CN101921865B - 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
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- CN101921865B CN101921865B CN2010102697055A CN201010269705A CN101921865B CN 101921865 B CN101921865 B CN 101921865B CN 2010102697055 A CN2010102697055 A CN 2010102697055A CN 201010269705 A CN201010269705 A CN 201010269705A CN 101921865 B CN101921865 B CN 101921865B
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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 (like 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 kinds of tilapias 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.Research confirms that Oreochromis aureus is a ZW sex determination type, and it possibly 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 following:
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. like 1 described primer, its sequence length is 18～28nt.
3. like 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. like 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. like 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 in female Oreochromis aureus genome, to amplify a female specific band; Having obtained a segment length through 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 present 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 preparatory 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 characteristic 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 characteristic 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) Hoxb7a gene and the Tumor-necrosis factor glycoproteins between the Hoxb6a gene of the fragment grown of 293bp and another kind of Callichthyidae (Cichlidae) fish Astatotilapia burtoni is higher altogether from 40bp to 332bp; Sequence identity is up to 93%; Its function still can not be known (SimoneHoegg etc.), and 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’
This validity of Oreochromis aureus check with 24 known sexes to primer; Characteristic band about 1500bp all appears in all tested female individuals DNA electrophoresis of PCR reaction and display, and does not all occur this characteristic 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 characteristic band about 1500bp all appears in all tested female individuals DNA electrophoresis, and this characteristic 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 preparatory sex change 5min; 35 circulations: 94 ℃ of 40sec, 62.3 ℃ of 50sec, 72 ℃ of 50sec; Then 72 ℃ are extended 7min; 4 ℃ of preservations.The agarose gel electrophoresis of PCR product warp 1.5%, EB dyeing is taken a picture, and the result is as shown in Figure 2:
Can find out from last figure; Characteristic band about 1500bp all appears in the DNA electrophoresis in all tested female individuals; And this characteristic 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 (3)
1. the primer of a pair of discriminating genetic sex of Oreochromis aureus is characterized in that sequence is the sequence shown in SEQ ID NO:2 and the SEQ ID NO:3.
One kind per sample the electrophoresis result of the PCR reaction product of DNA identify the genetic sex of Oreochromis aureus authentication method of sample sex; It is characterized in that its sequence of PCR primer that this method adopts is the sequence shown in SEQ ID NO:2 and the SEQ ID NO:3; The sample of the band that to occur a size in the PCR product electrophoresis result be 1488bp is female, and same area the sample of band not occur be male.
3. method according to claim 2 is characterized in that the PCR response procedures is 94 ℃ of preparatory sex change 5min, 94 ℃ of 40sec of 35 round-robin, 62.3 ℃ of 50sec, 72 ℃ of 50sec, and then 72 ℃ are extended 7min.
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| 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 |
| CN110055335B (en) * | 2018-12-14 | 2023-03-24 | 中山大学 | Microsatellite molecular marker primer, kit and rapid identification method for identifying female and male tilapia in stellera |
| CN110938626B (en) * | 2019-12-12 | 2023-07-18 | 西南大学 | Specific molecular marker of sex chromosome of Oreotilapia mossambica, genetic sex identification based on molecular marker and production method of unisex fish |
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| 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 |
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Patent Citations (4)
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| 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 |
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