CN105177143B - A kind of method of strain line hippocampus microsatellite Parentage determination - Google Patents

A kind of method of strain line hippocampus microsatellite Parentage determination Download PDF

Info

Publication number
CN105177143B
CN105177143B CN201510594893.1A CN201510594893A CN105177143B CN 105177143 B CN105177143 B CN 105177143B CN 201510594893 A CN201510594893 A CN 201510594893A CN 105177143 B CN105177143 B CN 105177143B
Authority
CN
China
Prior art keywords
ere
filial generation
hippocampus
primers
microsatellite
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.)
Active
Application number
CN201510594893.1A
Other languages
Chinese (zh)
Other versions
CN105177143A (en
Inventor
罗伟
林强
屈宏越
王信
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Sea Institute of Oceanology of CAS
Original Assignee
South China Sea Institute of Oceanology of CAS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by South China Sea Institute of Oceanology of CAS filed Critical South China Sea Institute of Oceanology of CAS
Priority to CN201510594893.1A priority Critical patent/CN105177143B/en
Publication of CN105177143A publication Critical patent/CN105177143A/en
Application granted granted Critical
Publication of CN105177143B publication Critical patent/CN105177143B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to fish breeding technical fields, and in particular to a kind of method of strain line hippocampus microsatellite Parentage determination.Its step includes：1）Strain line hippocampus family is built, and the filial generation of each family is individually raised；2）Parent and daughter DNA extraction；3）Microsatellite marker carries out PCR amplification in parent and filial generation；4）Genotyping is carried out to microsatellite using Genetic Analyser；5）The parental source of each filial generation is identified in data analysis；6）Family information according to filial generation hippocampus reality carries out reliability detection to this method.The method that the present invention establishes paternity test in strain line hippocampus for the first time, identification rate of accuracy reached to 97.37%.In this way, can control strain line hippocampus raise and train with the inbreeding phenomenon in selection and breeding, it is horizontal to improve germplasm, promotes that China's sea horse cultivation industry is sustainable, development of efficient and healthful.

