CN101921865A - Primer, fragment and method for identifying genetic sex of Oreochromis aureus - Google Patents
Primer, fragment and method for identifying genetic sex of Oreochromis aureus Download PDFInfo
- Publication number
- CN101921865A CN101921865A CN 201010269705 CN201010269705A CN101921865A CN 101921865 A CN101921865 A CN 101921865A CN 201010269705 CN201010269705 CN 201010269705 CN 201010269705 A CN201010269705 A CN 201010269705A CN 101921865 A CN101921865 A CN 101921865A
- Authority
- CN
- China
- Prior art keywords
- primer
- sequence
- oreochromis aureus
- sex
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular marker and specifically discloses a fragment, a group of primers and an identification method for identifying the genetic sex of Oreochromis aureus. The group of primers is used for performing PCR reaction so as to obtain an amplified fragment with length of 1488 bp from female Oreochromis aureus; if no amplified product exists in the female Oreochromis aureus, the fact is proved the primer is female specific, thereby indicating that the primer and the method can be applied to identification of the genetic sex of the Oreochromis aureus.
Description
Affiliated technical field the invention belongs to the molecular marking technique field, specifically one group of primer, fragment and method of differentiating genetic sex of Oreochromis aureus.
Usually require to differentiate its sex chromosome composition in genetics research of background technology fish and the breeding practice, but most of fish sex chromosome differentiation degrees are lower, do not have sex chromosome, the minority fish prove existence karyomit(e) through genetics experiments, but can not differentiate from cytogenetics.The fish sex differentiation is subject to such environmental effects (as temperature, illumination and food etc.) in embryo development procedure in addition, causes its sex phenotype and genotype inconsistent.Therefore, be necessary to seek a kind of short-cut method that can discern its genetic composition, and the molecule marker of sex-specific is undoubtedly a kind of simple and direct effective genetic sex discrimination method.
Oreochromis aureus (Oreochromis aureus) is one of higher kind of economic worth in several tilapias of introducing of China.Tilapia is male more than 40%, accurately differentiates therefore that its genetic sex has important Research Significance and economic worth for property control breeding than female fast growth.Studies confirm that Oreochromis aureus is a ZW sex determination type, it may be sex chromosome that 1 pair of special human chromosome is arranged in its karyotype, but this is similar to chromosome morphology, therefore also can't utilize traditional karyotyping to differentiate its genetic sex.
The molecular marking technique that development in recent years is got up (RAPD, AFLP, SSR etc.) can directly be sought the dna fragmentation relevant with objective trait from the molecular level, for the evaluation of fish genetic sex provides new effective way.The utilization molecular marking technique finds the sex dna fragmentation of being correlated with Cynoglossus semilaevis fish such as (Cynoglossus semilaevis), can be used for differentiating its genetic sex.
Still do not have about utilizing molecule marker successfully to differentiate the report of genetic sex of Oreochromis aureus both at home and abroad at present, the present invention has filled up the blank in this field.
Summary of the invention summary of the invention apportion is as follows:
1. one group of primer of differentiating genetic sex of Oreochromis aureus, the sequence that it is characterized in that this group primer is by sequences Design synthetic shown in the SEQID NO:1.
2. as 1 described primer, its sequence length is 18～28nt.
3. as 1 or 2 described primers, its sequence is by the sequences Design synthetic more than the continuous 300bp in the sequence shown in the SEQ ID NO:1.
4. as 1～3 described primer, wherein a pair of primer sequence is the sequence shown in SEQ ID NO:2 and the SEQ ID NO:3.
5. one section dna fragmentation of identifying genetic sex of Oreochromis aureus is characterized in that this fragments sequence comprises sequence shown in the SEQ ID NO:1.
6. a method of identifying genetic sex of Oreochromis aureus is characterized in that this method is the electrophoresis result evaluation sample sex of PCR reaction product per sample.
7. as 6 described methods, it is characterized in that the PCR primer is by the sequences Design synthetic more than the continuous 300bp in the sequence shown in the SEQ ID NO:1 in this method.
8. as 6 or 7 described methods, the sample that it is characterized in that occurring in the PCR product electrophoresis result in this method one section 300～1500bp size strip is female, and same area the sample of band not occur be male.
Specifically, the object of the invention provides one group of primer and the section of DNA fragment of differentiating genetic sex of Oreochromis aureus;
Another object of the present invention provides a kind of method of differentiating genetic sex of Oreochromis aureus.
