CN110106246A - A kind of molecular labeling and discrimination method identifying Hucho taimen genetic sex - Google Patents
A kind of molecular labeling and discrimination method identifying Hucho taimen genetic sex Download PDFInfo
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- CN110106246A CN110106246A CN201910512664.9A CN201910512664A CN110106246A CN 110106246 A CN110106246 A CN 110106246A CN 201910512664 A CN201910512664 A CN 201910512664A CN 110106246 A CN110106246 A CN 110106246A
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Abstract
A kind of molecular labeling and discrimination method identifying Hucho taimen genetic sex, it is related to the molecular labeling and discrimination method of a kind of fish identification genetic sex.Identify the nucleotide sequence of the molecular labeling of Hucho taimen gender as shown in SEQ ID NO:1.Discrimination method: one, genomic DNA is extracted；Two, PCR amplification, molecular labeling upstream primer is 5 '-TGTCAGGGTTGATTACAGTTCCT-3 ' in PCR amplification system, and molecular labeling downstream primer is 5 '-CTGCAGTCTGATTCCTCATCA-3 '；Three, gel electrophoresis, the appearance of band is not Hucho taimen raun, and 1 153bp band occur is Hucho taimen milter.The present invention identifies the gender of Hucho taimen using the method for molecular labeling PCR amplification, easy to operate, and identification can be completed in 3.5h, efficient quick, has important application value in producer face.
Description
Technical field
The present invention relates to molecular labelings and discrimination method that a kind of fish identify genetic sex.
Background technique
Hucho taimen Hucho taimen (pallas) is subordinate to salmon shape mesh (Salmoniformes), salmonidae (Salmonidae),
Taimen category (Hucho) is the rare rare cold water fishes in China, fine and tender taste, delicious flavour.Hucho taimen is arranged within 1998
Enter Chinese animals on the brink of extinction Red Data Book, threatened level is easily to endanger.The stock number of Hucho taimen constantly declines in recent years, has been difficult to see
It arrives, is put within 2004 Chinese species Red List.Hucho taimen is the maximum fish of individual in salmon fishes, and growth is rapid, by
4~5 years are needed in Hucho taimen first time sexal maturity, its gender can not be judged before sexal maturity by mode of appearance, after sexal maturity
Breeding period maturation male and female fish body is from abdomen to tail portion, including salmon pink nuptial coloration occur in abdomeinal fin and tail fin, and some individuals are wedded
Relation by marriage color is unobvious, is difficult to identify male and female by nuptial coloration, thus how quickly, the genetic sex of precise Identification Hucho taimen, in parent
Accomplish suitable female-male proportion in this cultivating process, save aquaculture cost, has become and restricted the important of Hucho taimen industry development
Factor.
Summary of the invention
Although can simply, quickly and accurately realize the identification of genetic sex based on molecular labeling, manpower and object are saved
Power, and application has been obtained in many species, but special point of correlated inheritance gender is not yet found in Hucho taimen
Son label.The object of the present invention is to provide a kind of molecular labelings and discrimination method that can be used for the identification of Hucho taimen genetic sex.
The present invention identifies the nucleotide sequence of the molecular labeling of Hucho taimen genetic sex as shown in SEQ ID NO:1.
The present invention is used to identify the molecular labeling primer of Hucho taimen genetic sex, and molecular labeling upstream primer is 5 '-
TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 '.
The present invention is used to identify the mitochondria accessory molecule labeled primer of Hucho taimen genetic sex, mitochondria accessory molecule mark
Note upstream primer is 5 '-CGTTCAACCTCACCACCTCT-3 ', and it is 5 '-that mitochondria accessory molecule, which marks downstream primer,
GCCTCAGAGCCAGTTTCAAG-3'.The nucleotide sequence of mitochondria accessory molecule label is as shown in SEQ ID NO:2.
A kind of identification Hucho taimen heredity method for distinguishing of the present invention:
One, sample gene group DNA to be identified is extracted；
Two, PCR amplification is carried out by template of step 1 genomic DNA, molecular labeling upstream primer is in PCR amplification system
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 '；
Three, PCR product gel electrophoresis, the appearance of band is not Hucho taimen raun, and 1 153bp band occurs in display
It is Hucho taimen milter.