Description

A kind of method of strain line hippocampus microsatellite Parentage determination
Technical field：
The invention belongs to fish breeding technical field, more particularly, to a kind of strain line hippocampus microsatellite Parentage determination Method.
Background technology：
Hippocampus belongs to Syngnathidae (Syngthidae), Hippocampus (Hippocampus), is a kind of rare marine Chinese medicine material, China is known as the title of " south ginseng ", have effects that tonifying kidney and strengthening yang, cardiac stimulant hasten parturition, hemostatic analgesia, tranquilizing and allaying excitement, while also very High ornamental value and the function of collection ornament.In the past few decades, due to long-term overfishing, environmental pollution and weather The factors such as variation, the threat that hippocampus germ plasm resource is seriously degenerated, some hippocampus kinds have even arrived the edge of extinction.In order to have The germ plasm resource of effect protection hippocampus, 38 kinds of hippocampus are put into《World Conservation Union》(IUCN) animals on the brink of extinction red in 2012 Register.Strain line hippocampus (Hippocampus erectus Perry, 1810) is mainly distributed on Atlantic Ocean west bank, is typical heat Band subtropical type hippocampus population, reproductive capacity is strong, individual is larger, build is graceful, and growth rate is most fast, has developed at present One of main breed variety in China.However, the production and operation under the plan of no scientific system, hippocampus is numerous through excessive generation After growing, phenomena such as build becomes smaller, sex premature, the speed of growth slow down, reproductive efficiency and premunition decline, germplasm are often shown The phenomenon that degeneration, is very serious.Team where inventor is during 2010-2014, and continuously the hippocampus coastal to China part provides After source and its aquiculture status carry out comprehensive survey, it is found that the breeding of China hippocampus and aquaculture model are extremely original, it is still very traditional Raise together, it is mixed hand over, genetic pedigree is unintelligible, and inbreeding depression is serious, directly results in most of hippocampus reproductive efficiency, Hou Daisheng Vigor and the speed of growth reduce.Found according to factual survey, the oviposition quantity of parent is more primary to have dropped 30-60%, the young into Motility rate only 15-25%, the speed of growth of young hippocampus is more primary to decline 30% or so.Therefore, in order to avoid or reduce to greatest extent The inbreeding depression of strain line hippocampus, clearly pedigree information is most important for the selection and breeding of family and the management of parent for holding.
In traditional aquatic livestock selection and use, different familys, which needs to divide, supports to maintain family information, and required water body is big And inconvenient management, it can be there are some differences in envirment factor moreover, each dividing between foster pond, the relevant genetic parameter to breeding Estimation can generate deviation.Family information is kept in mixed breed group, most of herdings are using physical markings as means, and physics Label for aquatic livestock there are it is cumbersome, have certain influence and the limitations such as larger to young injury on growth.Molecule mark The appearance of note make it possible raise together affiliation identification, the paternity test technology based on microsatellite is current aquatic products One of most widely used most reliable means during animal pedigree confirms.Have application to rainbow trout, Tilapia mossambica, Pelteobagrus fulvidraco, megalobrama amblycephala, big In the aquatic economic animals breeding such as brill and litopenaeus vannamei.At present, there is not yet microsatellite marker is applied to strain line hippocampus family The report of identification.
Invention content
Present invention solves the technical problem that being overcome the shortcomings of in existing strain line hippocampus Parentage determination, a kind of strain line is provided The method of hippocampus microsatellite Parentage determination.
The technical purpose of the present invention is achieved through the following technical solutions：
The present invention provides a kind of methods of strain line hippocampus microsatellite Parentage determination, include the following steps：
S1. strain line hippocampus family is built, and filial generation is individually raised；
S2. DNA extractions are carried out to the parent in step S1 and filial generation；
S3. the DNA of parent and filial generation of the microsatellite marker in S2 steps carry out PCR amplification；
S4. genotyping is carried out to the microsatellite PCR product of step S3 using Genetic Analyser；
S5. to the digital independent in step S4 and analysis, the parental source of each filial generation is identified；
S6. reliability detection is carried out to the analysis result of step S5 according to the family information of hippocampus filial generation reality；.
Preferably, the primer sequence of microsatellite marker used in the S3 is as follows：
H.ere-6 primers：F：ACCTGCTACCAACCACAA
R：AACAAGGCAACCACTCATT
H.ere-17 primers：F：GTTGATGCGACCCACGAT
R：GCCTTGCTCCTTCTCTACG
H.ere-18 primers：F：CCAGTTAGCATTGCGTCT
R：TCTTAGCCAGCGAGTGTT
H.ere-15 primers：F：TGATGGTTGTGTGAGAAGGA
R：GAGAGAAAAAGACGGCGA
H.ere-20 primers：F：CGGGGTAGCAAGGTGAGC
R：CAAGGGGACATTGAAGTAGG
H.ere-22 primers：F：TGCCATCATCGCTAACTAA
R：TGCCAAAGACACAAAAAGG
H.ere-30 primers：F：TGCCGAATGATGATACAC
R：AATGACGCAATGAGAACA
H.ere-14 primers：F：TGAGTCGGTGATGGTTGTG
R：AAAGACGGCGAGAGATAGG
H.ere-26 primers：F：AGTGGGTGTCTCTGTAAAC
R：CCTTCGCAGTATTCATTG.
Preferably, the primer of microsatellite marker is expanded in an amplification system in step S3, PCR amplification in S4 Product is mixed carries out genotyping again.
Preferably, the filial generation in step S1 is individually raised, keeps pedigree clear.
Preferably, DNA extractions are carried out to the fin ray of the parent in step S2 and filial generation using ammonium acetate/isoamyl alcohol method.
