CN101914529B - Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method - Google Patents
Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method Download PDFInfo
- Publication number
- CN101914529B CN101914529B CN2010102397296A CN201010239729A CN101914529B CN 101914529 B CN101914529 B CN 101914529B CN 2010102397296 A CN2010102397296 A CN 2010102397296A CN 201010239729 A CN201010239729 A CN 201010239729A CN 101914529 B CN101914529 B CN 101914529B
- Authority
- CN
- China
- Prior art keywords
- sex
- cynoglossus semilaevis
- seq
- female
- pcr
- 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.)
- Expired - Fee Related
Links
Images
Abstract
The invention relates to a cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method comprising a PCR method and a PCR testing method, wherein the PCR method is used for screening and testing of a cynoglossus semilaevis gunther sex-linked microsatellite marker, clone of female peculiar allelic gene, sequence analysis, peculiar primer design and cynoglossus semilaevis gunther genetics sex; and the PCR testing method is used for creating the cynoglossus semilaevis gunther genetics sex according to the obtained cynoglossus semilaevis gunther sex-linked microsatellite marker primer and female peculiar allelic gene. The invention ensures that the cynoglossus semilaevis gunther sex-linked microsatellite marker and the female peculiar allelic gene are screened for the first time, establishes the PCR technique tested by the cynoglossus semilaevis gunther genetics sex, has the characteristics of high efficiency, accuracy and stability, has important application value in the cynoglossus semilaevis gunther sex control and unisexuality seed production, and also has important application prospect in the sex determination gene research by cloning the cynoglossus semilaevis gunther.
Description
Technical field:
The invention belongs to the seawater fish genetic sex molecular markers for identification technology in the aquatic living things technical field, is a kind of and chain microsatellite marker and the genetic sex identification method of Cynoglossus semilaevis genetic sex.
Background technology:
Cynoglossus semilaevis (Cynoglossus semilaevis) belongs to Pleuronectiformes (P1euronectiformes), sole suborder (Soleoidei), Cynoglossidae (Cynoglossidae), tongue sole genus (Cynoglossus), three-way tongue sole subgenus (Arelia); Be the large-scale fishes in bothid and true plaice of shallow sea, warm temperate zone bottom; Distribute at most in the yellow Bohai Sea; And in the East Sea, the South Sea distributes lessly, is the seawater fishes in bothid and true plaice that has important breed prospect at present.
(Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science such as Zhou Liqing; Aquatic product journal, 2005,29:417-419) karyotype of Cynoglossus semilaevis is studied; The result shows that the diploid chromosome number order is 42; 2n=42, and in the metacinesis mutually of raun, have atypia karyomit(e), and in milter, do not have atypia karyomit(e).Further research based on karyomit(e) C-band and the analysis of G-band shows; Cynoglossus semilaevis is made up of 20 pairs of euchromosomes and 1 pair of sex chromosome (being respectively Z and W karyomit(e)), and Cynoglossus semilaevis belongs to ZW/ZZ sex determination system, female different joining; Male with joining (Wu et al.; Progress inNatural Sciences, 2006,16:29-32; Zhuang et al., Journal of Applied Ichthyology, 2006,22:437-440).Sophisticated Cynoglossus semilaevis female individuals is than the big 2-4 of male times, and both speeds of growth also differ 2-4 doubly, and the female individuals speed of growth is faster.Therefore, actively develop the research of chain molecule marker of Cynoglossus semilaevis sex or gene, find molecule marker or the gene chain, will have important significance for theories and using value Cynoglossus semilaevis sex controll and complete female seed rearing with sex.
At present, multinational scientist utilizes some molecular marking techniques based on full genome scanning theory, like AFLP, RAPD, RFLP etc., has screened the molecule marker relevant with sex of some economic fishs, but these are marked between kind and do not have versatility.(Marine Biotechnology such as the old pine forest of Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science; 2007; 9:273-280) be separated to the female specific AFLP mark of Cynoglossus semilaevis, change into the genetic sex that to identify Cynoglossus semilaevis behind the SCAR mark through simple PCR method through the AFLP labeling technique.But there is a bigger defective in the AFLP mark, can't distinguish homozygote and heterozygote exactly, is a kind of molecule marker of dominant inheritance.And microsatellite marker is the STR by 1-6 based composition, has the height polymorphum, and meets the Mendelian inheritance law of segregation, presents the characteristics of codominant inheritance, can distinguish homozygote and heterozygote.The flanking sequence of little satellite Tumor-necrosis factor glycoproteins is generally more conservative, generally in nearly edge species, also can stride kind of an amplification.If therefore can screen the chain microsatellite marker of Cynoglossus semilaevis sex, not only can set up the genetic sex identification method of precise and high efficiency, also will help to carry out the Study on Evolution of Cynoglossus semilaevis sex genes involved and the screening of WW superfemale fish.And relevant Cynoglossus semilaevis sex is chain or the screening study of special microsatellite marker, does not see any document or patent report so far both at home and abroad as yet.
