CN103525814A - Cynoglossus semilaevis gender specific SCAR mark and application method - Google Patents
Cynoglossus semilaevis gender specific SCAR mark and application method Download PDFInfo
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Abstract
The invention first provides a Cynoglossus semilaevis gender specific microsatellite marker, and comprises a microsatellite marker located in the Z chromosome, and the nucleotide sequence is SEQ ID NO:1. The invention also provides primers designed based on the above microsatellite marker, and the sequences of the upstream primer and the downstream primer are SEQ ID NO:2 and SEQ ID NO:3 respectively. The invention screens a Cynoglossus semilaevis gender specific microsatellite marker, and the Cynoglossus semilaevis gender specific microsatellite marker is subjected to SCAR conversion. Primers marked by SCAR are designed for Cynoglossus semilaevis heredity gender identification. Female, male and superfemale individuals of Cynoglossus semilaevis can be distinguished rapidly, accurately and effectively. Because the marker has characteristics that objective straps can be amplified in females and males and the objective straps can be distinguished with agarose electrophoresis, the time for accurate identification of Cynoglossus semilaevis heredity genders is shortened obviously, the on-site batch identification of Cynoglossus semilaevis heredity genders in a culturing farm of Cynoglossus semilaevis can be carried out, the detection cost is saved, and the work efficiency and accuracy of on-site detection of Cynoglossus semilaevis heredity genders in a culturing farm are raised.
Description
Technical field
The invention belongs to fish genetic sex identification and Sex Control field in aquatic living things technology, be specifically related to the special SCAR mark of a kind of Cynoglossus semilaevis sex and the application in genetic sex Rapid identification thereof.
Background technology
Cynoglossus semilaevis (Cynoglossus semilaevis) is the large-scale ground fish of China's distinctive coastal waters warm water, is distributed in yellow, Wei Zhu China's Coastal Areas, the Bohai Sea.Cynoglossus semilaevis fine and tender taste, delicious flavour, nutritious, have wide cultivation prospect, is important famous and precious marine fish.
Production practice show, Cynoglossus semilaevis is female, milter speed of growth difference is huge, the female speed of growth be male 2-4 doubly.Cynoglossus semilaevis milter grows slowly, individual little feature, has reduced the cultured output of Cynoglossus semilaevis, has increased aquaculture cost, has restricted the Sustainable development of cynoglossus semilaevis cultivation industry.Therefore, screening Cynoglossus semilaevis sex specific mark, sets up the fast method that genetic sex is identified, carry out Cynoglossus semilaevis sex control, improving the female ratio of cynoglossus semilaevis cultivation colony, and even develop complete female seed, is the effective way that further promotes the Sustainable development of cynoglossus semilaevis cultivation industry.And the Cynoglossus semilaevis sex identification mark of practicability and effectiveness is the essential tool of realizing this goal.
Cynoglossus semilaevis sex determination system is ZZ/ZW type, and male is ZZ, and female is ZW, has special-shaped W karyomit(e) (Zhou Liqing etc., 2005).Cynoglossus semilaevis can produce Z-type sperm and Z-type, two kinds of cells of W type.Under field conditions (factors), obtain the male and ZW female offspring of normal ZZ.By artificial gynogenesis, induce, can produce ZZ milter and WW superfemale fish, in theory, superfemale fish is the prerequisite of cultivating complete female seed.Therefore, identify the quick effective means of ZZ milter, ZW raun and WW superfemale fish, there is important scientific meaning and using value.
Aspect the screening of Cynoglossus semilaevis sex specific molecular marker and genetic sex evaluation, Huanghai Sea aquatic products institute has successively screened the female specific AFLP mark of Cynoglossus semilaevis (Chen et al., 2007) and gunther sex-linked microsatellite marker (Chen et al., 2012).But in actual applications, these two kinds of marks have the deficiency of self.AFLP mark, due to its dominant hereditary property, can not be distinguished ZW female individual with WW poly-x female, and likely there will be due to false negative in reality is identified, and the male and female generation erroneous judgement of ZW to ZZ.And although codominant micro-satellite sex specific mark is can precise Identification ZZ male, ZW is female and WW poly-x female individual, but because difference between its two DNA fragmentation bands is less, cannot differentiate with agarose gel electrophoresis, can only differentiate by the method for the higher polyacrylamide gel of resolving power (PAGE glue) electrophoresis, and the operation easier of PAGE gel electrophoresis is large, the operating time is long, to experimental installation and reagent etc. require high, increased cost, the difficulty that genetic sex identifies and the time of expending, be difficult to carry out Site Detection in cynoglossus semilaevis cultivation field.Therefore, the research of Cynoglossus semilaevis sex control and high female seed produce and also need a kind of molecular engineering that can adopt agarose electrophoresis method accurately to differentiate the genetic sex Rapid identification of ,Neng plant rig-site utilization.