Preferably, the primer of the microsatellite marker in step S3 carries out 5 ' end fluorophor modifications.
Preferably, the reaction system of the microsatellite marker PCR amplification in step S3 is 10 μ L, including：50ng genomes DNA, 1 × PCR buffer solution, Mg2+(1.5mmol/L), 1U Taq enzymes, dNTPs 0.2mmol/L, positive anti-primer are 0.25 μ mol/L。
Preferably, the PCR response procedures of the microsatellite marker PCR amplification in step S3 are：94 DEG C of pre-degeneration 5min；94℃ 30s is denaturalized, anneal 30s, 72 DEG C of extension 30s, 30 cycles；72 DEG C of extension 8min, last 4 DEG C of preservations.
Preferably, the method for genotyping is ABI3730XL Genetic Analyser Capillary Electrophoresis partings in step S4.
Preferably, GeneScan is used in step S5TM 500ROXTMSize Standard are used in combination as internal reference 4.0 softwares of GeneMapper read genotype；Read data use 3.0 softwares of CERVUS to parent and son in step S5 The genotype in generation is analyzed, and judges the parent of filial generation.
Compared with prior art, the invention has the advantages that：
The present invention establishes paternity test platform on strain line hippocampus using microsateilite markers for the first time, and identification is accurate Rate has reached more than 97%, and the strain line hippocampus Parentage determination method based on the microsateilite markers can quickly and accurately be reflected Other strain line hippocampus difference family provides strong work for the parental apolegamy of strain line hippocampus, genetic breeding and behavioral ecology research Tool；The application of the present invention, which may be such that, not to be needed to separately raise each family progeny in production, has saved cultivation equipment and management Cost；Largely reduce environmental error simultaneously, the estimation of genetic parameter is more accurate.
Specific embodiment
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that the present embodiment is served only for pair The present invention is further described, but it is not intended that limiting the scope of the invention, the person skilled in the art in the field Some nonessential modifications and adaptations can be made according to the content of the invention described above.
Unless stated otherwise, the reagent of the invention used, method and apparatus for the art conventional reagent, method and are set It is standby.
The acquisition and verification of embodiment 1, strain line hippocampus microsatellite marker and specific primer：
Strain line hippocampus sample collection and RNA extractions：
30 health, the good hippocampus of gonad development are selected from sea horse cultivation company, its dorsal rags of clip carry immediately Total RNA are taken, are as follows：(1) fin ray of strain line hippocampus about 50mg is put into the mortar of precooling, it is fast using liquid nitrogen Fast grind into powder is put into 2mL centrifuge tubes, and adds in 1mLTRIzol；(2) chloroform of 200 μ L is added in centrifuge tube, acutely 15s, the static 3min of room temperature are shaken, treats that solution is layered；(3) 4 DEG C, 12,000r/min centrifugation 15min, absorption supernatant is put into new In centrifuge tube；(4) it adds in and is placed at room temperature for 5-10min with the isometric isopropanol of supernatant, jog mixing；(5) 4 DEG C, 12,000r/ Min centrifuges 10min, discards supernatant, of short duration centrifugation sucks remaining isopropanol；(6) ethyl alcohol of 1mL 75% is added in, is fully washed Precipitation, 4 DEG C, 7,500r/min centrifugation 5min, of short duration centrifugation sucks remaining ethyl alcohol, is repeated once the step；(7) room temperature is dried Ethyl alcohol, adding in 20 μ LDEPC processing water makes RNA fully dissolve, and -80 DEG C save backup；(8) sample of 1uL or so is taken to carry out electrophoresis RNA mass is detected, and RNA concentration and quality are detected with NanoDropND-1000 UV detectors.
Sequencing, splicing and the assembling of strain line hippocampus transcript profile simultaneously search microsatellite locus：
Using SMARTTMCDNA library constructing technology, the double-strand that cDNA is carried out to RNA sample synthesize, and use Trimmer- Direct cDNA normalization kit carry out homogenization processing to full-length cDNA.Using Illumina Hi SeqTM2000 are sequenced, and obtain original series.The connector of original series and low quality sequence are removed, obtains high quality sequence, Using Trinity softwares from the beginning it is assembled, obtain the Unigenes of 117327 strain line hippocampus.
The lookup of microsatellite locus is carried out in Unigenes sequences with MISA softwares, obtains 39848 microsatellite locus, Number of repetition is 5~64 when microsatellite locus is searched, wherein 22582 mononucleotide repeat sequences, 9311 dinucleotides weights Complex sequences, 7218 trinucleotide repeats sequences, 645 tetranucleotide repeat, 59 fermentation by five tubes, 33 Hexanucleotide repetitive sequence.
Strain line hippocampus extracting genome DNA：
The preparation of strain line hippocampus DNA is carried out using ammonium acetate/isoamyl alcohol method, is as follows：(1) by strain line hippocampus Fin ray about 50mg is put into 2mL centrifuge tubes；(2) Tissue lysates (the Tris-HCl 100mM, pH of 600 μ L is added in centrifuge tube 8.0；EDTA 50mM, pH 8.0；SDS 1%, pH 8.0；NaCl125mM), tissue is shredded with clean scissors；(3)65℃ 2~4h of water-bath gently overturns mixing every half an hour；(4) it is cooled to room temperature；(5) 7.5M of 200 μ L is added in into centrifuge tube Ammonium acetate (NH4AC), mixing places 5~10min on ice；(6) 4 DEG C, 12000r/min centrifugation 10min, and shift supernatant In (about 500 μ L) to another clean 1.5mL centrifuge tubes；(7) isometric isopropanol is added in, jog places 1~2min；(8)4 DEG C, 12000r/min centrifugation 10min abandon supernatant；(9) it is primary that the ethyl alcohol washing DNA precipitations that 1ml 70% is pre-chilled are added in, it is natural With appropriate TE solution (200-500 μ L) dissolving DNA sample after drying, each DNA sample adds in 1 μ L of 10mg/mL RNA enzyme；(10) The sample of 1uL or so is taken to carry out electrophoresis detection DNA mass, and dense with NanoDropND-1000 UV detectors detection DNA Degree and quality；(11) obtained DNA is diluted to the working solution of 100ng/ μ L with distilled water, -20 DEG C spare.