The present invention uses the RAPD method to amplify a female specific band in female Oreochromis aureus genome, having obtained a segment length by clone and order-checking is the dna sequence dna of 1488bp, shown in SEQ ID NO:1, and designed one group of primer of differentiating genetic sex of Oreochromis aureus in view of the above, can produce the sequence partial design thing of the above amplified production of 300bp in the preferred SEQ ID NO:1 sequence.Primer length is 18～28nt, and non-specific band occurs during wherein less than 18nt, the easy self-polymerization of primer during greater than 28nt.The embodiment of the invention provides a pair of primer, and its sequence is shown in SEQID NO:2 and SEQ ID NO:3.
The invention provides a kind of method of differentiating genetic sex of Oreochromis aureus, it is characterized in that method is the sex of PCR reaction product electrophoresis result evaluation sample per sample.The PCR primer is no less than the sequences Design synthetic of 300bp at interval in this method by the sequence shown in the SEQ ID NO:1.The sample that occurs one section 300～1500bp left and right sides band in this method in the PCR product electrophoresis result is female, and same area the sample of band not occur be male.
When the PCR reaction system was 25 μ l, system contained 10 * Buffer2.5 μ l; MgCl
22 μ l; DNTP (each 2.5 μ mol/L) 2 μ l; Each 0.1～0.2 μ mol/L of female specificity upstream and downstream primer; Taq archaeal dna polymerase (TaKaRa) 1U; Genomic dna 20～30ng.Response procedures is 94 ℃ of pre-sex change 5min; 35 circulations: 94 ℃ of 30～40sec, 60～64 ℃ of 50～80sec, 72 ℃ of 50～80sec; Then 72 ℃ are extended 7～10min; 4 ℃ of preservations.
The present invention has used 800 RAPD random primers altogether, each primer has all amplified 6～9 polymorphic bandses, wherein primer RAPD71699 (GTGACGTAGG) can amplify a feature band about the about 1500bp of size in the female individuals genomic dna, and (Fig. 1) do not appear in the male genomic dna on the same position.
With above-mentioned feature band clone, order-checking, obtain sequence shown in SEQ ID NO:1.This sequence is found in the comparison of sequence and ncbi database, and the homology of Hoxba gene family (GcnBank:EF594310.1) the Hoxb7a gene of the fragment grown of 293bp and another kind of Callichthyidae (Cichlidae) fish Astatotilapia burtoni and the tumor-necrosis factor glycoproteins between the Hoxb6a gene is higher altogether from 40bp to 332bp, sequence identity is up to 93%, its function still unknowable (SimoneHoegg etc.), rest part is not found higher homology sequence.
By sequence SEQ ID NO:1 2 oligonucleotide chains of design construction (primer):
SEQ?ID?NO：25’-GTGACGTAGGTAGAATCATGGCGGC-3’
SEQ?ID?NO：35’-GTGACGTAGGGAGAGAGAACTTGTT-3’
Check this validity with the Oreochromis aureus of 24 known sexes to primer, feature band about 1500bp all appears in all tested female individuals DNA electrophoresis of PCR reaction and display, and does not all occur this feature band (Fig. 2) on all tested male corresponding positions.
In sum, the invention provides a segment length is the female special dna sequence dna of Oreochromis aureus of 1488bp, and according to the primer of this sequences Design, can be used for the evaluation of genetic sex of Oreochromis aureus.Embodiment has proved that primer and PCR method can 100% differentiate the genetic sex of Oreochromis aureus.
Description of drawings
Fig. 1, RAPD reaction result wherein have a specific band (position shown in the arrow) on the electrophoretic band of 7 female individuals (♀ sign), and do not have on 7 males (♂ sign) corresponding position.
Fig. 2, PCR reaction result, a feature band about 1500bp all appears in all tested female individuals DNA electrophoresis, and this feature band all do not occur on all tested male corresponding positions.