Summary of the invention:
The purpose of this invention is to provide a microsatellite marker and a genetic sex identification method that the Cynoglossus semilaevis sex is chain, for the screening of Cynoglossus semilaevis sex determining gene, sex controll and complete female seed rearing provide a kind of technique means.
The present invention realizes by following technical scheme: the present invention includes three aspects: one, the screening of the chain microsatellite marker of Cynoglossus semilaevis sex and primer sequence thereof; Two, the sequence of the female special microsatellite allele of Cynoglossus semilaevis and primer sequence thereof; Three, based on the Cynoglossus semilaevis genetic sex authentication method of microsatellite marker.
One, chain microsatellite marker of Cynoglossus semilaevis sex and primer sequence thereof: its technology contents comprises: 1, the screening of microsatellite sequence and clone; 2, the design of micro-satellite primers; 3, the checking of the chain microsatellite marker of sex.
1, the screening of microsatellite sequence and clone:
Extract the Cynoglossus semilaevis genomic dna, at first genomic dna is carried out restriction enzyme digestion, cut dna fragmentation with the T4 ligase enzyme to enzyme again and add top connection.Connect product as the pcr amplification template, and carry out PCR with the joint Auele Specific Primer and increase in advance.Amplified fragments and biotin labeled oligonucleotide probe are hybridized, and with the dna molecular of magnetic bead selective adsorption and biotinylated probe hybridization.Remove not and probe bonded dna fragmentation through 3 non-preciseness and 3 preciseness wash-outs at ambient temperature then, and magnetic bead-probe-DNA mixture is separated from damping fluid with magnet.Then add TE solution in the magnetic bead and 95 ℃ of water-bath sex change 5 minutes, with the fixing quick sucking-off supernatant behind the magnetic bead of magnet.When masterplate carries out pcr amplification, with pMD18-T carrier (Takara) at 16 ℃ be connected two hour after amplified production is purified with supernatant.Join and clone in the competent cell and smoothen on a LB flat board that contains acillin connecting product, after liquid-absorbent finishes, be inverted overnight cultures for 37 ℃.Select the positive colony of white and deliver to the order-checking of biological order-checking company.
2, the design of micro-satellite primers:, check wherein whether contain 1-6 base multiple unit according to the sequence that order-checking obtains.Choose the sequence that contains repeating unit, with the flanking sequence design primer of primer-design software to repeating unit.
3, the checking of the chain microsatellite marker of sex: at first every pair of micro-satellite primers is carried out pcr amplification with each 16 tail Cynoglossus semilaevis male and female individuals genome DNA sample; Amplified production is electrophoresis in 6% denaturing polyacrylamide gel; And dye through silver staining method, then relatively the distribution of microsatellite allele in the male and female individuality whether there were significant differences.It is SEQID NO:1 (tgaactgcaa gactgctcca) and SEQ ID NO:2 (catcagttgg tggcctgtaa) microsatellite marker and Cynoglossus semilaevis sex close linkage that finishing screen is chosen a primer sequence.Further use sexually matured Cynoglossus semilaevis 22 tail rauns of primer amplification and the 22 tail milter genomic dnas of sequence SEQ ID NO:1 and SEQID NO:2 then, amplify 2 allelotrope altogether, according to size order difference called after A, B allelotrope.According to comparing with molecular weight standard, A allelotrope size is about 244bp, and the allelic size of B is about 234bp.Wherein 22 tail milters and 22 tail rauns all amplify A allelotrope, and all 22 tail rauns all amplify B allelotrope, and all 22 tail milters all fail to amplify B allelotrope.Therefore B allelotrope is the female special allelotrope of Cynoglossus semilaevis.
Two, female special allelotrope sequence and primer sequence thereof of Cynoglossus semilaevis: its technology contents comprises: 1, female special allelic clone and order-checking; 2, female special allelic design of primers; 3, the checking of female specific mark.
1, female special allelic clone and order-checking:
In order to obtain female special allelotrope sequence; With the primer PCR of SEQ ID NO:1 (tgaactgcaagactgctcca) and SEQ ID NO:2 (catcagttgg tggcctgtaa) the sexually matured Cynoglossus semilaevis raun of the 1 tail DNA that increases; Agarose gel electrophoresis with 1.5% separates the PCR product; Downcut PCR product target electrophoretic band, with DNA purification kit purifying.Purified product is connected to pMD 18-T plasmid with the T4 dna ligase, and transformed competence colibacillus intestinal bacteria then, and smoothening on a LB flat board that contains acillin after liquid-absorbent finishes, are inverted overnight cultures for 37 ℃.Select 8 positive colonies and deliver to the order-checking of biological order-checking company.