Microsatellite locus PCR amplification and polymorphism verification：
Using the DNA of strain line hippocampus as template, randomly selected in acquired microsatellite locus, according to its two terminal sequence, Using the specific primer of Primer5 Software for Design microsatellite locus, PCR amplification is carried out, system is 10 μ L, including 50ng genes Group DNA, 1 × PCR buffer solutions, Mg2+(1.5mM), 1U Taq enzymes, dNTPs 0.2mM, positive anti-primer are 0.25 μM.PCR reacts Program is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, annealing 30s (optimum annealing temperature of each microsatellite locus is different), 72 DEG C of extension 30s, 30 cycles；72 DEG C of extension 8min, last 4 DEG C of preservations.
After the completion of PCR amplification, verify its polymorphism using 8% non denatured polyacrylate hydrogel electrophoresis, be as follows： (1) preparation of glue：Glass long slab and otic placode cleaned with tap water and detergent and is clamped after drying, prepares PAGE glue (pure water 41mL, 5 × TBE Buffer 14mL, 40% acrylamide 14mL, 10%APS650 μ L, TEMED65 μ L) and filled along encapsulating mouth Into among two plates, comb is gently plugged, polymerize at least 2h.(2) electrophoresis：Glass plate is put into electrophoresis tank, upper and lower slot adds respectively Enter 0.5 × TBE Buffer, comb is extracted and is loaded, 150V constant voltage electrophoresis 120min.(3) silver staining：It will after the completion of electrophoresis Glue takes out, and impregnates a moment with pure water；Use 0.1%AgNO3Impregnate simultaneously jog 20min or so；Washing is rocked with pure water；It weighs 10gNaOH and 0.4gNa2CO30.5L developing solutions are prepared, 1.5mL formaldehyde is measured and is added thereto, jog colour developing；Treat that colour developing finishes, item Band is clear, and glue is placed on color development stopping in pure water；(4) it is placed in gel imaging system and takes pictures, it is steady to filter out different loci amplification It is fixed, band clearly microsatellite amplified production (table 2).
The strain line hippocampus micro-satellite primers information that 2. embodiment 1 of table provides
The Parentage determination experiment of 2. strain line hippocampus of embodiment：
Strain line hippocampus family is established and sample collection：
30 pairs of health, the good hippocampus of gonad development are selected from sea horse cultivation company, it is male that 1 female 1 is put into each bucket Hippocampus, it induced to promote the sexual maturity under the conditions of artificial bionic state, laid eggs, is pregnant and producing children.After male hippocampus hatches young hippocampus, immediately The dorsal rags of female, the male hippocampus of the clip matched group, and the hippocampus being just born is collected, each family 10-15 items are stored in In 100% alcohol, the material as Parentage determination.
Strain line hippocampus parent and daughter DNA extraction：
The preparation of strain line hippocampus DNA is carried out using ammonium acetate/isoamyl alcohol method, is as follows：(1) by strain line parent's Fin ray about 50mg and whole young hippocampus are put into 2mL centrifuge tubes；(2) Tissue lysates (Tris- of 600 μ L is added in centrifuge tube HCl 100mM, pH 8.0；EDTA 50mM, pH 8.0；SDS 1%, pH 8.0；NaCl 125mM), with clean scissors by group It knits and shreds；(3) 65 DEG C of 2~4h of water-bath gently overturn mixing every half an hour.(4) it is cooled to room temperature；(5) into centrifuge tube Add in the ammonium acetate (NH of the 7.5M of 200 μ L4AC), mixing places 5~10min on ice；(6) 4 DEG C, 12000r/min centrifugations 10min, and shift in supernatant (about 500 μ L) to another clean 1.5mL centrifuge tubes；(7) isometric isopropanol is added in, gently It shakes, places 1~2min；(8) 4 DEG C, 12000r/min centrifugation 10min abandon supernatant；(9) ethyl alcohol that 1ml 70% is pre-chilled is added in It is primary to wash DNA precipitations, is added in after naturally dry with appropriate TE solution (200-500 μ L) dissolving DNA sample, each DNA sample 1 μ L of 10mg/mL RNA enzyme.(10) sample of 1uL or so is taken to carry out electrophoresis detection DNA mass, and purple with NanoDropND-1000 Outer spectrophotometer detection DNA concentration and quality；(11) obtained DNA is diluted to the working solution of 100ng/ μ L with distilled water ,- 20 DEG C spare.
Microsatellite locus PCR amplification and genotyping：
Using parent and the DNA of filial generation as template, with 9 pairs of micro-satellite primers (H.ere-6,17,18,15,20,22,30,14, 26) it (refers to table 2) and PCR amplification is carried out to all parents and filial generation, system is 10 μ L, and including 50ng genomic DNAs, 1 × PCR delays Fliud flushing, Mg2+(1.5mM), 1U Taq enzymes, dNTPs0.2mM, positive anti-primer are 0.25 μM.PCR response procedures are：94 DEG C of pre- changes Property 5min；94 DEG C of denaturation 30s, annealing 30s (optimum annealing temperature and fluorescent decoration group of each microsatellite locus are shown in Table 2), 72 DEG C of extension 30s, 30 cycles；72 DEG C of extension 8min, last 4 DEG C of preservations.
After the completion of PCR amplification, 0.5 μ L are sucked out from the PCR product in each site of each individual, according to the size of segment With the color of fluorescent marker, according to shown in the combination of table 2, the PCR product in each site is mixed, as upper machine examination The sample of survey.The PCR product sample for mixing 1.5 μ L is added in 96 orifice plates of ABI3730 Genetic Analysers configuration, Mei Gekong The interior GeneScan for adding in 0.5 μ LTM350ROXTMStandard segment (ABI) in addition adds in the Genescan of 8 μ L in each hole Hi-DiTM96 orifice plates are put into PCR instrument and are denaturalized 10min in 95 DEG C, are immediately placed on after deformation on ice, so by formamide (ABI) After be loaded to ABI3730XL types Genetic Analyser analysis.After treating Genetic Analyser electrophoresis, with software GeneMapper4.0 Genotyping is done, reads genotype of each individual in each microsatellite locus.
Data analysis：