Embodiment
Embodiment:
Extract the respectively genomic dna of 12 Oreochromis aureus of male and female respectively with conventional DNA extraction method, be diluted to 10ng/ μ l and use in order to pcr amplification;
The PCR primer adopts the Auele Specific Primer of the present invention according to the female DNA fragment specific design of Oreochromis aureus:
5’-GTGACGTAGGTAGAATCATGGCGGC-3’
5’-GTGACGTAGGGAGAGAGAACTTGTT-3’
The PCR reaction system is 25 μ l, and system contains 10 * Buffer2.5 μ l; MgCl
22 μ l; DNTP (each 2.5 μ mol/L) 2 μ l; Each 0.2 μ mol/L of female specificity upstream and downstream primer; Taq archaeal dna polymerase (TaKaRa) 1U; Genomic dna 20ng.Response procedures is 94 ℃ of pre-sex change 5min; 35 circulations: 94 ℃ of 40sec, 62.3 ℃ of 50sec, 72 ℃ of 50sec; Then 72 ℃ are extended 7min; 4 ℃ of preservations.The PCR product is through 1.5% agarose gel electrophoresis, and EB dyeing is taken a picture, the result as shown in Figure 2:
From the graph as can be seen, feature band about 1500bp all appears in the DNA electrophoresis in all tested female individuals, and this feature band does not all appear on all tested male corresponding positions, confirmed that thus according to the female DNA fragment specific design of the Oreochromis aureus that obtains synthetic primer be female special, shown that also primer provided by the invention, fragment and method can be applied to the evaluation of genetic sex of Oreochromis aureus simultaneously.
Claims (8)
1. one group of primer of differentiating genetic sex of Oreochromis aureus, the sequence that it is characterized in that this group primer is by sequences Design synthetic shown in the SEQID NO:1.
2. primer according to claim 1, its sequence length are 18～28nt.
3. primer according to claim 1 and 2, its sequence are by the sequences Design synthetic more than the continuous 300bp in the sequence shown in the SEQ ID NO:1.
4. according to the described primer of claim 1～3, wherein a pair of primer sequence is the sequence shown in SEQ ID NO:2 and the SEQ IDNO:3.
5. one section dna fragmentation of identifying genetic sex of Oreochromis aureus is characterized in that this fragments sequence comprises sequence shown in the SEQ ID NO:1.
6. a method of identifying genetic sex of Oreochromis aureus is characterized in that this method is the electrophoresis result evaluation sample sex of PCR reaction product per sample.
7. method according to claim 6 is characterized in that the PCR primer is by the sequences Design synthetic more than the continuous 300bp in the sequence shown in the SEQ ID NO:1 in this method.
8. according to claim 6 or 7 described methods, the sample that it is characterized in that occurring in the PCR product electrophoresis result in this method one section 300～1500bp size strip is female, and same area the sample of band not occur be male.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102697055A CN101921865B (en) | 2010-09-02 | 2010-09-02 | Primer, fragment and method for identifying genetic sex of Oreochromis aureus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102697055A CN101921865B (en) | 2010-09-02 | 2010-09-02 | Primer, fragment and method for identifying genetic sex of Oreochromis aureus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101921865A true CN101921865A (en) | 2010-12-22 |
| CN101921865B CN101921865B (en) | 2012-09-19 |
Family
ID=43337080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102697055A Expired - Fee Related CN101921865B (en) | 2010-09-02 | 2010-09-02 | Primer, fragment and method for identifying genetic sex of Oreochromis aureus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101921865B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561332A (en) * | 2015-01-20 | 2015-04-29 | 黑龙江省林业科学研究所 | SSR molecular marker for identifying aspen gender and application of SSR molecular marker |
| CN110055335A (en) * | 2018-12-14 | 2019-07-26 | 中山大学 | A kind of microsatellite molecular marker primer, kit and rapid identification method identifying the female milter of star continent red tilapia |
| CN110938626A (en) * | 2019-12-12 | 2020-03-31 | 西南大学 | Specific molecular marker of sex chromosome of Oreochromis aureus, genetic sex identification based on molecular marker and method for producing unisex fish |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240348A (en) * | 2008-01-23 | 2008-08-13 | 中国水产科学研究院黄海水产研究所 | Rapid identification method for half-smooth tongue-sole genetic sex |
| CN101270389A (en) * | 2008-05-09 | 2008-09-24 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis special numerator mark and uses thereof |
| CN100436599C (en) * | 2006-05-12 | 2008-11-26 | 中国水产科学研究院黄海水产研究所 | Female-specific AFLP fragment of Cynoglossus semilaevis Gunther and PCR method for identification of genetic sex |
| CN100469896C (en) * | 2007-12-01 | 2009-03-18 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
-
2010