2, female special allelic design of primers: according to sequencing result; 8 positive colonies check order successfully; Obtain 8 sequences altogether, wherein size is 6 of the sequences of 244bp, and size is 2 of 234bp sequences; Difference corresponding A, two allelotrope of B, B allelotrope is the female special allelotrope of Cynoglossus semilaevis.According to A, the allelic sequence alignment result of B, except there is the disappearance of 2 GT the iteron, with respect to A allelotrope, B allelotrope (being female special allelotrope) has the disappearance of 6 bases at 5 ' end.Therefore with the position of primer-design software to 6 bases of disappearance; Designed a special primer of SEQ ID NO:3 (tctgcatgac attggaaag); The primer of this primer and SEQ ID NO:2 (catcagttgg tggcctgtaa) the pairing size that is used to increase be the female specific DNA fragment of Cynoglossus semilaevis of 204bp, carries out the Cynoglossus semilaevis genetic sex detection.
Female special allelotrope (size is the B allelotrope of 234bp) sequence is SEQ ID NO:4, and its sequence is following:
tgaactgcaa?gactgctcca?gggcagtgtc?tctgcatgac?attggaaagc?tgtgtgtgga
tgcatgtgtg?tgtgcgtgta?atgttctcca?gctatggcct?acgctggctg?cagcctggag
ccgctctgtg?gtgtggaaaa?gagagcagat?gcctcttcac?tggagtgcat?caggccacca
aatcgcaaat?atcactgatc?atctcccaat?tcaattacag?gccaccaact?gatg。
3, the checking of female specific mark: the primer with SEQ ID NO:2 (catcagttgg tggcctgtaa) and SEQ ID NO:3 (tctgcatgac attggaaag) carries out pcr amplification to each 10 tail Cynoglossus semilaevis is male with the female individuals genome DNA sample; Amplified production is electrophoresis in 1.5% sepharose, EB dyeing.The result shows, all can amplify size in all female individuals and be the dna fragmentation of 204bp, and not have amplified production in the male, and this shows the genetic sex that can accurately differentiate Cynoglossus semilaevis through the molecule marker of female special allelotrope conversion.
Three, the application method of identifying based on the Cynoglossus semilaevis genetic sex of microsatellite marker: can identify: 1, according to the primer PCR amplification Cynoglossus semilaevis genomic dna of sequence SEQ ID NO:1 and SEQ ID NO:2 according to the incompatible Cynoglossus semilaevis genetic sex that carries out of following 2 kinds of primer sets; Amplified production is electrophoresis in 6% denaturing polyacrylamide gel; And dye through silver staining method; Analytical electrophoresis collection of illustrative plates then: amplify A, the allelic individuality of B and must be heredity and go up and be female individuality, only amplify the allelic individuality of A and must be heredity and go up and be male individuality.2, according to sequence be the primer PCR amplification Cynoglossus semilaevis genomic dna of SEQ ID NO:2 and SEQ ID NO:3; Amplified production is electrophoresis in 1.5% sepharose; EB dyeing; Analytical electrophoresis collection of illustrative plates then: amplify size for the dna fragmentation of 204bp must be that heredity is gone up and is female individuality, and the individuality that does not have an amplified production must to be heredity go up is male individuality.
The present invention and prior art contrast are characterized in:
Screened the chain microsatellite marker of Cynoglossus semilaevis sex first; Found female special allelotrope; And obtained this special allelic gene order, and designed to this allelic special primer, set up genetic sex identification method based on micro-satellite labeling technique.The present invention has efficient, accurate and stable characteristics, in Cynoglossus semilaevis sex controll and the production of unisexuality seed, has significant application value, in the research of clone's Cynoglossus semilaevis sex determining gene, also has the important application prospect simultaneously.
Description of drawings:
Fig. 1, with the chain microsatellite marker primer SEQ ID NO:1 of sex with SEQ ID NO:2 amplification Cynoglossus semilaevis is female and the electrophoretogram of male:
1-22: Cynoglossus semilaevis female individuals, 23-44: Cynoglossus semilaevis male; A: the allelotrope that Cynoglossus semilaevis male and female individuality can both amplify; B: the female special allelotrope of Cynoglossus semilaevis; M: molecular weight standard.
The electrophoretogram of Fig. 2, and male female with female special allelic primer SEQ ID NO:2 and SEQ ID NO:3 amplification Cynoglossus semilaevis.
1-10: Cynoglossus semilaevis female individuals, 11-20: Cynoglossus semilaevis male; M: molecular weight standard
Embodiment:
Below through embodiment and combine accompanying drawing that technology contents of the present invention is described in detail:
The present invention includes three aspects: one, the screening of the chain microsatellite marker of Cynoglossus semilaevis sex and primer sequence thereof; Two, the sequence of the female special microsatellite allele of Cynoglossus semilaevis and primer sequence thereof; Three, the application method of identifying based on the Cynoglossus semilaevis genetic sex of microsatellite marker.