In strain line hippocampus breeding parent Genetic Analyser electricity is treated through Genetic Analyser in the PCR product of 9 microsatellite locus After swimming, according to the color of fluorescent marker, genotyping is done with software GeneMapper 4.0 (ABI), reads each individual In the genotype (clip size) of each microsatellite locus.
The genotype of parent and filial generation in 9 microsatellite locus is counted with Microsoft Excel, using software CERVUS 3.0 calculate gene frequency, expectation heterozygosity (H of the filial generation on each microsatellite seatE), observation heterozygosity (HO) and it is polymorphic The parameters such as information content (PIC), average probability of exclusion, Hardy-Weinberg balances and amorph frequency are specific to tie Fruit is shown in Table 3.With the Parent according to LOD value (the Logarithm conversion value of paternity test likelihood ratio) identification 152 tails individual.
The result of identification is compared with the practical family information of hippocampus, 148 tails are by precise Identification, accuracy rate 97.37%. Wherein 4 tails are not by precise Identification, the reason is that due to DNA quality problems and operation, microsatellite locus which has It could not enough successful parting.
Detected value of 9 micro-satellite primers in strain line hippocampus family in 3. embodiment 2 of table
Micro-satellite primers n Na HO HE HW Excl 1 Excl 2 F(null)
H.ere-6 152 5 0.763 0.668 ** 0.255 0.289 +0.001
H.ere-17 152 6 0.822 0.785 NS 0.315 0.404 +0.003
H.ere-18 152 6 0.842 0.761 ** 0.324 0.375 +0.004
H.ere-15 152 7 0.888 0.767 NS 0.432 0.368 -0.001
H.ere-20 152 5 0.789 0.678 NS 0.268 0.289 +0.012
H.ere-22 152 6 0.739 0.682 NS 0.366 0.368 +0.008
H.ere-30 152 6 0.645 0.612 ** 0.322 0.354 -0.007
H.ere-14 152 8 0.743 0.674 ** 0.452 0.486 +0.005
H.ere-26 152 4 0.895 0.822 ** 0.218 0.225 +0.011
Totalexclusion 5.89 0.995 0.999
Note：Na is allele number, and n is detects family progeny individual amount, HOTo observe heterozygosity, HEIt is miscellaneous it is expected Right, HW is examined for hardy weinberg equilibrium, and NS expressions meet, and * * represent to deviate significantly, and Excl 1 represents male parent elimination factor, Excl 2 represents maternal elimination factor, and F (null) represents amorph frequency, and Total exclusion represent that accumulation excludes Rate.
Comparative example 1：
Method with embodiment 2, unlike the primer that uses for H.ere-1,2,3,4,5,8,9,24 and 25 in table 2, Its result shows such as table 4：
Detected value of 9 micro-satellite primers in strain line hippocampus family in 4. comparative example 1 of table
Micro-satellite primers n Na HO HE HW Excl 1 Excl 2 F(null)
H.ere-1 152 2 0.691 0.732 ** 0.078 0.058 +0.005
H.ere-2 152 3 0.711 0.756 ** 0.189 0.228 +0.024
H.ere-3 152 2 0.809 0.821 ** 0.112 0.125 +0.004
H.ere-4 152 2 0.842 0.867 ** 0.145 0.142 -0.008
H.ere-5 152 3 0.724 0.762 ** 0.215 0.252 +0.002
H.ere-8 152 3 0.513 0.622 ** 0166 0.168 +0.014
H.ere-9 152 2 0.559 0.582 ** 0.085 0.096 -0.0015
H.ere-24 152 2 0.737 0.789 ** 0.0788 0.105 +0.0000
H.ere-25 152 2 0.888 0.856 ** 0.112 0.124 +0.0004
Totalexclusion 2.33 0.435 0.576
It can be seen from the table, Parentage determination, allele quantity, male parent and female parent are carried out using 9 pairs of micro-satellite primers Elimination factor well below this 9 pairs of microsatellite markers of H.ere-6 of the present invention, 17,18,15,20,22,30,14 and 26 Elimination factor.In practical applications, the accuracy rate for carrying out Parentage determination using 9 pairs of micro-satellite primers is only 54.25%, remote low In the accuracy rate of this 9 pairs of microsatellite markers of H.ere-6 of the present invention, 17,18,15,20,22,30,14 and 26 (97.37%).Therefore, when carrying out strain line hippocampus Parentage determination, micro-satellite primers of the present invention are far superior to other Primer.
Meanwhile it is a discovery of the invention that 9 pairs of primers provided by the present invention are needed after applied to PCR amplification by whole PCR Product is mixed, and could obtain best Parentage determination effect.
By this experiment, the 9 high, amplifications to (H.ere-6,17,18,15,20,22,30,14,26) polymorphism have been screened The few microsatellite marker of stable, miscellaneous band；And other microsatellite markers cannot achieve the effect that the Parentage determination.
Wherein, the microsatellite sequence such as SEQ.ID.NO of acquisition is expanded respectively according to 2 each primer of embodiment:Shown in 1~9.
SEQUENCE LISTING
<110>Chinese Academy of Science Nanhai Ocean Research Institute
<120>A kind of method of strain line hippocampus microsatellite Parentage determination
<130>
<160> 45
<170> PatentIn version 3.3
<210> 1
<211> 511
<212> DNA
<213> 1
<400> 1
atccaagaga agacatttga atgttgcacc gctacaattg tcttgcaacc gcagaatgtc 60
acgtcgagcc acctttcgtt cgctgcgtat gtgcagaaat gaagcgttgg taaacagacg 120
cgtatggaat gtcagcacct cagtaaatgt gaatttacac atttaattta tacattcagt 180
tctattgtac ctgctaccaa ccacaaacat tgatatgtca tttctctagt tgtgagtgtg 240
tgtgtgtgta tatatactgt atattctaag ctggctacat ggatgggaga gcaccatctt 300
ctgtagcctt ttgtcagtga cagttttgtc attctgggca atatattatc aacaaagtga 360
acctaaatat ttacattccc tccgttactg aacagtggag ttaaaccggt agtctattga 420
ggtgacaaaa atccacattt catcaatgag tggttgcctt gttcgcgact cagtggtgac 480
tgatgctgct ggtccggcct ggctgtctct t 511
<210> 2
<211> 498
<212> DNA
<213> 2
<400> 2
gcgaagaatc tcttggaatt gaggtttaac cttggtgttg gtaaaaactt tgccaaaggt 60
tcgacagaac ctccaaaaaa agcgcgagaa ctcatgaacg caggcggacg aggtcacgtt 120
gatgcgaccc acgatctcta tgagctgagg aagccttcag aggtagcata aaaaaaagga 180
acgaatgaat ttgatgtctg aatacactta cactgtgtgt gtgtgtgtgt gtgtgtgtgt 240
gtgtgtgtgt gtgtgtgcgt ctcacagctg gttgaccacc caaagcaagc tttcccatcc 300
ggtggtccct tcatgctcta gttggtggtc gtagagaagg agcaaggcac tcagcatctc 360
cctgctgccg atgatggtgg ccacatcctg gaggggggaa gccggccttg gaaagcgagt 420
cactacaatg gcaaatacat gctaacagca actgaacaca aagctgaaac aagaaaccaa 480
caggctgaag tttcatcc 498
<210> 3
<211> 423
<212> DNA
<213> 3
<400> 3
ccccaaaaac aattcaaaca gaaaaaaaca gaggcgtatt tggcaaatgg atccacactg 60
tatgcttaaa tgtcttctag acgttgagaa gcagctgtgg gcgtggccac agctagcgat 120
gctatcatga gccagttagc attgcgtctc acatgcccac aaaaaacccc cccaacaaca 180