Summary of the invention
The object of this invention is to provide a kind of Cynoglossus semilaevis sex-specific SCAR mark and application method thereof, it is the special micro-satellite SCAR mark of a kind of Cynoglossus semilaevis sex, and set up and a kind ofly can, in cynoglossus semilaevis cultivation field by the method for agarose electrophoresis, quick and precisely identify the molecular method of Cynoglossus semilaevis ZZ milter, ZW raun and WW superfemale fish.
First the present invention provides a kind of Cynoglossus semilaevis sex special microsatellite marker, be positioned on Z chromosome, total length 169bp, its sequence is: CCTAAATGATGGATGTAGATTCTGTCCTTGTTGCCTGTTGTTCTTACTCAGACCAG GCCTGCATGGGTGTATGTGTGTGTGCGTGTGTGTGTGCGTGTGTGTGCGCGCACGT GCATGTTCTGTGCTGATGTCCTTGTATAGTCAGGCCTGGGTTTATTTTCTCTGGAT C(SEQ ID NO:1).
Above-mentioned microsatellite marker also comprises the sequence on the W karyomit(e) corresponding with SEQ ID NO:1.
The present invention also provides a pair of SCAR labeled primer, is that the microsatellite marker based on SEQ ID NO:1 designs, and can pass through female, the male specific DNA fragment of the clear resolution of agarose gel electrophoresis, identifies male and female genetic sex, and wherein upstream and downstream primer sequence is respectively:
5′-CCTAAATGATGGATGTAGATTCTGTC-3′；SEQ?ID?NO:2
5′-GATCCAGAGAAAATAAACCCAGG-3′；SEQ?ID?NO:3
By above-mentioned SCAR labeled primer, for Cynoglossus semilaevis genetic sex Rapid identification, its step comprises the extraction of Cynoglossus semilaevis genomic dna, the agarose electrophoresis of the pcr amplification of microsatellite sequence, amplified production detects.It is characterized by and in male, amplify the DNA fragmentation that a kind of size is 169bp; In female individuals, amplify the DNA fragmentation that two kinds of sizes are respectively 169bp and 134bp; In poly-x female individuality, amplify the DNA fragmentation that a kind of size is 134bp.
The present invention screens the special microsatellite marker of a kind of Cynoglossus semilaevis sex, according to the difference of its 35bp on Z chromosome and W karyomit(e), design SCAR labeled primer carries out Cynoglossus semilaevis genetic sex evaluation, and quick, accurate and effective differentiation Cynoglossus semilaevis is female, male and poly-x female is individual.Because this is marked at the feature that all can amplify object band and can differentiate by agarose electrophoresis in male and female, obviously shortened the time of precise Identification Cynoglossus semilaevis genetic sex, can carry out at scene, cynoglossus semilaevis cultivation field the mass of Cynoglossus semilaevis genetic sex identifies, save testing cost, and improved working efficiency and the operability of Liao plant Site Detection Cynoglossus semilaevis genetic sex.The present invention is to the evaluation of Cynoglossus semilaevis superfemale fish and screening, the evaluation of pseudo-milter and large-scale production and the cultivation of screening and high female cultured population, for improving significant and industry using values such as cultured output and economic benefit, promotion cynoglossus semilaevis cultivation industry sustainable and healthy development etc.
Accompanying drawing explanation
Fig. 1: the present invention 1% agarose concentration electrophoresis result figure, 1-8 swimming lane is respectively Marker, ZW female individuals * 3, ZZ male * 3, Marker;
Fig. 2: the present invention 2% agarose concentration electrophoresis result figure, 1-8 swimming lane is respectively Marker, ZW female individuals * 3, ZZ male * 3, Marker;
Fig. 3: the present invention 4% agarose concentration electrophoresis result figure, 1-8 swimming lane is respectively Marker, ZW female individuals * 3, ZZ male * 3, Marker;
Fig. 4: the actual effect figure of the present invention in sex identification, 1-14 swimming lane is respectively Marker, ZW female individuals * 6, ZZ male * 6, Marker;
Fig. 5: the application of the present invention in poly-x female Individual identification, 1-14 swimming lane is respectively Marker, WW type poly-x female individuality * 4, ZW type female individuals * 4, ZZ type male * 4, Marker;
Wherein the swimming lane of electrophoresis picture is all arranged from left to right.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
One, the screening of the special microsatellite marker of Cynoglossus semilaevis sex
The present invention's microsatellite marker sequence used derives from the Cynoglossus semilaevis genome sequencing that Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science's aquatic products genome and cell engineering research department complete, 159 special microsatellite markers of sex that screen from 3000 microsatellite locus subsequently.Wherein contain 122 Type Type B sites (a female band, male not band) and 37 Type A type sites (female two bands, a male band).
Each 4, female, the male Cynoglossus semilaevis DNA sample of the known sex identified of learning from else's experience, adopts polyacrylamide gel electrophoresises to carry out gene type to 37 Type A type sites, to differentiate male and female band difference.The primer that one of them A phenotypic marker is corresponding is 5 '-GTTGTGACCTTGTCTCTCAAAGTGT-3 ' (SEQ ID NO:4) and 5 '-GCACAGCTGTAACCTTCGAGC-3 ' (SEQ ID NO:5) primer, from the result of polyacrylamide gel electrophoresis, can find out, the male and female difference of this microsatellite marker is maximum.
Two, the design of the SCAR of the special microsatellite marker of Cynoglossus semilaevis sex conversion and SCAR primer
Electrophoretic separation and the recovery of the special microsatellite marker of 1 Cynoglossus semilaevis sex: the Cynoglossus semilaevis of getting known sex is female, milter DNA, and the SEQ ID NO:4,5 of take is primer, carries out 50 μ L reaction system PCR, wherein distilled water 33.7 μ L; DNTP2.7 μ L; 10 * Buffer5.4 μ L; RTap enzyme 0.5 μ L; Each 2 μ L of upstream and downstream primer; Template DNA 3.7 μ L.Vortex, centrifugal it is fully mixed, carries out pcr amplification, amplification program be 95 ℃ 10 minutes, carry out subsequently sex change (95 ℃ 30 seconds), renaturation (57.5 ℃ 30 seconds), extend (72 ℃ 30 seconds) 35 circulations, 72 ℃ are extended 10 minutes, 4 ℃ of preservations.Amplified production carries out 1% agarose electrophoresis, and object band is cut with blade, uses glue to reclaim test kit and reclaims.
The clone of 2 selection marquee male and female bands
The PCR product of recovery is connected to pMD18-T carrier, is converted in competent cell, picking positive colony after cultivating, delivers to the order-checking of order-checking company.According to sequencing result, obtained the special SCAR flag sequence of Z chromosome, the large 35bp of corresponding fragment than it on W karyomit(e).
The design of 3.SCAR labeled primer
By separation, recovery and the order-checking of microsatellite marker, thereby the special microsatellite marker of selected sex is converted into SCAR mark, and then has designed primer SEQ ID NO:2 and the SEQ ID NO:3 of SCAR mark.Adopt SEQ ID NO:2 and SEQ ID NO:3 primer from ZZ milter genomic dna, to amplify the DNA band of 169bp, amplify 2 DNA bands of 169bp and 134bp from ZW raun genomic dna, wherein the DNA band of 134bp is positioned on W karyomit(e).
The foundation of 4.PCR amplified production deposition condition
Use the SCAR labeled primer that sequence is SEQ ID NO:2 and SEQ ID NO:3 to carry out pcr amplification, reaction system 15 μ L, wherein 10 * buffer1.5 μ L, dNTP0.8 μ L, each 0.6 μ L of upstream and downstream primer, template DNA 1 μ L, ddH
2o11.3 μ L, finally adds rTaq enzyme 0.2 μ L.Response procedures be 95 ℃ 10 minutes, 35 circulations are extended in 95 ℃ of sex change, 54 ℃ of renaturation, 72 ℃, 72 ℃ 10 minutes, 4 ℃ of preservations.