One, chain microsatellite marker of Cynoglossus semilaevis sex and primer sequence thereof: comprising: 1, the screening of microsatellite sequence and clone; 2, the design of micro-satellite primers; 3, the checking of the chain microsatellite marker of sex.
1, the screening of microsatellite sequence and clone:
Choose the about 5mm of fin ray of 1 tail Cynoglossus semilaevis
2Put into the 1.5mL centrifuge tube, treat that ethanol volatilization back shreds it with clean surgical scissors.Add 700 μ L extraction buffers (damping fluid component: 10mmol/LTris-HCl, pH=8.0,5mmol/L EDTA, 0.5%SDS) and 5 μ L 1mg/mL Proteinase Ks, 55 ℃ of abundant digestion 4-6h.The sample that the digestion of cool to room temperature is good adds isopyknic phenol-chloroform-primary isoamyl alcohol (phenol: chloroform: primary isoamyl alcohol is 25: 24: 1) extracting twice, the 10min that at every turn vibrates gently, centrifugal 10min in 4 ℃ of refrigerated centrifuges of 10000rpm.Adopt the suction nozzle gentle aspiration supernatant of clip, change in the new centrifuge tube.Adding 6 μ L concentration is that 25mg/mL RNaseA digests 2-3h in 37 ℃ of water-baths.Repeat a phenol-chloroform extracting then.In supernatant, add isopyknic chloroform (chloroform: primary isoamyl alcohol is 24: 1) extracting 1 time again.The 10min that vibrates gently, centrifugal 10min in 4 ℃ of refrigerated centrifuges of 10000rpm.Adopt the suction nozzle gentle aspiration supernatant of clip.The absolute ethyl alcohol deposit D NA that in supernatant, adds-20 ℃ of precoolings of 2 times of volumes then.The DNA deposition is chosen with suction nozzle, the washing with alcohol twice with 70%, dry under the room temperature, use 100 μ L TE damping fluids (10mmol/LTris-HCl, 1mmol/L EDTA, pH=8.0) dissolving DNA deposition then.The centrifuge tube of about 100ng Cynoglossus semilaevis genome DNA of extracting being put into the restriction endonuclease MseI (New England Biolabs) that contains 5U carries out endonuclease reaction.Endonuclease reaction mixture TV is 25 μ L, is that temperature is bathed 2h in 37 ℃ the steady circulation appearance of constant temperature heat in water temperature.Endonuclease reaction product and the MseI AFLP joint (5 '-TACTCAGGACTCAT-3 '/5 '-GACGATGAGTCCTGAG-3 ') of getting 10 μ L are the ligation (15 ℃ bath temperatures) of carrying out 6h in the 30 μ L reaction mixtures at TV, wherein add the T4 dna ligase (TaKaRa) of 10U.Utilize the connection product to carry out the PCR reaction as masterplate; Contain the 5 μ L enzymes of dilution after 10 times in the PCR reaction mixture of 25 μ L and cut-connect the MseI-N primer that product, final concentration are 0.4 μ M (5 '-GATGAGTCCTGAGTAAN-3 '), 1 * PCR damping fluid (Biostar contains Mg2+), the dNTP of 0.2 μ M and the Taq polysaccharase (Takara) of 1U.PCR is reflected on the MJ Research PTC-200 PCR appearance and carries out.Reaction conditions is: 94 ℃ of preparatory sex change 5min at first, and then at 94 ℃ of sex change 30s, 53 ℃ of annealing 1min, 72 ℃ are extended 1min and carry out 17 circulations, and last 72 ℃ are extended 10min eventually.Biotinylated probes (GT) 13 hybridization that preparatory amplified production and the 5 μ L concentration of 25 μ L behind PCR product purification test kit purifying are 20mM.Hybridization buffer is 6 * SSC+0.1%SDS, and the reaction TV is 100 μ L.Hybridization conditions is: 95 ℃ of following sex change 5min, hybridize 60min down at 55 ℃ then, and hybridization carries out in the PCR appearance.The hybridization product that is cooled to room temperature adds the TEN100 of 300 μ l (100mM NaCl, pH 7.5 for 10mM Tris-HCl, 1mMEDTA) solution.The magnetic bead that is coated with the affine mycin of chain of 1-2mg at room temperature washs 3 times with 300 μ L TEN100, then with PCR product-biotinylated probes complex body bulk crossing 30min at room temperature, needs frequently stirring gently and piping and druming therebetween, to avoid the magnetic bead deposition.With the fixing magnetic bead of magnet, remove the hybridization mixed solution.At first carry out non-preciseness wash-out 3 times: use 400 μ L damping fluid TEN1000 (10mM Tris-HCl; 1mM EDTA, 1M NaCl, pH 7.5) washing PCR product-biotinylated probes under room temperature-magnetic bead mixture 3 times; Each 5 minutes, stirring frequently or blow and beat with pipettor gently.And then carry out 3 times the preciseness wash-out then: use 400 μ L, 0.2 * SSC+0.1%SDS under room temperature, to wash 3 times, each 5 minutes, stirring frequently or blow and beat with pipettor gently.All need use fixedly magnetic bead of magnet after each washing, siphon away supernatant with pipettor and throw away.In centrifuge tube, add 50 μ L TE (pH=8.0), inhale with pipettor and beat evenly, then in 95 ℃ of following water-baths 5 minutes.Stir or blow and beat evenly gently with pipettor frequently during