acaacaacaa tgaaggatac acaatgagga gtaaggttat aacactcgct ggctaagagc 240
acttttcgaa cgtggagagc tagcatagac tagcgaagca tagcatcacc tcaccaaatg 300
attacaacgg caagctcttg tgtctgtttt cgtcagtgcg caacaagtga gaacttgtgg 360
aaaaaagaaa caatctttat gattccaaac atacatacag acttttccgt tacacatttt 420
cta 423
<210> 4
<211> 498
<212> DNA
<213> 4
<400> 4
gcttcgaccc cgcccactca ctctccacag cctccggctg ccccgcctcc ggtgccatcc 60
ccgacatcct cggccacgca aaacgccctc gaccagcaga gggagatggc gcgccgtcgt 120
gagcaggaga ggcgcaggag ggaggcgatg gccgacacca ttgacattaa cttccagagc 180
gacctgatgg ccatttttga agagaatctc ttctgagtcg gtgatggttg tgtgagaagg 240
acctacacac acacacacac acacactgag gtggtgagct tatatacagt acatgccccc 300
acatgatcat ccgcatacgg actacatagt acctgtcccc tatctctcgc cgtctttttc 360
tctcggagcg cagatacgtt ttggtgtctg acgtgatgtt tatagttgtt ggctcgcaaa 420
gtggagttta aaaaaaaaaa agtcctataa aaggcagagt catagtcaag gggggtgcga 480
caaaactaaa ctgaagaa 498
<210> 5
<211> 507
<212> DNA
<213> 5
<400> 5
aaaaacaggt taaaacgaaa aagtgtctca ccattgtatg agttaaaaaa accttaaaac 60
aataacaaag gggttttact gtgtcgaggg gcggggtagc aaggtgagcg cttacctgga 120
tggtggcagt gctgcttcgg ctcaaggata atcagtcctc cagatttaga gcatattatg 180
tccgcacgtg gggtgtctcg ccgaattgag tagtctccca agcactgtaa ttaggcaccc 240
aatattccgg taccagatcg tgtgtgtgtg tgtgtttgga aaaacgtaat tccaaaactt 300
gttagccagg cccagtagtc tcttctttat gggcgtggta tcatagacat gaaacctact 360
tcaatgtccc cttgagcgtt tggcaacgtt gctaaaatac gccagcgcgc ccgccatatt 420
ggatgtgtcc tctctcatcc gtaggtaaat acaacctgat gggcaaaata ccaagcgcaa 480
taaaatgaat taatataacc cgattac 507
<210> 6
<211> 491
<212> DNA
<213> 6
<400> 6
cagcaaccgt tggtggacgc aggagggttg gggaggcacg accccctcac ctccaaaaac 60
aagacaccag gatgagactc cacccggacg aggtcgcctc cgctgaaggg aagcgggccg 120
atgccaacga cgggggttgc gggaccgcgc gcatgccatc atcgctaact aaacagatgt 180
acacacaaca cacccataga acaaaagcac acacccacag tcacacacac atggaacaaa 240
agcacacaca aacacgcaca catccacaca cacacacgag tggatcggca ggacgagttt 300
gtccacgccg gacatcctcc caagtccacg cgaggctttt tcgtggcctt tttgtgtctt 360
tggcaaatct gagggtgagc gaggaccctg cagtggtcgg gtgacttgga agattgaatt 420
cctcactatt tctttccttg ctgaggaaga cgacgaggac gtctaaacat gtaaatgcgc 480
aaaagatggg a 491
<210> 7
<211> 289
<212> DNA
<213> 7
<400> 7
cttgccgaat gatgatacac cctgcacgtg tagcacatct gcgcgcacgc acacacacac 60
acacacacac acgcatgcat gcagcagaca cacaaactcc aacctcatta cacttaaaag 120
tgtaaaaata aaataaacac aagtcgtgtc agttctgccc aaaaactcaa acaggagtcc 180
cggctccgct tgggcacttt gacccattta gtgtgctttg ttctcattgc gtcatttaag 240
gtgcaagctt ccttttcttt taaaacacag ctcaacccgt tccatacaa 289
<210> 8
<211> 498
<212> DNA
<213> 8
<400> 8
gcttcgaccc cgcccactca ctctccacag cctccggctg ccccgcctcc ggtgccatcc 60
ccgacatcct cggccacgca aaacgccctc gaccagcaga gggagatggc gcgccgtcgt 120
gagcaggaga ggcgcaggag ggaggcgatg gccgacacca ttgacattaa cttccagagc 180
gacctgatgg ccatttttga agagaatctc ttctgagtcg gtgatggttg tgtgagaagg 240
acctacacac acacacacac acacactgag gtggtgagct tatatacagt acatgccccc 300
acatgatcat ccgcatacgg actacatagt acctgtcccc tatctctcgc cgtctttttc 360
tctcggagcg cagatacgtt ttggtgtctg acgtgatgtt tatagttgtt ggctcgcaaa 420
gtggagttta aaaaaaaaaa agtcctataa aaggcagagt catagtcaag gggggtgcga 480
caaaactaaa ctgaagaa 498
<210> 9
<211> 510
<212> DNA
<213> 9
<400> 9
ggatcagttt caccctgaag tcattttgtg acggatcaga aggcgtccga gttggatgga 60
ggtgaatgga acacaagtag caggatgcga cgtgtgccat atgtgcgagg ccattgcatt 120
gcgacgcctc tgtctgtctg tctgtctgtg agtgggtgtc tctgtaaaca cgtgtgcggc 180
ttacacgggg tgtggaattc accaacgaat gagaaagcac tgtgcagggt gcggccttat 240
tgcttactaa gggcaggccc ttttgtttgt gtgtgtgtgt gtgtgtgtgt gagtgcgtgt 300
gcgtgtgtcg tactatgcgt atgactaaat gtagacatca ctgaatgagg aatgtacagc 360
aatgaatact gcgaagggaa tattaacttg cactaaaaaa aaaataaaaa aaagattaaa 420
aaaacccaac aaaactgaaa aaaaaaatgg acacacactc acacacacaa tacttgattc 480
catgagtcag ttttatcata ggtctccgtc 510
<210> 10
<211> 18
<212> DNA
<213> 10
<400> 10
acctgctacc aaccacaa 18
<210> 11
<211> 19
<212> DNA
<213> 11
<400> 11
aacaaggcaa ccactcatt 19
<210> 12
<211> 18
<212> DNA
<213> 13
<400> 12
gttgatgcga cccacgat 18
<210> 13
<211> 19
<212> DNA
<213> 13
<400> 13
gccttgctcc ttctctacg 19
<210> 14
<211> 18
<212> DNA
<213> 14
<400> 14
ccagttagca ttgcgtct 18
<210> 15
<211> 18
<212> DNA
<213> 15
<400> 15
tcttagccag cgagtgtt 18
<210> 16
<211> 20
<212> DNA
<213> 16
<400> 16
tgatggttgt gtgagaagga 20
<210> 17
<211> 18
<212> DNA
<213> 17
<400> 17
gagagaaaaa gacggcga 18
<210> 18
<211> 18
<212> DNA
<213> 18
<400> 18
cggggtagca aggtgagc 18
<210> 19
<211> 20
<212> DNA
<213> 19
<400> 19
caaggggaca ttgaagtagg 20
<210> 20
<211> 19
<212> DNA
<213> 20
<400> 20
tgccatcatc gctaactaa 19
<210> 21
<211> 19
<212> DNA
<213> 21
<400> 21
tgccaaagac acaaaaagg 19
<210> 22
<211> 18
<212> DNA
<213> 22
<400> 22
tgccgaatga tgatacac 18
<210> 23
<211> 18
<212> DNA
<213> 23
<400> 23
aatgacgcaa tgagaaca 18
<210> 24
<211> 19
<212> DNA
<213> 24
<400> 24
tgagtcggtg atggttgtg 19
<210> 25
<211> 19
<212> DNA
<213> 25
<400> 25
aaagacggcg agagatagg 19
<210> 26
<211> 19
<212> DNA
<213> 26
<400> 26
agtgggtgtc tctgtaaac 19
<210> 27
<211> 18
<212> DNA
<213> 27
<400> 27
ccttcgcagt attcattg 18
<210> 28
<211> 19
<212> DNA
<213> 28
<400> 28
tcggcataga ttttgaggc 19
<210> 29
<211> 18
<212> DNA
<213> 29
<400> 29
gacccgctga cattttcg 18
<210> 30
<211> 20
<212> DNA
<213> 30
<400> 30
ccagccagta gtctttcctt 20
<210> 31
<211> 18
<212> DNA
<213> 31
<400> 31
accctccctt gctttcat 18
<210> 32
<211> 20
<212> DNA
<213> 32
<400> 32
tccaccaaac attgagtaac 20
<210> 33
<211> 18
<212> DNA
<213> 33
<400> 33
gccagcagga ccataaag 18
<210> 34
<211> 19
<212> DNA
<213> 34
<400> 34
cccacaggaa atgccaatc 19
<210> 35
<211> 18
<212> DNA
<213> 35
<400> 35
gacccaacca gggaacca 18
<210> 36
<211> 18
<212> DNA
<213> 36
<400> 36
agagcgggaa ggatgtga 18
<210> 37
<211> 20
<212> DNA
<213> 37
<400> 37
tgtggaatga ggaggaaggt 20
<210> 38
<211> 20
<212> DNA
<213> 38
<400> 38
atgttgcctc ttttgatttg 20
<210> 39
<211> 18
<212> DNA
<213> 39
<400> 39
ctctggtgcg atgtggtg 18
<210> 40
<211> 19
<212> DNA
<213> 40
<400> 40
cggaagccac atctaaaca 19
<210> 41
<211> 18
<212> DNA
<213> 41
<400> 41
catccacatc cacccact 18
<210> 42
<211> 18
<212> DNA
<213> 42
<400> 42
acaaaagcca agtgacca 18
<210> 43
<211> 21
<212> DNA
<213> 43
<400> 43
agcagttccg tgtaagtaat c 21
<210> 44
<211> 18
<212> DNA
<213> 44
<400> 44
cctgctggca aatctgtt 18
<210> 45
<211> 19
<212> DNA
<213> 45
<400> 45
ggagtggtcc tcggtaaag 19

