5. The method of claim 4, wherein the lysate has proteinase K concentration of 0.5mg/ml, tris concentration of 10mM, KCl concentration of 50mM, tween20 concentration of 0.3% (w/v), NP-40 concentration of 0.3% (w/v).
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| CN110172519A (en) * | 2019-06-13 | 2019-08-27 | 中国水产科学研究院黑龙江水产研究所 | A method of identifying Hucho taimen and fine-scaled graphite |
| CN111748639A (en) * | 2020-08-07 | 2020-10-09 | 集美大学 | Molecular marker for identifying sex of haliotis discus hannai and application thereof |
| CN114480671B (en) * | 2022-02-16 | 2024-01-26 | 中国水产科学研究院黑龙江水产研究所 | Microsatellite marker primer pair for identifying genetic sex of brachymystax lenok and sex identification method |
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Non-Patent Citations (3)
| Title |
|---|
| The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): Repetitive sequences in the control region and phylogenetic implications for Salmonidae;Ying Wang et al;《Marine Genomics》;20110930;第4卷(第3期);全文 * |
| 利用AFLP技术筛选与哲罗鱼Hucho taimen(Pallas)性别相关的分子标记;张超等;《东北农业大学学报》;20100525(第05期);摘要，第97页左栏第2段以及1.2方法，第99页2.2，第101-102页3讨论与结论 * |
| 哲罗鲑、细鳞鲑及杂交种(哲罗鲑♀×细鳞鲑♂)遗传结构的SRAP分析;许凌雪等;《江西农业大学学报》;20111220(第06期);全文 * |
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