this time.Fixedly behind the magnetic bead, the sucking-off supernatant is used for pcr amplification behind DNA purification kit purifying rapidly with magnet.5 μ L target DNA wash-out fragments behind the purifying are used for pcr amplification, and amplification condition is consistent with the preparatory amplification condition of top PCR.The purified back of PCR product is connected two hours with pMD18-T carrier (Takara) at 16 ℃.In-70 ℃ refrigerator, take out a pipe TG1 competent cell, be positioned in the ice bath immediately.Add plasmid in the competent cell and connect mixed solution 2 μ L, be positioned over 90s in 42 ℃ of water-baths behind the ice bath 30min, then ice bath 2min.Add liquid nutrient medium at last to 1mL, in 37 ℃ shaking table with speed recovery 60min less than 225 rev/mins.With the centrifugal 1min sedimentation cell of the speed of 10000rpm, only stay 200 μ L substratum with re-suspended cell.Add IPTG and X-Gal, mix.It is dull and stereotyped that per 100 μ L suspension cell liquid smoothen a LB who contains acillin, after liquid-absorbent finishes, is inverted overnight cultures for 37 ℃.The clone who selects white places the centrifuge tube of the 0.5mL LB liquid nutrient medium that contains acillin, and 3-4h is cultivated in 37 ℃ of vibrations (300rpm).The clone is through the method screening of PCR.The TV of PCR reaction is 12.5 μ L, includes 1 μ L culture as template DNA, the primer of 0.2 μ M (MseI-N primer or M13F/R order-checking universal primer), the Taq enzyme of 0.5U, the dNTP of 0.1 μ M, 1 * PCR damping fluid.Except that cycle index is 35 times, other PCR reaction conditions is with top consistent.The positive colony bacterium liquid that screens is transferred to the big genome company of Beijing China and is checked order with 3730 sequenators.
2, the design of micro-satellite primers: with ' Tandem Repeats Finder ' software is searched Tumor-necrosis factor glycoproteins, and is aided with hand inspection and checks.According to the microsatellite DNA flanking sequence, utilize online primer-design software ' Primer 3 ' design primer.
3, the checking of the chain microsatellite marker of sex: at first every pair of micro-satellite primers is carried out pcr amplification with each 16 tail Cynoglossus semilaevis male and female individuals genome DNA sample; Little satellite amplification PCR reaction conditions is following: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s; At 55-60 ℃ of annealing 40s, 72 ℃ are extended 40s, carry out extending 10min eventually after 38 circulations.Amplification reaction system is 12.5 μ L, comprises the total DNA of about 20ng, primer 0.2 μ mol/L, 0.1mmol/L dNTP, 0.5U Taq polysaccharase, 1 * PCR damping fluid.Pcr amplification product carries out electrophoresis on 6% denaturing polyacrylamide gel, silver dyes and shows band.It is following that silver dyes step: 1. fixing: the sheet glass that is stained with gel is immersed in about 10min in 1% acetic acid aqueous solution; 2. rinsing: change sheet glass over to and rinse acetate in the distilled water; 3. dyeing: in staining fluid (0.1% Silver Nitrate, 0.06% Superlysoform), soak 30min; 4. rinsing once more: the about 5s of rinsing in distilled water; 5. colour developing: the gel glass plate with rinsing changes colour developing liquid (0.5-1.0% sodium hydroxide, 0.06% Superlysoform) over to immediately, and is rocking on the shaking table till showing band; 6. color development stopping: change sheet glass in the 1% acetate stationary liquid over to the color development stopping reaction.Dry then and preserve and take a picture with digital camera.Relatively whether there were significant differences in the distribution of microsatellite allele in the male and female individuality.Finishing screen is chosen microsatellite marker and the Cynoglossus semilaevis sex close linkage that primer sequence is SEQ ID NO:1 and SEQ ID NO:2.Further then sexually matured Cynoglossus semilaevis 22 tail rauns of pcr amplification and 22 tail milter genomic dnas; The PCR reaction conditions is following: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s are at 61.5 ℃ of annealing 40s; 72 ℃ are extended 40s, carry out extending 10min eventually after 38 circulations.Amplification reaction system is 12.5 μ L, comprises the total DNA of about 20ng, primer 0.2 μ mol/L, 0.1mmol/L dNTP, 0.5U Taq polysaccharase, 1 * PCR damping fluid.Pcr amplification product carries out electrophoresis on 6% denaturing polyacrylamide gel, silver dyes and shows band.Experimental result shows, amplifies 2 allelotrope altogether, according to size order difference called after A, B allelotrope.The comparison molecular weight standard, A allelotrope size is 244bp, B allelotrope size is 234bp.Can only amplify A allelotrope in all 22 tail milters, and 22 all tail rauns can both amplify A, two allelotrope of B (Fig. 1).