Claims (8)

  1. A kind of 1. method of strain line hippocampus microsatellite Parentage determination, which is characterized in that include the following steps：
    S1. strain line hippocampus family is built, and filial generation is individually raised；
    S2. DNA extractions are carried out to the parent in step S1 and filial generation；
    S3. the DNA of parent and filial generation of the microsatellite marker in S2 steps carry out PCR amplification；
    S4. genotyping is carried out to the microsatellite PCR amplified production of step S3 using Genetic Analyser；
    S5. to the digital independent in step S4 and analysis, the parental source of each filial generation is identified；
    S6. reliability detection is carried out to the analysis result of step S5 according to the family information of hippocampus filial generation reality；
    Wherein, the primer of microsatellite marker is as follows in the S3：
    H.ere-6 primers：F：ACCTGCTACCAACCACAA
    R：AACAAGGCAACCACTCATT
    H.ere-17 primers：F：GTTGATGCGACCCACGAT
    R：GCCTTGCTCCTTCTCTACG
    H.ere-18 primers：F：CCAGTTAGCATTGCGTCT
    R：TCTTAGCCAGCGAGTGTT
    H.ere-15 primers：F：TGATGGTTGTGTGAGAAGGA
    R：GAGAGAAAAAGACGGCGA
    H.ere-20 primers：F：CGGGGTAGCAAGGTGAGC
    R：CAAGGGGACATTGAAGTAGG
    H.ere-22 primers：F：TGCCATCATCGCTAACTAA
    R：TGCCAAAGACACAAAAAGG
    H.ere-30 primers：F：TGCCGAATGATGATACAC
    R：AATGACGCAATGAGAACA
    H.ere-14 primers：F：TGAGTCGGTGATGGTTGTG
    R：AAAGACGGCGAGAGATAGG
    H.ere-26 primers：F：AGTGGGTGTCTCTGTAAAC
    R：CCTTCGCAGTATTCATTG.
  2. 2. according to the method described in claim 1, it is characterized in that, pcr amplification product is mixed and carries out base again in step S4 Because of type parting.
  3. 3. according to the method described in claim 1, it is characterized in that, using ammonium acetate/isoamyl alcohol method to the parent in step S2 DNA extractions are carried out with the fin ray of filial generation.
  4. 4. according to the method described in claim 1, it is characterized in that, the 5 ' end of primer progress of the microsatellite marker in step S3 is glimmering Light base group modification.
  5. 5. the according to the method described in claim 1, it is characterized in that, reactant of the microsatellite marker PCR amplification in step S3 Be for 10 μ L, including：50 ng genomic DNAs, 1 × PCR buffer solutions, the Mg of a concentration of 1.5 mmol/L2+, 1 U Taq enzymes, 0.2 mmol/L of dNTPs, positive anti-primer are 0.25 μm of ol/L.
  6. 6. the according to the method described in claim 1, it is characterized in that, PCR reactions of the microsatellite marker PCR amplification in step S3 Program is：94 DEG C of 5 min of pre-degeneration；94 DEG C of 30 s of denaturation, anneal 30 s, 72 DEG C of extension 30s, 30 cycles；72 DEG C of extensions 8 Min, last 4 DEG C of preservations.
  7. 7. according to the method described in claim 1, it is characterized in that, the method for genotyping is ABI3730XL in step S4 Genetic Analyser Capillary Electrophoresis parting.
  8. 8. according to the method described in claim 1, it is characterized in that, use GeneScan in step S5TM 500 ROXTM Size Standard reads genotype as internal reference, and with 4.0 softwares of GeneMapper；Read data use in step S5 3.0 softwares of CERVUS analyze the genotype of parent and filial generation, judge the parent of filial generation.
CN201510594893.1A 2015-09-16 2015-09-16 A kind of method of strain line hippocampus microsatellite Parentage determination Active CN105177143B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510594893.1A CN105177143B (en) 2015-09-16 2015-09-16 A kind of method of strain line hippocampus microsatellite Parentage determination

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510594893.1A CN105177143B (en) 2015-09-16 2015-09-16 A kind of method of strain line hippocampus microsatellite Parentage determination

Publications (2)

Publication Number Publication Date
CN105177143A CN105177143A (en) 2015-12-23
CN105177143B true CN105177143B (en) 2018-06-26

Family

ID=54899565

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510594893.1A Active CN105177143B (en) 2015-09-16 2015-09-16 A kind of method of strain line hippocampus microsatellite Parentage determination

Country Status (1)

Country Link
CN (1) CN105177143B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105603097A (en) * 2016-02-05 2016-05-25 中国水产科学研究院南海水产研究所 Microsatellite marker primers used for pinctada fucata martensii microsatellite family identification, identification method and application

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834517B (en) * 2017-03-23 2020-07-17 辽宁省海洋水产科学研究院 SSR (simple sequence repeat) labeled primer group for parent-child identification of parva and application of SSR labeled primer group
CN107439428A (en) * 2017-09-04 2017-12-08 天津农学院 A kind of selection of strain line hippocampus high yield family
CN109182546B (en) * 2018-10-19 2021-11-30 广东省科学院动物研究所 SSR fluorescence labeling primer for paternity test of pangolin scales and application thereof
CN117025788B (en) * 2023-08-29 2024-03-26 山东省海洋科学研究院(青岛国家海洋科学研究中心) Microsatellite marker related to growth traits of Hippocampus kelloggi and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102653785A (en) * 2011-03-03 2012-09-05 华中农业大学 Identifying method of megalobrama amblycephala family by microsatellite
CN103789441A (en) * 2014-02-19 2014-05-14 华中农业大学 SSR (Simple Sequence Repeat) molecular marker method for identifying two hippocampus populations
CN104357553A (en) * 2014-10-14 2015-02-18 华中农业大学 Pelteobagrus fulvidraco microsatellite family identification method
CN104531870A (en) * 2014-12-25 2015-04-22 中国科学院南海海洋研究所 Specific primer and identification method for microsatellite markers of sea horses in coastal waters of China

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102653785A (en) * 2011-03-03 2012-09-05 华中农业大学 Identifying method of megalobrama amblycephala family by microsatellite
CN103789441A (en) * 2014-02-19 2014-05-14 华中农业大学 SSR (Simple Sequence Repeat) molecular marker method for identifying two hippocampus populations
CN104357553A (en) * 2014-10-14 2015-02-18 华中农业大学 Pelteobagrus fulvidraco microsatellite family identification method
CN104531870A (en) * 2014-12-25 2015-04-22 中国科学院南海海洋研究所 Specific primer and identification method for microsatellite markers of sea horses in coastal waters of China

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Isolation of microsatellite markers for the endangered Knysna seahorse Hippocampus capensis and their use in the detection of a genetic bottleneck;PETER等;《Molecular Ecology Notes》;20070731;第7卷(第4期);第638-640页 *
Microsatellite luci to determine population structure in the yellow seahorse(Hippocampus kuda)and the three-spotted seahorse(H.trimaculatus);Singh等;《MARINE BIODIVERSITY》;20121231;第42卷(第4期);第481-488页 *
Novel microsatellite loci in the threatend European long-snouted seahorse(Hippocampus guttulatus)for genetic diversity and parentage analusis;Pardo等;《CONSERVATION GENETICS》;20071231;第8卷(第5期);第1243-1245页 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105603097A (en) * 2016-02-05 2016-05-25 中国水产科学研究院南海水产研究所 Microsatellite marker primers used for pinctada fucata martensii microsatellite family identification, identification method and application
CN105603097B (en) * 2016-02-05 2020-01-14 中国水产科学研究院南海水产研究所 Microsatellite marker primer for identifying pinctada fucata microsatellite families as well as identification method and application

Also Published As

Publication number Publication date
CN105177143A (en) 2015-12-23

Similar Documents

Publication Publication Date Title
CN105177143B (en) A kind of method of strain line hippocampus microsatellite Parentage determination
CN106434949A (en) Acipenser dabryanus microsatellite marker as well as screening method and application of acipenser dabryanus microsatellite molecular marker
CN106591429B (en) Method for screening sex reversal genetic control sites of cynoglossus semilaevis, kit and application
CN114836543A (en) Female molecular marker primer of channa maculata, application thereof and method for identifying sex of channa maculata
CN104351096A (en) Paramisgurnus dabryanus selective breeding method
CN115261489A (en) Catharanthus roseus microsatellite marker primer group and application and evaluation method thereof
CN105177142B (en) A kind of strain line hippocampus microsatellite marker and its screening technique
CN110564867B (en) SNP molecular marker of Qinchuan cattle CFL1 gene and detection method thereof
CN111996261A (en) Macrobrachium rosenbergii sex molecular marker primer and application thereof
CN111254203A (en) Saline-alkali-resistant molecular marker C325 of portunus trituberculatus and application thereof
CN106701930B (en) Method for detecting sheep FTH-1 gene insertion deletion polymorphism by using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and application thereof
CN111286545A (en) Saline-alkali-resistant molecular marker C0 of portunus trituberculatus and application thereof
CN109439771A (en) A method of hybridization porgy family is identified using microsatellite marker
CN113736890B (en) SNP molecular marker related to healthy number and living number rate and application thereof
CN111286546B (en) Saline-alkali-resistant molecular marker C1480 of portunus trituberculatus and application thereof
CN113684280A (en) Apostichopus japonicus high temperature resistant breeding low-density 12K SNP chip and application
CN111269993A (en) Saline-alkali-resistant molecular marker C261 of portunus trituberculatus and application thereof
CN110724749A (en) Molecular marker C104 of portunus trituberculatus resistant vibrio parahaemolyticus and application thereof
CN110747281A (en) Low-salt-resistant molecular marker C62 of portunus trituberculatus and application thereof
CN117683911B (en) Primer for detecting SNP molecular markers related to protein utilization characteristics of garrupa feed and application of primer
CN110724750B (en) Molecular marker C242 of portunus trituberculatus parahaemolyticus and application thereof
CN113981113B (en) InDel marker C142 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof
CN117487931B (en) Sillago sihama hypoxia tolerance character related SNP molecular marker and application thereof
CN111286544B (en) Saline-alkali-resistant molecular marker C72 of portunus trituberculatus and application thereof
CN109837346B (en) Cynoglossus semilaevis sex reversal genetic control site, kit containing same 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
GR01 Patent grant
GR01 Patent grant