- 2010-09-02 CN CN2010102697055A patent/CN101921865B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100436599C (en) * | 2006-05-12 | 2008-11-26 | 中国水产科学研究院黄海水产研究所 | Female-specific AFLP fragment of Cynoglossus semilaevis Gunther and PCR method for identification of genetic sex |
| CN100469896C (en) * | 2007-12-01 | 2009-03-18 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
| CN101240348A (en) * | 2008-01-23 | 2008-08-13 | 中国水产科学研究院黄海水产研究所 | Rapid identification method for half-smooth tongue-sole genetic sex |
| CN101270389A (en) * | 2008-05-09 | 2008-09-24 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis special numerator mark and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《水产养殖》 20031231 董江水等 罗非鱼的SRY基因PCR扩增分析 25-29 1-8 第24卷, 第6期 2 * |
| 《遗传》 20090131 马洪雨等 半滑舌鳎雌性特异AFLP 标记CseF783的克隆及其在遗传性别鉴定中的应用 88-94 6，8 第31卷, 第1期 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561332A (en) * | 2015-01-20 | 2015-04-29 | 黑龙江省林业科学研究所 | SSR molecular marker for identifying aspen gender and application of SSR molecular marker |
| CN104561332B (en) * | 2015-01-20 | 2016-10-05 | 黑龙江省林业科学研究所 | A kind of SSR molecular marker identifying Populus davidiana sex and application thereof |
| CN110055335A (en) * | 2018-12-14 | 2019-07-26 | 中山大学 | A kind of microsatellite molecular marker primer, kit and rapid identification method identifying the female milter of star continent red tilapia |
| CN110938626A (en) * | 2019-12-12 | 2020-03-31 | 西南大学 | Specific molecular marker of sex chromosome of Oreochromis aureus, genetic sex identification based on molecular marker and method for producing unisex fish |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101921865B (en) | 2012-09-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2012245004A5 (en) | ||
| US20140170657A1 (en) | Probe for Detection of Polymorphism in EGFR Gene, Amplification Primer, and Use Thereof | |
| Braga et al. | Five hypermethylated microRNA genes as potential markers of ovarian cancer | |
| CN101921865B (en) | Primer, fragment and method for identifying genetic sex of Oreochromis aureus | |
| CN105713990B (en) | Wheat molecular marker and its application in identification wheat yield correlated traits | |
| CN101818195B (en) | Genetic marker by taking pig miR-27a precursor flanking sequence SNP as trait of litter size of pig and application | |
| CN116732157A (en) | Universal molecular marker for sex and variety identification of snakeheads, macula maculata and hybrid snakeheads | |
| CN103667489A (en) | DNA (Deoxyribonucleic Acid) marking method for identifying domestic duck, muscovy duck and mule duck | |
| CN105368948A (en) | Primer for sex identification of Nile tilapia and PCR (polymerase chain reaction) identification method | |
| CN103525822B (en) | The molecular mark detection method that a kind of piglet diarrhea resistance is relevant and application | |
| CN108913798A (en) | The method of the freeze proof correlation CAPS label of Wheat Seedling and identification Wheat Seedling frost resistance | |
| CN104975012A (en) | Microsatellite molecular marker sites of platichthys stellatus and primers | |
| Pawlas-Opiela et al. | Molecular comparison of Gasterophilus intestinalis and Gasterophilus nasalis from two distinct areas of Poland and Italy based on cox1 sequence analysis | |
| CN102899323A (en) | Sex-linked microsatellite marker for oplegnathus fasciatus and genetic sex determination method for oplegnathus fasciatus | |
| KR101514431B1 (en) | Single Nucleotide Polymorphic DNA markers for discriminating habitat of the albino swamp eel, Monopterus albus and a method using the same | |
| Kostrzewska-Poczekaj et al. | Recurrent amplification in the 22q11 region in laryngeal squamous cell carcinoma results in overexpression of the CRKL but not the MAPK1 oncogene | |
| CN113373241B (en) | Microsatellite marker of fishes in loach, and amplification primer and application thereof | |
| CN104313164A (en) | Method for identifying cotton D genome and D sub-genome complete chromosome | |
| KR101270091B1 (en) | DNA markers for detecting increase of porcine muscle fiber type I and meat quality containing SNP in 3' UTR of PPARGC1A gene | |
| Gruszczyńska et al. | Application of chicken microsatellite markers to molecular monitoring of the experimental population of Japanese quail (Coturnix japonica). | |
| KR101508689B1 (en) | Markers for origin discrimination of the northern mauxia shrimp(Acetes chinensis) | |
| CN103642907B (en) | The tobacco reference gene utilizing gene chip to screen in conjunction with Real time PCR and method thereof | |
| Minh et al. | Association between selected microRNA SNPs and breast cancer risk in a Vietnamese population | |
| CN103181361B (en) | Method for breeding Ili horses, and kit employed in method | |
| CN106701747A (en) | Universal PCR (polymerase chain reaction) primer pair and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120919 Termination date: 20130902 |