Two, female special allelotrope sequence and primer sequence thereof of Cynoglossus semilaevis: its technology contents comprises: 1, female special allelic clone and order-checking; 2, female special allelic design of primers; 3, the checking of female specific mark.
1, female special allelic clone and order-checking:
With the primer PCR of sequence SEQ ID NO:1 (tgaactgcaa gactgctcca) and SEQ ID NO:2 (catcagttggtggcctgtaa) the sexually matured Cynoglossus semilaevis raun of the 1 tail DNA that increases; Agarose gel electrophoresis with 1.5% separates the PCR product; Downcut PCR product target electrophoretic band, with DNA purification kit purifying.Purified product is connected to pMD 18-T plasmid with the T4DNA ligase enzyme, and transformed competence colibacillus intestinal bacteria then, and smoothening on a LB flat board that contains acillin after liquid-absorbent finishes, are inverted overnight cultures for 37 ℃.Select 8 positive colonies and deliver to the order-checking of biological order-checking company.
2, female special allelic design of primers: according to sequencing result; 8 positive colonies check order successfully; Obtain 8 sequences altogether, wherein size is 6 of the sequences of 244bp, and size is 2 of 234bp sequences; Difference corresponding A, two allelotrope of B, B allelotrope is the female special allelotrope of Cynoglossus semilaevis.Utilize software MEGA4.0 comparison A, the allelic sequence of B, the result finds, except there is the disappearance of 2 GT the iteron, with respect to A allelotrope, B allelotrope (being female special allelotrope) has the disappearance of 6 bases at 5 ' end.Therefore be directed against the position of 6 bases of disappearance; ' Primer 3 ' has designed a special primer of SEQ ID NO:3 (tctgcatgac attggaaag) to utilize online primer-design software; This primer can be the female specific DNA fragment of Cynoglossus semilaevis of 204bp with the primer of the SEQ ID NO:2 pairing size that be used to increase, and carries out the Cynoglossus semilaevis genetic sex detection thereby set up simple PCR method.Female special allelotrope sequence is SEQ ID NO:4 (seeing sequence table).
3, the checking of female specific mark: the primer with SEQ ID NO:2 and SEQ ID NO:3 carries out pcr amplification to each 10 tail Cynoglossus semilaevis is male with the female individuals genome DNA sample; The PCR response procedures is following: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s; At 60 ℃ of annealing 40s, 72 ℃ are extended 40s, carry out extending 10min eventually after 38 circulations.Amplification reaction system is 12.5 μ L, comprises the total DNA of about 20ng, primer 0.2 μ mol/L, 0.1mmol/L dNTP, 0.5U Taq polysaccharase, 1 * PCR damping fluid.Amplified production is electrophoresis in 1.5% sepharose, EB dyeing.The result shows, all can amplify size in all female individuals and be the dna fragmentation of 204bp, and not have amplified production (Fig. 2) in the male, and this shows that female specific mark can accurately differentiate the genetic sex of Cynoglossus semilaevis.