Known larger agarose concentration is easier to differentiate the minute differences of small pieces segment DNA.Under same deposition condition (150V, 15min), agarose concentration gradient is set, grope to differentiate the suitable agarose concentration of 35bp difference DNA fragmentation.To grope gradient and be made as 1%, 2% and 4%, applied sample amount is 10 μ L, and result as shown in Figure 1, Figure 2, Figure 3 shows.Empirical tests, under 4% concentration, can two kinds of DNA fragmentations of clear differentiation, and then differentiate genetic sex.
Three, the application of SCAR mark
1. under laboratory condition, Cynoglossus semilaevis genetic sex is identified
Use SCAR labeled primer and ordinary method, detect Cynoglossus semilaevis genetic sex.PCR reaction system 15 μ L, wherein each 0.6 μ L, template DNA 1 μ L(50ng/ μ L of 10 * buffer1.5 μ L, dNTP0.8 μ L, upstream and downstream primer), ddH
2o11.3 μ L, finally adds rTaq enzyme 0.2 μ L.Response procedures be 95 ℃ 10 minutes, 35 circulations are extended in 95 ℃ of sex change, 54 ℃ of renaturation, 72 ℃, 72 ℃ 10 minutes, 4 ℃ of preservations.
Add 6 * Loading Buffer3 μ L, 4% agarose electrophoresis, 150V, 15 minutes, records qualification result under gel imaging instrument.Can female 169bp and the two kinds of DNA bands of 134bp of containing of clear explanation ZW, ZZ is female only 169bp band, as shown in Figure 4.
The evaluation of 2.WW poly-x female individuality
Authentication method is identical with the conventional sex appraisal method in above-mentioned laboratory, and acquired results poly-x female individuality only can obtain the DNA band of 134bp.As shown in Figure 5.
3. the on-the-spot sex identification under plant's condition
For obtaining the high female ratio cultured population of Cynoglossus semilaevis, in parent fish rearing and seed breeding process, reject the pseudo-male parent population of ZW type, filter out the male parent population of ZZ type high-quality, most important for the physiology raun ratio improving in semi-smooth tongue sole offspring breed.And in screening process, require fast, accurately, corresponding one by one, to guarantee when identifying high-quality milter, reduce as far as possible the damage to parent population.The genetic sex evaluation work of this fish of will seeking a marriage alliance must be carried out at scene, cynoglossus semilaevis cultivation field.
3.1 the preparation of situ appraisal
Preparing experiment instrument PCR instrument, whizzer, electrophoresis apparatus, electrophoresis chamber, water-bath, potentiostat, microwave oven, gel imaging instrument and test required all ingredients, instrument, consumptive material.In plant, arrange laboratory.
The collection of 3.2 parent population fin rays to be identified
Centrifuge tube and the two numbering of label of being ready in advance numbering are corresponding one by one.At parent population cultivating workshop, the fin ray of a small amount of parent population to be identified of collection in worksite, is positioned in numbering centrifuge tube one by one.The parent population of adopting fin ray is put into cylindrical plastic bag, add seawater and and oxygen, the two ratio is 1:2, sticks the label corresponding with centrifuge tube after sealing, is placed in temporary transient preservation the in the shady and cool place of lucifuge.
The extraction of 3.3 genomic dnas
In each is equipped with the centrifuge tube of fin ray, add 500 μ L lysates and 15 μ L Proteinase Ks, 55 ℃ of water-bath cracking 1 hour, during jog for several times to accelerate cracking.Cracked centrifuge tube is taken out, add 500 μ L phenol: chloroform: primary isoamyl alcohol, put upside down jog 10min, 12000 leave heart 10min, getting supernatant liquor 450 μ L adds and is ready in advance reference numeral and is equipped with in the centrifuge tube of 500 μ L alcohol, jog 30 times, 12000 leave heart 5min, and visible white DNA is deposited in centrifuge tube bottom.Outwell the liquid in centrifuge tube, DNA precipitation is dried, add 100 μ LddH
2o, vortex makes for twice DNA fully dissolve.
The pcr amplification of 3.4 parent population DNA to be measured and detection
Adopt sex identification SCAR mark to carry out pcr amplification to the genomic dna extracting, PCR reaction system is 15 μ L, comprises 10 * buffer1.5 μ L, dNTP0.8 μ L, each 0.6 μ L of upstream and downstream primer, template DNA 1 μ L, ddH
2o11.3 μ L, finally adds rTaq enzyme 0.2 μ L.Response procedures be 95 ℃ 10 minutes, 35 circulations are extended in 95 ℃ of sex change, 54 ℃ of renaturation, 72 ℃, 72 ℃ cooling after 10 minutes.