Another identification Hucho taimen heredity method for distinguishing of the invention:
One, sample gene group DNA to be identified is extracted；
Two, PCR amplification is carried out by template of step 1 genomic DNA, molecular labeling upstream primer is in PCR amplification system
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 ',
It is 5 '-CGTTCAACCTCACCACCTCT-3 ' that accessory molecule, which marks upstream primer, and it is 5 '-that accessory molecule, which marks downstream primer,
GCCTCAGAGCCAGTTTCAAG-3'；
Three, PCR product gel electrophoresis, display occur 1 251bp band be Hucho taimen raun, display occur 153bp and
Two band of 251bp is Hucho taimen milter.
The present invention identifies the gender of Hucho taimen using the method for molecular labeling PCR amplification, easy to operate, and can be in 3.5h
Interior completion identification, efficient quick have important application value in producer face.
The present invention identifies the molecular labeling of Hucho taimen genetic sex, provides scientific basis for Hucho taimen sex identification.
It joined mitochondrial DNA as reference in second of discrimination method of the invention, and to mitochondrial DNA primer and specifically
Property sequence primer ratio be optimized, both make the clear band of amplified band, whether can accurately distinguish due to amplification
The missing of band caused by the reasons such as process fault, DNA degradation, improves the accuracy of identification.
Detailed description of the invention
Fig. 1 is the gel electrophoresis identification figure that 4 rauns and 4 milters are chosen in embodiment 1.
Fig. 2 is the gel electrophoresis identification figure that 5 rauns and 5 milters are chosen in embodiment 2.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: present embodiment identifies Hucho taimen heredity method for distinguishing:
One, sample gene group DNA to be identified is extracted；
Two, PCR amplification is carried out by template of step 1 genomic DNA, molecular labeling upstream primer is in PCR amplification system
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 '；
Three, PCR product gel electrophoresis, the appearance of band is not Hucho taimen raun, and 1 153bp band occurs in display
It is Hucho taimen milter.
Specific embodiment 2: the difference of present embodiment and specific embodiment one is: PCR amplification is anti-in step 2
Answering system is 20 μ l:2 × PCR Taq mix, 10 μ l, and 2 μ l of sample gene group DNA to be identified, molecular labeling primer concentration is 10
μM, each 1 μ l, Yu Qiyong ultrapure water of molecular labeling upstream and downstream primer supplies；PCR reaction are as follows: then 95 DEG C of initial denaturation 3min are carried out
30 circulations, 95 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 30sec；Last 72 DEG C of extensions 5min.Other and implementation
Mode one is identical.
Specific embodiment 3: the difference of present embodiment and specific embodiment one or two is:
Step 1: Hucho taimen 0.4cm to be identified is taken2Fin ray sample, with 100 μ l lysates crack, then disappeared with PCR instrument
Change sample, digestion process is 55 DEG C of digestion 50min, 98 DEG C of digestion 10min；It mixes later, 1000~2000rpm is centrifuged 1- again
2min takes supernatant to obtain sample gene group DNA.It is other identical as embodiment one or two.
Specific embodiment 4: the difference of present embodiment and one of specific embodiment one to three is: in lysate
It is 10mM, KCl concentration be 20 concentration of 50mM, Tween is 0.3% that proteinase K concentration, which is the Tris concentration of 0.5mg/ml, pH8.0,
(w/v), NP-40 concentration is 0.3% (w/v).It is other identical as one of embodiment one to three.
Specific embodiment 5: present embodiment identifies Hucho taimen heredity method for distinguishing:
One, sample gene group DNA to be identified is extracted；
Two, PCR amplification is carried out by template of step 1 genomic DNA, molecular labeling upstream primer is in PCR amplification system
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 ',
It is 5 '-CGTTCAACCTCACCACCTCT-3 ' that accessory molecule, which marks upstream primer, and it is 5 '-that accessory molecule, which marks downstream primer,
GCCTCAGAGCCAGTTTCAAG-3'；
Three, PCR product gel electrophoresis, display occur 1 251bp band be Hucho taimen raun, display occur 153bp and
Two band of 251bp is Hucho taimen milter.
Specific embodiment 6: the difference of present embodiment and specific embodiment five is: PCR amplification is anti-in step 2
Answering system is 20 μ l:2 × Taq PCR mix, 10 μ l, and 2 μ l of sample gene group DNA to be identified, molecular labeling primer concentration is 10
μM, each 1 μ l of molecular labeling upstream and downstream primer, accessory molecule labeled primer concentration be 1 μM, accessory molecule mark upstream and downstream primer it is each
1μl；Yu Qiyong ultrapure water is supplied；PCR reaction are as follows: 95 DEG C of initial denaturation 3min, then carry out 30 circulations, 95 DEG C of denaturation 30sec,
60 DEG C of annealing 30sec, 72 DEG C of extension 30sec；Last 72 DEG C of extensions 5min.It is other identical as embodiment five.
Specific embodiment 7: the difference of present embodiment and specific embodiment five or six is:
Step 1: Hucho taimen 0.4cm to be identified is taken2Fin ray sample, with 100 μ l lysates crack, then disappeared with PCR instrument
Change sample, digestion process is 55 DEG C of digestion 50min, 98 DEG C of digestion 10min；It mixes later, 1000~2000rpm is centrifuged 1- again
2min takes supernatant to obtain sample gene group DNA.It is other identical as embodiment five or six.
Specific embodiment 8: the difference of present embodiment and one of specific embodiment five to seven is: in lysate
It is 10mM, KCl concentration be 20 concentration of 50mM, Tween is 0.3% that proteinase K concentration, which is the Tris concentration of 0.5mg/ml, pH8.0,
(w/v), NP-40 concentration is 0.3% (w/v).It is other identical as one of embodiment five to seven.
Embodiment 1
1, the group of known gender obtains
Heilongjiang Inst. of Aquatic Products, Chinese Academy of Aquatic Products Scie's Bohai Sea cold water fish experiment centre identifies male and female, male and female when laying eggs
Each 30 tail of fish sample, clip fin ray sample, which is attached on filter paper, to dry in the shade.
2, sample gene group DNA to be identified is extracted:
Take Hucho taimen 0.4cm to be identified2Fin ray sample, with 100 μ l lysates crack, then with PCR instrument digest sample,
Digestion process is 55 DEG C of digestion 50min, 98 DEG C of digestion 10min；Later with Vortex mix, again 1000~2000rpm centrifugation 1~
2min takes supernatant to obtain sample gene group DNA.Proteinase K concentration is that the Tris of 0.5mg/ml, pH8.0 are dense in lysate
Degree be 10mM, KCl concentration be 20 concentration of 50mM, Tween be 0.3% (w/v), NP-40 concentration be 0.3% (w/v).
3, PCR amplification:
PCR amplification is carried out by template of previous step genomic DNA, molecular labeling upstream primer is in PCR amplification system
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 '；
Pcr amplification reaction system is 20 μ l:2 × Taq PCR mix, 10 μ l, 2 μ l of sample gene group DNA to be identified, and molecular labeling draws
Object concentration is 10 μM, each 1 μ l, Yu Qiyong ultrapure water of molecular labeling upstream and downstream primer is supplied；PCR reaction are as follows: 95 DEG C of initial denaturations
Then 3min carries out 30 circulations, 95 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 30sec；Last 72 DEG C of extensions
5min。
4, PCR product gel electrophoresis takes 8 μ l PCR products to be analyzed with 1.5% agarose gel electrophoresis, and 30 rauns do not have
There is the appearance of band, 30 milters amplify the band of 1 153bp.
Wherein the gel electrophoresis identification result of 4 rauns (swimming lane 1~4) and 4 milters (swimming lane 5~8) is as shown in Figure 1.
The method of the present invention identifies conclusion and fits like a glove with actual conditions, it was demonstrated that the method for the present invention is accurate.
Embodiment 2
1, the group of known gender obtains
Heilongjiang Inst. of Aquatic Products, Chinese Academy of Aquatic Products Scie's Bohai Sea cold water fish experiment centre identifies male and female, male and female when laying eggs
Each 50 tail of fish sample, clip fin ray sample, which is attached on filter paper, to dry in the shade.
2, sample gene group DNA to be identified is extracted:
Take Hucho taimen 0.4cm to be identified2Fin ray sample, with 100 μ l lysates crack, then with PCR instrument digest sample,
Digestion process is 55 DEG C of digestion 50min, 98 DEG C of digestion 10min；Later with Vortex mix, again 1000~2000rpm centrifugation 1~
2min takes supernatant to obtain sample gene group DNA.Proteinase K concentration is that the Tris of 0.5mg/ml, pH8.0 are dense in lysate
Degree be 10mM, KCl concentration be 20 concentration of 50mM, Tween be 0.3% (w/v), NP-40 concentration be 0.3% (w/v).
3, PCR amplification:
PCR amplification is carried out by template of previous step genomic DNA, molecular labeling upstream primer is in PCR amplification system
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 ',
It is 5 '-CGTTCAACCTCACCACCTCT-3 ' that accessory molecule, which marks upstream primer, and it is 5 '-that accessory molecule, which marks downstream primer,
GCCTCAGAGCCAGTTTCAAG-3'；Pcr amplification reaction system is 20 μ l:2 × PCR mix, 10 μ l, sample gene to be identified
Group 2 μ l of DNA, molecular labeling primer concentration are 10 μM, each 1 μ l of molecular labeling upstream and downstream primer, accessory molecule labeled primer concentration
Each 1 μ l of upstream and downstream primer is marked for 1 μM, accessory molecule；Yu Qiyong ultrapure water is supplied；PCR reaction are as follows: 95 DEG C of initial denaturation 3min,
Then 30 circulations, 95 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 30sec are carried out；Last 72 DEG C of extensions 5min.
4, PCR product gel electrophoresis takes 8 μ l PCR products to be analyzed with 1.5% agarose gel electrophoresis, and 50 rauns are expanded
Increase the band of 1 251bp of shaping, 50 milters amplify two band of 1 251bp and 1 153bp.
Wherein the gel electrophoresis identification result of 5 rauns (swimming lane 1~5) and 5 milters (swimming lane 6~10) is as shown in Figure 2.
The method of the present invention identifies conclusion and fits like a glove with actual conditions, it was demonstrated that the method for the present invention is accurate.
Sequence table
<110>Heilongjiang Inst. of Aquatic Products, Chinese Academy of Aquatic Products Scie
<120>a kind of molecular labeling and discrimination method for identifying Hucho taimen genetic sex
<130>line 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 153
<212> DNA
<213>taimen category (Hucho)
<400> 1
tgtcagggtt gattacagtt cctaaggcat ttgcatttta tctcatggta gtggttgtgt 60
cctgcagcct cccaacagcc ttgtcgttct gtggagttca tgtgggatgt atatcaatca 120
tggctggagg tgtgatgagg aatcagactg cag 153
<210> 2
<211> 251
<212> DNA
<213>taimen category (Hucho)
<400> 2
cgttcaacct caccacctct tgttttcccc gcctatatac caccgtcgtc agcttaccct 60
gtgaaggccc tatagtaagc aaaatgggca aaacccaaaa cgtcaggtcg aggtgtagcg 120
catggggtgg gaagaaatgg gctacattct ctaaattaga gcactacgaa ccacgctgtg 180
aaaccagcgt ccgaaggtgg atttagcagt aaacagaaaa cagagagttc tcttgaaact 240
ggctctgagg c 251
Claims (9)
1. a kind of molecular labeling for identifying Hucho taimen genetic sex, it is characterised in that the nucleotide sequence of the molecular labeling such as SEQ
Shown in ID NO:1.
2. for identifying the molecular labeling primer of Hucho taimen genetic sex, it is characterised in that the molecular labeling upstream primer is
5 '-TGTCAGGGTTGATTACAGTTCCT-3 ', the molecular labeling downstream primer are 5 '-
CTGCAGTCTGATTCCTCATCA-3’。
3. the mitochondria accessory molecule labeled primer for identifying Hucho taimen genetic sex, it is characterised in that the mitochondria is auxiliary
Helping molecular labeling upstream primer is 5 '-CGTTCAACCTCACCACCTCT-3 ', and the mitochondria accessory molecule label downstream is drawn
Object is 5 '-GCCTCAGAGCCAGTTTCAAG-3 '.
4. a kind of identification Hucho taimen heredity method for distinguishing, it is characterised in that this method identifies Hucho taimen heredity according to the following steps
Other:
One, sample gene group DNA to be identified is extracted；
Two, PCR amplification is carried out by template of step 1 genomic DNA, molecular labeling upstream primer is 5 '-in PCR amplification system
TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 '；
Three, PCR product gel electrophoresis, the appearance of band is not Hucho taimen raun, and display 1 153bp band occurs and is
Hucho taimen milter.
5. the identification Hucho taimen heredity method for distinguishing stated according to claim 4, it is characterised in that pcr amplification reaction in step 2
System is 20 μ l:2 × Taq PCR mix10 μ l, 2 μ l of sample gene group DNA to be identified, molecular labeling primer concentration is 10 μM,
Each 1 μ l, Yu Qiyong ultrapure water of molecular labeling upstream and downstream primer is supplied；PCR reaction are as follows: then 95 DEG C of initial denaturation 3min carry out 30
95 DEG C of denaturation 30sec of a circulation, 60 DEG C of annealing 30sec, 72 DEG C of extension 30sec；Last 72 DEG C of extensions 5min.
6. a kind of identification Hucho taimen heredity method for distinguishing, it is characterised in that this method identifies Hucho taimen heredity according to the following steps
Other:
One, sample gene group DNA to be identified is extracted；
Two, PCR amplification is carried out by template of step 1 genomic DNA, molecular labeling upstream primer is 5 '-in PCR amplification system
TGTCAGGGTTGATTACAGTTCCT-3 ', molecular labeling downstream primer are 5 '-CTGCAGTCTGATTCCTCATCA-3 ', auxiliary
Molecular labeling upstream primer is 5 '-CGTTCAACCTCACCACCTCT-3 ', and it is 5 '-that accessory molecule, which marks downstream primer,
GCCTCAGAGCCAGTTTCAAG-3'；
Three, PCR product gel electrophoresis, display occur 1 251bp band be Hucho taimen raun, display occur 153bp and
Two band of 251bp is Hucho taimen milter.
7. the identification Hucho taimen heredity method for distinguishing stated according to claim 6, it is characterised in that pcr amplification reaction in step 2
System is 20 μ l:2 × Taq PCR mix10 μ l, 2 μ l of sample gene group DNA to be identified, molecular labeling primer concentration is 10 μM,
Each 1 μ l of molecular labeling upstream and downstream primer, accessory molecule labeled primer concentration is 1 μM, accessory molecule marks each 1 μ of upstream and downstream primer
l；Yu Qiyong ultrapure water is supplied；PCR reaction are as follows: then 95 DEG C of initial denaturation 3min carry out 30 circulations 95 DEG C of denaturation 30sec, 60
DEG C annealing 30sec, 72 DEG C of extension 30sec；Last 72 DEG C of extensions 5min.
8. according to identification Hucho taimen heredity method for distinguishing described in claim 4,5,6 or 7, it is characterised in that step 1: take
Hucho taimen 0.4cm to be identified2Fin ray sample, cracked with 100 μ l lysates, then digest sample with PCR instrument, digestion process is
55 DEG C of digestion 50min, 98 DEG C of digestion 10min；Mixing later, 1000~2000rpm is centrifuged 1-2min again, and supernatant is taken to obtain
Sample gene group DNA.
9. identification Hucho taimen heredity method for distinguishing according to claim 8, it is characterised in that Proteinase K is dense in lysate
It is 10mM, KCl concentration be 50mM, Tween20 concentration is 0.3% (w/v), NP- that degree, which is the Tris concentration of 0.5mg/ml, pH8.0,
40 concentration are 0.3% (w/v).
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877445A (en) * | 2021-03-08 | 2021-06-01 | 河南牧业经济学院 | Microsatellite marker and primer for identifying genetic sex of hucho taimen, and identification method and application |
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| CN101736007A (en) * | 2009-10-29 | 2010-06-16 | 中国水产科学研究院黑龙江水产研究所 | Mitochondrion complete genome sequence of hucho taimen and amplification primers thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101736007A (en) * | 2009-10-29 | 2010-06-16 | 中国水产科学研究院黑龙江水产研究所 | Mitochondrion complete genome sequence of hucho taimen and amplification primers thereof |
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| Title |
|---|
| YINGWANG ET AL: "The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): repetitive sequences in the control region and phylogenetic implications for Salmonidae", 《MARINE GENOMICS》 * |
| 匡友谊等: "线粒体序列分析黑龙江流域哲罗鲑的种群遗传结构", 《生态学报》 * |
| 周丽青等: "基于遗传连锁图谱筛选虾夷扇贝性别相关AFLP分子标记的方法", 《渔业科学进展》 * |
| 张超等: "利用AFLP技术筛选与哲罗鱼Hucho taimen(Pallas)性别相关的分子标记", 《东北农业大学学报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877445A (en) * | 2021-03-08 | 2021-06-01 | 河南牧业经济学院 | Microsatellite marker and primer for identifying genetic sex of hucho taimen, and identification method and application |
| CN112877445B (en) * | 2021-03-08 | 2023-10-24 | 河南牧业经济学院 | Microsatellite marker and primer for genetic sex identification of hucho taimen as well as identification method and application |
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