Three, the application method of identifying based on the Cynoglossus semilaevis genetic sex of microsatellite marker: can carry out Cynoglossus semilaevis genetic sex according to following 2 kinds of combination of primers and identify: 1, according to the primer (combination of primers 1) of sequence SEQ ID NO:1 (tgaactgcaa gactgctcca) and SEQ ID NO:2 (catcagttgg tggcctgtaa); Pcr amplification Cynoglossus semilaevis genomic dna; The PCR response procedures is following: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s; At 61.5 ℃ of annealing 40s, 72 ℃ are extended 40s, carry out extending 10min eventually after 38 circulations.Amplification reaction system is 12.5 μ L, comprises the total DNA of about 20ng, primer 0.2 μ mol/L, 0.1mmol/L dNTP, 0.5U Taq polysaccharase, 1 * PCR damping fluid.Pcr amplification product carries out electrophoresis on 6% denaturing polyacrylamide gel, silver dyes and shows band.Analytical electrophoresis collection of illustrative plates then: amplify A, two allelic individualities of B must be female individuals, only amplify A allelic individual and be decided to be male.2, according to the primer (combination of primers 2) of sequence SEQ ID NO:2 (catcagttgg tggcctgtaa) and SEQ ID NO:3 (tctgcatgac attggaaag); Pcr amplification Cynoglossus semilaevis genomic dna; The PCR response procedures is following: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s are at 60 ℃ of annealing 40s; 72 ℃ are extended 40s, carry out extending 10min eventually after 38 circulations.Amplification reaction system is 12.5 μ L, comprises the total DNA of about 20ng, primer 0.2 μ mol/L, 0.1mmol/L dNTP, 0.5U Taq polysaccharase, 1 * PCR damping fluid.Pcr amplification product carries out electrophoresis on 1.5% sepharose, EB dyeing.Analytical electrophoresis collection of illustrative plates then: amplifying size must be female individuals for the individuality of the dna fragmentation of 204bp, and the individuality one of the DNA product that can not increase is decided to be male.
Claims (2)
1. microsatellite marker sequence that the Cynoglossus semilaevis sex is chain, said sequence is shown in the SEQ ID NO:4, concrete sequence is:
SEQ?ID?NO:4： tgaactgcaa?gactgctcca?gggcagtgtc?tctgcatgac?attggaaagc?tgtgtgtgga?tgcatgtgtg?tgtgcgtgta?atgttctcca?gctatggcct?acgctggctg?cagcctggag?ccgctctgtg?gtgtggaaaa?gagagcagat?gcctcttcac?tggagtgcat?caggccacca?aatcgcaaat?atcactgatc?atctcccaat?tcaattacag?gccaccaact?gatg。
2. the method identified of a Cynoglossus semilaevis genetic sex; The method that it is characterized in that it is: the Cynoglossus semilaevis genomic dna of extracting genetic sex to be measured; Chain microsatellite marker and the female special allelic sequence SEQ ID NO:4 of Cynoglossus semilaevis sex according to obtaining designs 3 special primers: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; With SEQ ID NO:1 and SEQ ID NO:2 the Cynoglossus semilaevis genomic dna is carried out pcr amplification, pcr amplification product carries out electrophoresis on 6% denaturing polyacrylamide gel, and silver dyes and shows band; Analytical electrophoresis collection of illustrative plates then: amplifying the individuality that A, two allelotrope sizes of B be respectively 244bp and 234bp must be female individuals, only amplifies A allelotrope size and is decided to be male for the individuality one of 244bp; With SEQ ID NO:2 and SEQ ID NO:3 the Cynoglossus semilaevis genomic dna is carried out pcr amplification, pcr amplification product carries out electrophoresis on 1.5% sepharose, EB dyeing; Analytical electrophoresis collection of illustrative plates then: amplifying size must be female individuals for the individuality of the dna fragmentation of 204bp, and the individuality one of the DNA product that can not increase is decided to be male.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102397296A CN101914529B (en) | 2010-07-29 | 2010-07-29 | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102397296A CN101914529B (en) | 2010-07-29 | 2010-07-29 | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101914529A CN101914529A (en) | 2010-12-15 |
| CN101914529B true CN101914529B (en) | 2012-03-14 |
Family
ID=43322173
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102397296A Expired - Fee Related CN101914529B (en) | 2010-07-29 | 2010-07-29 | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101914529B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134593B (en) * | 2010-12-23 | 2012-02-15 | 中国水产科学研究院黄海水产研究所 | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis |
| CN103525814B (en) * | 2013-10-28 | 2016-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of special SCAR mark of Cynoglossus semilaevis sex and application method |
| CN109161603B (en) * | 2018-10-31 | 2021-06-04 | 江苏省淡水水产研究所 | Microsatellite marker primer linked with channel catfish gender and genetic gender identification method |
| 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 |
| 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 |
| CN112011601A (en) * | 2020-08-19 | 2020-12-01 | 广东省生物资源应用研究所 | Identification primer, kit and identification method for sex-related microsatellite markers of scarab beetles |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005123121A2 (en) * | 2004-06-22 | 2005-12-29 | Forinnova As | Use of dna molecule as vaccine adjuvant |