Add 6 * Loading Buffer3 μ L, 4% agarose electrophoresis, 150V 15 minutes, observes under gel imaging instrument.A record corresponding reaction result of numbering, occurs that the fish of 1 DNA band is the male parent population of ZZ type high-quality, can be used for breeding of high female ratio seed, and the fish that occurs two DNA bands is the pseudo-milter of ZW type, should eliminate.
According to gained qualification result, by temporary transient parent population classification of preserving in workshop, and put back in corresponding culturing pool.About 6 hours consuming time of whole process, can guarantee to detect survival and the ensuing normal breeding of parent population.
SCAR labeled primer of the present invention screens for the male parent population of Cynoglossus semilaevis high-quality in units such as Haiyang Shandong Huanghai Sea aquatic products company limited and Changyi, Shandong three new aquatic products Miao Ye testing ground, thereby by the male parent population of screening high-quality, the physiology raun ratio of semi-smooth tongue sole offspring breed has been improved to 20% left and right, produced significant economic and social benefit.
Claims (6)
1. the special microsatellite marker of Cynoglossus semilaevis sex, is characterized in that, the nucleotides sequence of described microsatellite marker is classified SEQ ID NO:1 as.
2. the application of microsatellite marker claimed in claim 1 in detecting Cynoglossus semilaevis genetic sex.
3. for detection of a SCAR labeled primer for Cynoglossus semilaevis genetic sex, described SCAR labeled primer is that Accessory Right requires to design on the microsatellite marker described in 1.
4. SCAR labeled primer as claimed in claim 3, is characterized in that, the upstream and downstream primer sequence of described primer is respectively SEQ ID NO:2, SEQ ID NO:3.
5. the application of primer as claimed in claim 4 in identifying Cynoglossus semilaevis genetic sex.
6. application as claimed in claim 5, is characterized in that, described primer amplifies the DNA fragmentation that a kind of size is 169bp in male; In female individuals, amplify 2 DNA fragmentations that size is respectively 169bp and 134bp; In poly-x female individuality, amplify the DNA fragmentation that a kind of size is 134bp.
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| CN109136388A (en) * | 2018-09-03 | 2019-01-04 | 天津渤海水产研究所 | The microRNA label of Cynoglossus semilaevis differential expression and application |
| CN109536624A (en) * | 2019-01-22 | 2019-03-29 | 天津渤海水产研究所 | For screening the fluorescent molecule tagging and testing method of Cynoglossus semilaevis true and false milter property |
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| CN106011266B (en) * | 2016-06-30 | 2019-05-28 | 福建农林大学 | Distinguish the specific primer and method of the microsatellite marker of water coconut palm illiciumverum armor gender |
| CN109055520A (en) * | 2018-09-03 | 2018-12-21 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis excretion body Sexual differentially expressed label and kit |
| CN109136388A (en) * | 2018-09-03 | 2019-01-04 | 天津渤海水产研究所 | The microRNA label of Cynoglossus semilaevis differential expression and application |
| CN109136388B (en) * | 2018-09-03 | 2021-05-14 | 天津渤海水产研究所 | MicroRNA label of cynoglossus semilaevis differential expression and application |
| CN109536624A (en) * | 2019-01-22 | 2019-03-29 | 天津渤海水产研究所 | For screening the fluorescent molecule tagging and testing method of Cynoglossus semilaevis true and false milter property |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
| CN109536624B (en) * | 2019-01-22 | 2021-09-28 | 天津渤海水产研究所 | Fluorescent molecular marker and detection method for discriminating true and false male fish of cynoglossus semilaevis |
| CN109825565B (en) * | 2019-01-22 | 2022-04-15 | 天津渤海水产研究所 | Cynoglossus semilaevis true and false male fish screening method based on fluorescent molecular marker system |
| CN111471774A (en) * | 2019-01-23 | 2020-07-31 | 安徽微分基因科技有限公司 | Codominant long INDE L molecular marker for sex determination of cynoglossus semilaevis and method |
| CN111471774B (en) * | 2019-01-23 | 2023-03-14 | 安徽微分基因科技有限公司 | Co-dominant long INDEL molecular marker for sex discrimination of cynoglossus semilaevis and method |
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| C06 | Publication | ||
| PB01 | Publication | ||
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