| JP2006238888A (en) * | 2006-05-08 | 2006-09-14 | Eiken Chem Co Ltd | Primer for discriminating sex of cattle embryo and method for discriminating sex of cattle by using the same |
| CN101120660A (en) * | 2007-08-28 | 2008-02-13 | 中国水产科学研究院黄海水产研究所 | Method for inducing female nucleus growth with heterologous frozen sperm |
| CN101225437A (en) * | 2007-12-01 | 2008-07-23 | 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 |
| CN101690469A (en) * | 2009-10-14 | 2010-04-07 | 中国水产科学研究院黄海水产研究所 | Method for inducing cleavage gynogenesis of fish fry of cynoglossus semilaevis |
-
2010
- 2010-07-29 CN CN2010102397296A patent/CN101914529B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005123121A2 (en) * | 2004-06-22 | 2005-12-29 | Forinnova As | Use of dna molecule as vaccine adjuvant |
| JP2006238888A (en) * | 2006-05-08 | 2006-09-14 | Eiken Chem Co Ltd | Primer for discriminating sex of cattle embryo and method for discriminating sex of cattle by using the same |
| CN101120660A (en) * | 2007-08-28 | 2008-02-13 | 中国水产科学研究院黄海水产研究所 | Method for inducing female nucleus growth with heterologous frozen sperm |
| CN101225437A (en) * | 2007-12-01 | 2008-07-23 | 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 |
| CN101690469A (en) * | 2009-10-14 | 2010-04-07 | 中国水产科学研究院黄海水产研究所 | Method for inducing cleavage gynogenesis of fish fry of cynoglossus semilaevis |
Non-Patent Citations (2)
| Title |
|---|
| Acquadro A ET-AL.Development and characterization of microsatellite markers in Cynara cardunculus L..《Genome》.2005,第48卷(第2期),217-225. * |
| 廖小林.长江流域几种重要鱼类的分子标记筛选开发及群体遗传分析.《中国优秀博士学位论文全文数据库》.2006,1-2. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101914529A (en) | 2010-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101914529B (en) | Cynoglossus semilaevis gunther sex-linked microsatellite marker and genetics sex testing method | |
| Lee et al. | Genetic and physical mapping of sex-linked AFLP markers in Nile tilapia (Oreochromis niloticus) | |
| Lysak et al. | Analysis of plant meiotic chromosomes by chromosome painting | |
| CN105316412A (en) | Method for identification or auxiliary identification of day age of pig at 100kg weight and special kit of method | |
| CN107151690B (en) | Molecular marker for detecting day age of pigs with weight of 100kg and application thereof | |
| CN100532571C (en) | PCR-RFLP method for checking goat luteotropin gene mononucleotide polymorphism | |
| CN109161603A (en) | The microsatellite marker primer and genetic sex identification method of channel catfish gunther sex-linked | |
| CN103866032B (en) | A kind of method and application thereof detecting goat STAT3 gene mononucleotide polymorphism | |
| CN104878099A (en) | Method for detecting single-nucleotide polymorphism of goat ATBF1 gene and application of goat ATBF1 gene | |
| CN101633943A (en) | Identification method taking growth hormone receptor gene as Chinese grassland red bull excellent slaughter trait molecular marker and application thereof | |
| CN101148668B (en) | Clone for pork generation character related gene BTG1 of pig and application thereof in pig molecule mark auxiliary selection | |
| CN103725790A (en) | Molecular marker relevant to growth of fenneropenaeus chinensis and application of molecular marker | |
| CN110438260A (en) | A kind of African swine fever virus nucleic acid test strips detection kit | |
| CN113502335B (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN101921852B (en) | Method for detecting single nucleotide polymorphism of cattle AdPLA gene | |
| CN110241212B (en) | Primer set for sequencing and detecting BRCA1 and BRCA2 gene amplicons and application thereof | |
| CN104232776A (en) | Molecular marker related to milk production traits of dairy cows and application of molecular marker | |
| CN106566872B (en) | The analysis method in the pig SNP marker site based on sequencing genotyping technique | |
| CN104109669B (en) | Genetic markers and application of the pig AMPD1 gene promoter regions SNP as hog on hook character | |
| CN110205391A (en) | SNP marker relevant to Luchuan fat thickness at back of pig and its primer and application | |
| Kretschmer et al. | FISH—in Birds | |
| Nani et al. | Location of low copy genes in chromosomes of Brachiaria spp. | |
| CN107151693B (en) | Molecular marker for detecting day age of pigs reaching 100kg body weight based on MEG3 gene and application | |
| CN115323061A (en) | Pig intramuscular fat content character related ADIG gene haplotype variation genetic marker and application | |
| CN109207605B (en) | Method and kit for detecting IGF-1 allele expression of Ornithoia mossambica |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liao Xiaolin Inventor after: Chen Songlin Inventor after: Xu Genbo Inventor after: Liu Haijin Inventor after: Xu Ying Inventor before: Liao Xiaolin Inventor before: Chen Songlin Inventor before: Xu Genbo Inventor before: Xu Ying |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: LIAO XIAOLIN CHEN SONGLIN XU GENBO XU YING TO: LIAO XIAOLIN CHEN SONGLIN XU GENBO LIU HAIJIN XU YING |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120314 Termination date: 20170729 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |