CN109536620A - The method and kit of yellow tail silver xenocypris paternity test - Google Patents
The method and kit of yellow tail silver xenocypris paternity test Download PDFInfo
- Publication number
- CN109536620A CN109536620A CN201811588161.1A CN201811588161A CN109536620A CN 109536620 A CN109536620 A CN 109536620A CN 201811588161 A CN201811588161 A CN 201811588161A CN 109536620 A CN109536620 A CN 109536620A
- Authority
- CN
- China
- Prior art keywords
- seq
- loc
- silver xenocypris
- paternity test
- tail silver
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012360 testing method Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 18
- 241001222098 Xenocypris Species 0.000 title claims description 55
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims description 53
- 229910052709 silver Inorganic materials 0.000 title claims description 53
- 239000004332 silver Substances 0.000 title claims description 53
- 241001600434 Plectroglyphidodon lacrymatus Species 0.000 title claims description 42
- 108020004414 DNA Proteins 0.000 claims abstract description 60
- 108091092878 Microsatellite Proteins 0.000 claims abstract description 47
- 238000012408 PCR amplification Methods 0.000 claims abstract description 8
- 238000009395 breeding Methods 0.000 claims abstract description 8
- 230000001488 breeding effect Effects 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 8
- 108700028369 Alleles Proteins 0.000 claims abstract description 6
- 230000003321 amplification Effects 0.000 claims abstract description 6
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 6
- 239000000284 extract Substances 0.000 claims abstract description 4
- 230000004720 fertilization Effects 0.000 claims abstract description 4
- 230000012447 hatching Effects 0.000 claims abstract description 4
- 239000003550 marker Substances 0.000 claims description 13
- 238000004458 analytical method Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 102000054765 polymorphisms of proteins Human genes 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 230000004087 circulation Effects 0.000 claims description 2
- 238000012106 screening analysis Methods 0.000 claims description 2
- 230000007423 decrease Effects 0.000 abstract description 3
- 238000009399 inbreeding Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 241000277323 Pangasius pangasius Species 0.000 abstract 4
- 241001233037 catfish Species 0.000 abstract 1
- 238000012252 genetic analysis Methods 0.000 abstract 1
- 238000013537 high throughput screening Methods 0.000 abstract 1
- 238000012165 high-throughput sequencing Methods 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 230000007717 exclusion Effects 0.000 description 9
- 238000009825 accumulation Methods 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000252210 Cyprinidae Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000282985 Cervus Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241001133085 Xenocypris davidi Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及黄尾鲴亲子鉴定的方法与试剂盒，方法包括：（1）提取黄尾鲴DNA；（2）通过高通量测序，将获得的59对微卫星标记进行PCR扩增，筛选分离出多态性高的引物进一步进行温度梯度筛选分析；（3）筛选得到的16对多态性高且能稳定扩增的微卫星标记，分别标上两种荧光引物，PCR扩增后，读取等位基因值；（4）挑选一组黄尾鲴个体单独进行受精孵化养殖，提取30个个体DNA作为子代群体，同时选取繁殖亲本33尾用于候选亲本建立亲子鉴定体系，进行亲子鉴定分析。本发明利用微卫星建立黄尾鲴亲子鉴定技术，准确度高，可以用于黄尾鲴后期个体识别，进行增殖放流的效果评估；也可进行群体遗传学分析，对有效防止其近亲繁殖和种质资源的衰退。The invention relates to a method and kit for paternity identification of the yellowtail catfish. The method includes: (1) extracting DNA of the yellowtail catfish; (2) performing PCR amplification on the obtained 59 pairs of microsatellite markers by high-throughput sequencing, screening and separating The primers with high polymorphism were further screened and analyzed by temperature gradient; (3) 16 pairs of microsatellite markers with high polymorphism and stable amplification obtained from the screening were marked with two fluorescent primers respectively. After PCR amplification, read Take the allele value; (4) Select a group of yellow-tailed catfish individuals for fertilization, hatching and breeding, and extract DNA from 30 individuals as a progeny group. At the same time, 33 breeding parents are selected for candidate parents to establish a paternity test system and conduct paternity test. analyze. The invention utilizes microsatellites to establish a paternity identification technology for the yellowtail catfish, has high accuracy, can be used for individual identification of the yellowtail catfish in the later stage, and evaluates the effect of proliferation and release; it can also carry out population genetic analysis to effectively prevent its inbreeding and breeding. decline in quality resources.
Description
Technical field
The invention belongs to aquaculture fields, are related to the method and kit of yellow tail silver xenocypris paternity test.
Background technique
Yellow tail silver xenocypris (Xenocypris davidi) it is under the jurisdiction of Cyprinidae (Cyprinidae), silver xenocypris subfamily
(Xenocyprininae), Xenocypris (Xenocypris) is a kind of middle-size and small-size economic freshwater fish, is distributed widely in each of China
In big water system, streams.Feeding habits are ominivore-fish, organic debris of mainly ingesting, Filamentous algae, diatom and green alga；Secondary food
For zooplankter, " street cleaner " referred to as in water body is widely used in the enhancement releasing of the natural water areas such as reservoir, lake.
Microsatellite DNA is also known as simple sequence repeats (Simple Sequence Repeats, SSR) or Short tandem repeatSTR
Sequence (Short Tandem Repeats, STR), generally by the guarantor of the core recurring unit of 1 ~ 6 bp and repeat region both ends
Sequence two parts composition is kept, different repetition numbers and repeatable position are to form the basis of microsatellite locus polymorphism, microsatellite
Its plurality of advantages of molecular labeling and huge application prospect, are widely used in various biological studies, in aquatic livestock
Main application group's science of heredity, Relationship iden- tification and individual identification, the building of genetic map and molecular mark etc.
Aspect.
Yellow tail silver xenocypris mainly releases fish as the whole nation, and the recruitment evaluation of the enhancement releasing in later period is that Fishery Resources Enhancement is put
Most noticeable problem during stream, it will determine " what (releasing type) put " " putting how many (releasing quantity) " and " how
Put and (release mode) ", the key for answering these problems then needs to be evaluated with reliable means such as molecular labeling etc..
Summary of the invention
The present invention provides a kind of method of yellow tail silver xenocypris paternity test, establishes yellow tail silver xenocypris paternity test technology using microsatellite,
Accuracy is high, can be used for yellow tail silver xenocypris later period individual identification, carries out the recruitment evaluation of enhancement releasing；It can also carry out Population Genetics
Analysis, the decline to its inbreeding and germ plasm resource is effectively prevent.
The method of yellow tail silver xenocypris paternity test, comprising:
(1) Huang tail silver xenocypris individual DNA is extracted: extracting yellow tail silver xenocypris parent and daughter DNA is spare；
(2) by high-flux sequence, 59 pairs of microsatellite markers of acquisition the screening of yellow tail silver xenocypris microsatellite marker: are subjected to PCR expansion
Increase, the high primer further progress temperature gradient screening analysis of polymorphism is isolated in screening；
(3) synthesis and PCR amplification of yellow tail silver xenocypris micro-satellite primers fluorescent marker: the 16 pairs of polymorphisms screened are high and can stablize
The microsatellite marker of amplification, puts on two kinds of fluorescent primers respectively, after PCR amplification, reads allele value；
(4) it the foundation of yellow tail silver xenocypris paternity test technology: selects one group of Huang tail silver xenocypris individual and individually carries out fertilization and hatching cultivation, extract
30 individual DNA choose breeding 33 tail of parent and establish paternity test system for candidate parent as progeny population, carry out
Paternity test analysis.
1 16 pairs of table yellow tail silver xenocypris microsatellite marker features
Preferably, step (3) Huang tail silver xenocypris paternity test PCR reaction system are as follows:
Preferably, yellow tail silver xenocypris paternity test PCR reaction condition are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45s, anneal 45s under the annealing temperature of respective primer, 72 DEG C of extension 90s, and 35
A circulation；72 DEG C re-extend 10 min, 4 DEG C of preservation PCR products.
The present invention also provides yellow tail silver xenocypris paternity test kits, and the paternity test of yellow tail silver xenocypris may be implemented.
Kit (100 specifications) forms as follows:
The invention has the benefit that the present invention establishes yellow tail silver xenocypris paternity test technology using microsatellite, accuracy is high, Ke Yiyong
It is assessed in the enhancement effect in yellow tail silver xenocypris later period, inbreeding and the decline of germ plasm resource can also be effectively prevent.
Specific embodiment
It is specifically described below to further appreciate that the contents of the present invention, feature:
The method of yellow tail silver xenocypris paternity test, comprising:
One) Huang tail silver xenocypris individual DNA is extracted
Yellow 33 tail of tail silver xenocypris parent population tail fin sample of acquisition, 30 tail of filial generation, absolute alcohol room temperature preservation are spare.Reagent is extracted using DNA
Box extracts DNA, carries out DNA concentration measurement by NanoDrop2000, after be diluted to 100ng/ul and save backup.
Two) screening of yellow tail silver xenocypris microsatellite marker:
By high-flux sequence, 59 pairs of microsatellite markers of acquisition are subjected to PCR amplification, utilize 12% polyacrylamide gel electricity
Swimming carries out preliminary screening separation, obtains 16 pairs of polymorphisms height and can stablize the microsatellite marker of amplification, further progress temperature
Degree gradient screens and carries out population analysis；
Three) synthesis and PCR amplification of yellow tail silver xenocypris micro-satellite primers fluorescent marker:
Screening obtains 16 pairs of polymorphisms height and can stablize the microsatellite marker (table 1) of amplification, puts on two kinds of fluorescent primer (5- respectively
HEX and 5-FAM), after PCR reaction system amplification in table 2, product is read after 1% agarose gel electrophoresis Preliminary detection
Take allele value.
Four) foundation of yellow tail silver xenocypris paternity test technology
Yellow tail silver xenocypris whole artificial propagation offspring selects one group of individual and individually carries out fertilization and hatching breeding, and 30 tails are chosen in the filial generation of generation
DNA extraction is carried out, while being used for paternity test Establishing for other breeding 31 tails of parent as candidate parent.Utilize cervus
3.0 software statistics allele categories, heterozygosity (H 0 ), expectation heterozygosity (He), polymorphism information content (PIC), average elimination factor
With accumulative elimination factor.It see the table below 3.
3:16 microsatellite marker number of alleles of table, observation heterozygosity (Ho), expectation heterozygosity (He), polymorphism information contain
It measures (PIC), the accumulation probability of exclusion of the first parent, the second parent accumulation exclusion generally and parents accumulates probability of exclusion.
Using 30 individuals as progeny population, it is assumed that 33 tail candidate parents are the parent of this 30 offspring individuals, with sieve
The yellow tail silver xenocypris microsatellite of 16 pairs selected carries out simulation paternity test, samples 10000 times, does not know to carry out modeling point with Parent
Analysis, while choosing other breeding 33 tails of parent individual as candidate parent for establishing paternity test system, respectively with maternal or
Male parent is known, Parent is known and the unknown modeling of Parent carries out paternity test analysis, and PRELIMINARY RESULTS is shown, the first parent
Accumulation probability of exclusion (combined exclusion probability of the first parent, CE-1P), second
Parent accumulate probability of exclusion (combined exclusion probability of the second parent, CE-2P) and
Parents accumulate probability of exclusion (combined exclusion probability of a parent pair, CE-PP)
0.99807593,0.999983 and 0.99999999.30 offspring individuals accurately find parent, and paternity test result is shown in
Table 4.
Shown in above-mentioned 30 individual paternity test results, wherein LOD value is parent-offspring's index (paternity index)
Logarithm, meaning of the LOD greater than 0 is that compared with any parent, candidate parent is most likely to be true parental generation.Above-mentioned knot
Fruit shows that 30 individuals accurately have found real male parent and maternal respectively YM1 and YF1, and the above analysis result confirms
It is feasible that this 16 yellow tail silver xenocypris microsatellite markers, which are used for yellow tail silver xenocypris paternity test,.
The above method is utilized in summary, and the paternity test of yellow tail silver xenocypris may be implemented, formed according to agents useful for same in method
Kit for yellow tail silver xenocypris paternity test.Kit (100 specifications), such as the following table 5.
Sequence table
<110>Zhejiang Institute of Fresh Water Aquatic Products
<120>method and kit of yellow tail silver xenocypris paternity test
<160> 48
<170> SIPOSequenceListing 1.0
<210> 1
<211> 195
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(195)
<223>microsatellite sequence 5LOC
<400> 1
gacctgtcac ttttttagtg cacactacag ccattgttgc ctggcataat agaaccagag 60
ctttaaaaaa cttacttttt tttagttata agacacagta aggttgttgt tattattatt 120
attattatta ttattattat tattatagta tacttttttt cattcattta attcatgcac 180
agccatgaca gaact 195
<210> 2
<211> 284
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(284)
<223>microsatellite sequence 8LOC
<400> 2
tttccaccat aaacacctta tttattgaat ataatactta tgcaaactct tattgttctg 60
tttcagagcc cagattcagt gatgttggcg ttgaaactca acagctccac attactggag 120
cgaaagatca gggtgaagcg ctccatgaag aaggagaaag agaagaaatc acatccaggt 180
cgtcagtccg aaggaaaaga gcgatggaga gcaggaggag gaggaggagg attcagaggt 240
ccaaagcagg agttcagaaa catgacggga agaactcagt ccat 284
<210> 3
<211> 281
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(281)
<223>microsatellite sequence 13LOC
<400> 3
attttgttgt ttaaaataac tttttctaat ttaatatatt ttaaaatgta atttattcca 60
ttcctctagc cttcagtgtc acataatcct tcagaaatca ttctaacatt ctggtgctca 120
aaataacatt tattattatt attattatta ttattattat aagtgttgaa aacagttatg 180
ctcatcaata tgtttttgtg gaaaccatga tttatataat gttttttttt aatgatcttt 240
tacagatgga tgaataatgg ctggtcatga ataatggtct c 281
<210> 4
<211> 228
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(228)
<223>microsatellite sequence 14LOC
<400> 4
aatgcactca attgattcca tttcaaggaa tctgttgcat cacttcagaa aagaaagata 60
agtagcctac tatgtggctg gactatttaa ggtactagat ccacacagaa taataataat 120
aataataata ataataataa agctcttttg tttgagtcag ggcgctctac cgaactgcaa 180
agagctcata actcaaccaa cccaggaaga tgactacaaa aattgtaa 228
<210> 5
<211> 214
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(214)
<223>microsatellite sequence 18LOC
<400> 5
tgtaggtcac agtagagaac ttcctacgag ttctgacagg gcgcctcccc cccagtaccc 60
ctcgatccaa gcgtttgcta tcagacgacc gcagcaacat tctcatctac ctgacaggtc 120
agaccagtaa cagtttatta ttattattat tattcctcct ttttcctgat tccatgactc 180
tccattttca caggtcatgg tggaaatggc ttcc 214
<210> 6
<211> 278
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(278)
<223>microsatellite sequence 20LOC
<400> 6
actgtcatgg tgaccagggg acgagggatg tttggcacct ggtggcgtgt gagctggacg 60
cctggttctt tcagaacgct gaggagatga tgatgatgat gaagataagg caggaggtga 120
acgtgttctt gttggaagtt gattgaggct ccttctagac atgttggatg ggagctccac 180
ttaagaaagg aaaaaaagaa aaaagtttga gcatcctgca agatggcaca caaccaaacc 240
acaagaaatt caatatttgc aaacagcaga aggacaga 278
<210> 7
<211> 217
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(217)
<223>microsatellite sequence 21LOC
<400> 7
tggagcggtg ctcgtccagc ttcctgtgct tgctgtcact gtagtacctg caaaacgagt 60
cattgatgat gaggatgatg atgatgatga tgatgatgat gatgatacaa ccaagctgga 120
cattttactg atacaacaaa acaacctgaa attctcacac taatttaaag atggtaaaaa 180
cacatgcata acaaatgctg aaataagatt ttactaa 217
<210> 8
<211> 214
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(214)
<223>microsatellite sequence 24LOC
<400> 8
tgtggtaacc caacatcaga acctcgactc tcaaaatcat acaattataa ttaaatatat 60
atatatatat atatatatat atatatatat attaccaaga tctgcttagg cccaacaata 120
tttaaaattt gctaagtatt tagtttctaa atggtttgat gctctcagca gcattattaa 180
cctttagcat agtttatgag aacaaaacac acct 214
<210> 9
<211> 279
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(279)
<223>microsatellite sequence 28LOC
<400> 9
aaatccaact acattaatta aaaaatatct aactgaacat ctatgttgct taaaataaaa 60
taaaatccag ttgagcttct acattctaca tgtctggtca gcgaagaagg ttgtccaacc 120
cattgtcaca tccttcagac caccaagatt tcgatcctca aagagaatat cgctccatat 180
tacaacaatg atggacacac acacacacac acacacacag tgtaagtgat agcttatgtg 240
ctgtcagcag cttcaagtga gagcttagag ctttgtgca 279
<210> 10
<211> 204
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(204)
<223>microsatellite sequence 29LOC
<400> 10
ggaaaatatt actaaagaaa tcattgtggc actatatgtg gttacatgat ggtgccaaag 60
tttgttatct cccaaataat aataataata ataataataa taaagactgc atctgatctg 120
ccactatatt aaataattaa aactgaatta acactagctg tatgcagttg gtgaccagat 180
tttctaaaaa ttgaaaatcc caca 204
<210> 11
<211> 224
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(224)
<223>microsatellite sequence 30LOC
<400> 11
taataatcca gaacaagtaa acggatgaat ggtgcagcag tcttttaaag ttgattttta 60
atgtaaatgt ataataataa taataataat aataataata atgctaacac tttgtagtaa 120
cggtttcatt agcattgaac taacaataag caatacattt gttactgtat ttgttaatct 180
ttgttatcat tatttaaaaa tacaattgtt tattgtttgt tcat 224
<210> 12
<211> 243
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(243)
<223>microsatellite sequence 37LOC
<400> 12
ttcagttgaa tcaaaaatat ggattaaaca acgatggatg tcgctttcaa gttatttaag 60
agaaaattct ggattcatct attatcaggt ggcaacaatt atgtccacct cttcatttaa 120
taataataat aataataata ataataaaca tttatttgta tagcactttt cataaatgta 180
atgaaactca aagagcttca caagcataaa aacaaataat acaatttcac acaatgtcaa 240
tat 243
<210> 13
<211> 277
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(277)
<223>microsatellite sequence 39LOC
<400> 13
aaggggtctt gatcaccaac tttgacttgt ttagtagttt gaaaattgtg cagcacactc 60
cactcctgag gtgcagagaa tgacattcaa gtatgaactg aataataata ataataataa 120
taaaatgaat aaatgcaatt taaacaagta aaattcacca cctaatctga ccaagacatg 180
ttctcaaatt agtatgtgag actgacctga tggcttttgg tcatggcaag gcaaggccat 240
tttatttgta tagcacattt catacacaat ggtaatt 277
<210> 14
<211> 227
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(227)
<223>microsatellite sequence 41LOC
<400> 14
catgcattta tttaagttgg ggtaacagat ccactgaatt acattttaga gtaagggata 60
gtgatatata ggaatatttc tttctttctt tctttctttc tttctttctt tcttttaaat 120
cacaattcta aaacattcta agacaattcg ttgaatcaga tgattatctt gtccgctaac 180
ctgatttcaa atcaatttgt aaaccagaag atcaatgtgt tttttct 227
<210> 15
<211> 243
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(243)
<223>microsatellite sequence 51LOC
<400> 15
gaatggaatt tgtgaaaaat gaagcagaat tcaagaacaa acgctgctgg atttccactc 60
actcacctct gacacactct gaactctctc tggatctcta cgagctgatg atcttcaaga 120
taacctgcag aaaacatcat catcatcatc atcatcatca tcatcatcac agacacattc 180
gtctctcact gcagtgtgta tttctgtgat tattgtcctt tattgtgcat tctttaatgt 240
ttt 243
<210> 16
<211> 227
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(227)
<223>microsatellite sequence 52LOC
<400> 16
agaaaaagac aaggaagcag ttcaatgaat caaactttaa agaaaacata gaaactcaat 60
gaaacaaact gtaaatcagt tttcaaataa taatgaaaaa atatatataa taatgagaaa 120
ataataataa taataataat aataataata ataataataa taataataat aataatacaa 180
aaatacagac tatgaatatc catggggaaa aaatgaaaga gaaagct 227
<210> 17
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>5LOC primer 1
<400> 17
cactacagcc attgttgcct 20
<210> 18
<211> 23
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(23)
<223>5LOC primer 2
<400> 18
tgtgttctgt catggctgtg cat 23
<210> 19
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>8LOC primer 1
<400> 19
cagattcagt gatgttggcg 20
<210> 20
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>8LOC primer 2
<400> 20
tgaactcctg ctttggacct 20
<210> 21
<211> 21
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(21)
<223>13LOC primer 1
<400> 21
ccattcctct agccttcagt g 21
<210> 22
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>13LOC primer 2
<400> 22
ccattattca tgaccagcca 20
<210> 23
<211> 21
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(21)
<223>14LOC primer 1
<400> 23
ccatttcaag gaatctgttg c 21
<210> 24
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>14LOC primer 2
<400> 24
tcttcctggg ttggttgagt 20
<210> 25
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>18LOC primer 1
<400> 25
cctacgagtt ctgacagggc 20
<210> 26
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>18LOC primer 2
<400> 26
gaagccattt ccaccatgac 20
<210> 27
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>20LOC primer 1
<400> 27
cctggttctt tcagaacgct 20
<210> 28
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>20LOC primer 2
<400> 28
cttgtggttt ggttgtgtgc 20
<210> 29
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>21LOC primer 1
<400> 29
cctgtgcttg ctgtcactgt 20
<210> 30
<211> 22
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(22)
<223>21LOC primer 2
<400> 30
ttcagcattt gttatgcatg tg 22
<210> 31
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>24LOC primer 1
<400> 31
aacccaacat cagaacctcg 20
<210> 32
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>24LOC primer 2
<400> 32
ctgctgagag catcaaacca 20
<210> 33
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>28LOC primer 1
<400> 33
tctggtcagc gaagaaggtt 20
<210> 34
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>28LOC primer 2
<400> 34
gctctcactt gaagctgctg 20
<210> 35
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>29LOC primer 1
<400> 35
tgatggtgcc aaagtttgtt 20
<210> 36
<211> 21
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(21)
<223>29LOC primer 2
<400> 36
aaatctggtc accaactgca t 21
<210> 37
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>30LOC primer 1
<400> 37
taaacggatg aatggtgcag 20
<210> 38
<211> 22
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(22)
<223>30LOC primer 2
<400> 38
agttcaatgc taatgaaacc gt 22
<210> 39
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>37LOC primer 1
<400> 39
taaacaacga tggatgtcgc 20
<210> 40
<211> 22
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(22)
<223>37LOC primer 2
<400> 40
tgcttgtgaa gctctttgag tt 22
<210> 41
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>39LOC primer 1
<400> 41
acactccact cctgaggtgc 20
<210> 42
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>39LOC primer 2
<400> 42
ccaaaagcca tcaggtcagt 20
<210> 43
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>41LOC primer 1
<400> 43
tggggtaaca gatccactga 20
<210> 44
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>41LOC primer 2
<400> 44
tgaaatcagg ttagcggaca 20
<210> 45
<211> 20
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(20)
<223>51LOC primer 1
<400> 45
agaacaaacg ctgctggatt 20
<210> 46
<211> 21
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(21)
<223>51LOC primer 2
<400> 46
ctgcagtgag agacgaatgt g 21
<210> 47
<211> 22
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(22)
<223>52LOC primer 1
<400> 47
ggaagcagtt caatgaatca aa 22
<210> 48
<211> 23
<212> DNA
<213>artificial sequence
<220>
<222> (1)..(23)
<223>52LOC primer 2
<400> 48
tccccatgga tattcatagt ctg 23
Claims (7)
1. the method for yellow tail silver xenocypris paternity test, which is characterized in that
(1) Huang tail silver xenocypris individual DNA is extracted: extracting yellow tail silver xenocypris parent and daughter DNA is spare；
(2) by high-flux sequence, 59 pairs of microsatellite markers of acquisition the screening of yellow tail silver xenocypris microsatellite marker: are subjected to PCR expansion
Increase, the high primer further progress temperature gradient screening analysis of polymorphism is isolated in screening；
(3) synthesis and PCR amplification of yellow tail silver xenocypris micro-satellite primers fluorescent marker: the 16 pairs of polymorphisms screened are high and can stablize
The microsatellite marker of amplification, puts on two kinds of fluorescent primers respectively, after PCR amplification, reads allele value；
(4) it the foundation of yellow tail silver xenocypris paternity test technology: selects one group of Huang tail silver xenocypris individual and individually carries out fertilization and hatching cultivation, extract
30 individual DNA choose breeding 33 tail of parent and establish paternity test system for candidate parent as progeny population, carry out
Paternity test analysis.
2. the method for yellow tail silver xenocypris paternity test according to claim 1, which is characterized in that 16 pairs of microsatellite marker sequences are
5LOC: as shown in SEQ ID NO.1,8 LOC: as shown in SEQ ID NO.2,13 LOC: as shown in SEQ ID NO.3,
14LOC: as shown in SEQ ID NO.4,18 LOC: as shown in SEQ ID NO.5,20LOC: as shown in SEQ ID NO.6,
21LOC: as shown in SEQ ID NO.7,24 LOC: as shown in SEQ ID NO.8,28LOC: as shown in SEQ ID NO.9,29
LOC: as shown in SEQ ID NO.10,30LOC: as shown in SEQ ID NO.11,37LOC: as shown in SEQ ID NO.12,
39LOC: as shown in SEQ ID NO.13,41 LOC: as shown in SEQ ID NO.14,51 LOC: as shown in SEQ ID NO.15,
52 LOC: as shown in SEQ ID NO.16.
3. the method for yellow tail silver xenocypris paternity test according to claim 2, which is characterized in that 16 pairs of yellow tail silver xenocypris microsatellite markers
Primer sequence are as follows: 5LOC: as shown in SEQ ID NO.17-18,8 LOC: as shown in SEQ ID NO.19-20,13 LOC: such as
Shown in SEQ ID NO.21-22,14LOC: as shown in SEQ ID NO.23-24,18 LOC: as shown in SEQ ID NO.25-26,
20LOC: as shown in SEQ ID NO.27-28,21LOC: as shown in SEQ ID NO.29-30,24 LOC: such as SEQ ID
Shown in NO.31-32,28LOC: as shown in SEQ ID NO.33-34,29 LOC: as shown in SEQ ID NO.35-36,30LOC:
As shown in SEQ ID NO.37-38,37LOC: as shown in SEQ ID NO.39-40,39LOC: such as SEQ ID NO.41-42 institute
Show, 41 LOC: as shown in SEQ ID NO.43-44,51 LOC: as shown in SEQ ID NO.45-46,52 LOC: such as SEQ ID
Shown in NO.47-48.
4. the method for yellow tail silver xenocypris paternity test according to claim 3, which is characterized in that step (3) Huang tail silver xenocypris paternity test
PCR reaction system is 25 uL:2 of total volume × Taq Master Mix12.5 uL, primer each 1.0 uL, Template(100
Ng/ μ L) 4 uL, ddH2O6.5 uL.
5. the method for yellow tail silver xenocypris paternity test according to claim 4, which is characterized in that step (3) Huang tail silver xenocypris paternity test
PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45s, anneal 45s under the annealing temperature of respective primer, and 72 DEG C
Extend 90s, 35 circulations；72 DEG C re-extend 10 min, 4 DEG C of preservation PCR products.
6. the method for yellow tail silver xenocypris paternity test according to claim 1, which is characterized in that used by step (3) two kinds it is glimmering
Light is 5-HEX and 5-FAM.
7. a kind of Huang tail silver xenocypris paternity test kit, which is characterized in that include 16 pairs as claimed in claim 3 yellow tail silver xenocypris microsatellites
Labeled primer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811588161.1A CN109536620B (en) | 2018-12-25 | 2018-12-25 | Method and kit for paternity test of xenocypris davidi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811588161.1A CN109536620B (en) | 2018-12-25 | 2018-12-25 | Method and kit for paternity test of xenocypris davidi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109536620A true CN109536620A (en) | 2019-03-29 |
| CN109536620B CN109536620B (en) | 2021-09-10 |
Family
ID=65857593
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811588161.1A Active CN109536620B (en) | 2018-12-25 | 2018-12-25 | Method and kit for paternity test of xenocypris davidi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109536620B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877447A (en) * | 2021-04-21 | 2021-06-01 | 贵州大学 | Parent-child identification method for Qinling mountain brachymystax lenok |
| CN113774152A (en) * | 2021-10-21 | 2021-12-10 | 中国水产科学研究院南海水产研究所 | A kind of primer and method for parent-child identification based on microsatellite marker |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955934A (en) * | 2010-10-25 | 2011-01-26 | 浙江省淡水水产研究所 | Hemibarbus maculates microsatellite sites and primers |
| WO2012155084A1 (en) * | 2011-05-12 | 2012-11-15 | Netbio, Inc. | Methods and compositions for rapid multiplex amplification of str loci |
| CN104726555A (en) * | 2015-02-03 | 2015-06-24 | 浙江省海洋水产研究所 | Nibea japonica microsatellite DNA markers |
| CN108611405A (en) * | 2018-04-25 | 2018-10-02 | 浙江省淡水水产研究所 | A method of sticking up mouth Culter paternity test microsatellite Multiplex fluorescent PCRs |
-
2018
- 2018-12-25 CN CN201811588161.1A patent/CN109536620B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955934A (en) * | 2010-10-25 | 2011-01-26 | 浙江省淡水水产研究所 | Hemibarbus maculates microsatellite sites and primers |
| WO2012155084A1 (en) * | 2011-05-12 | 2012-11-15 | Netbio, Inc. | Methods and compositions for rapid multiplex amplification of str loci |
| CN104726555A (en) * | 2015-02-03 | 2015-06-24 | 浙江省海洋水产研究所 | Nibea japonica microsatellite DNA markers |
| CN108611405A (en) * | 2018-04-25 | 2018-10-02 | 浙江省淡水水产研究所 | A method of sticking up mouth Culter paternity test microsatellite Multiplex fluorescent PCRs |
Non-Patent Citations (2)
| Title |
|---|
| INÉS GONZÁLEZ-CASTELLANO ET AL.: ""Isolation and characterization of 21 polymorphic microsatellite loci for the rockpool shrimp Palaemon elegans using Illumina MiSeq sequencing"", 《SCIENNTIFIC REPORTS》 * |
| 张燕萍等: ""基于RNA-seq的黄尾鲴肝脏转录组测序与分析"", 《水生态学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877447A (en) * | 2021-04-21 | 2021-06-01 | 贵州大学 | Parent-child identification method for Qinling mountain brachymystax lenok |
| CN113774152A (en) * | 2021-10-21 | 2021-12-10 | 中国水产科学研究院南海水产研究所 | A kind of primer and method for parent-child identification based on microsatellite marker |
| CN113774152B (en) * | 2021-10-21 | 2023-07-14 | 中国水产科学研究院南海水产研究所 | A primer and method for paternity identification of sea bass based on microsatellite markers |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109536620B (en) | 2021-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106811540B (en) | Microsatellite marker and specific primer for identifying male and female individuals of Pseudobagrus ussuriensis and application of microsatellite marker and specific primer | |
| CN115261489A (en) | A primer set for Changchun bream microsatellite marker and its application and evaluation method | |
| CN104498613A (en) | SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application | |
| Parpinelli et al. | MRJP microsatellite markers in Africanized Apis mellifera colonies selected on the basis of royal jelly production | |
| Mahmoodi et al. | Sequence analysis of 12S rRNA and 16S rRNA mitochondrial genes in Iranian Afshari sheep. | |
| CN117431324B (en) | A high-density SNP chip in the whole genome of dairy cows and its application | |
| CN112029868B (en) | Microsatellite marker of portunus trituberculatus and application of microsatellite marker in growth trait association analysis | |
| CN109536620A (en) | The method and kit of yellow tail silver xenocypris paternity test | |
| Kánainé Sipos et al. | Comparative genetic analysis of natural and farmed populations of pike-perch (Sander lucioperca) | |
| Bekaert et al. | Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis | |
| CN110438242B (en) | A Primer for Portunus trituberculatus Microsatellite Marker and Its Application | |
| CN114317770B (en) | SNP molecular marker related to growth of Yangtze river coilia ectenes as well as detection primer and application thereof | |
| CN111286546B (en) | A salt-tolerant molecular marker C1480 of Portunus trituratus and its application | |
| Emil et al. | Genetic characterization of Romanian local breeds using microsatellite markers | |
| CN111286545B (en) | A Molecular Marker C0 for Salt-Alkali Tolerance of Portunus trituratus and its Application | |
| CN118406780A (en) | Primer pairs for SNP markers related to pig chromosome 2 and piglet number born alive and their application | |
| CN117965752A (en) | A SNP marker primer pair related to the pig vulva width trait and its application | |
| Sharma et al. | Simple sequence repeat (SSR) genotypic data reveal high genetic diversity in Rajasthan donkey of India | |
| Syaifudin et al. | DNA barcoding of wild and culture tilapia based on cytochrome c oxidase subunit I (COI) gene | |
| Rossetti et al. | Genetic investigation for the characterization of three indigenous pig breeds of southern Italy: advantages and prospects. | |
| KR102017236B1 (en) | The primer set for analysis of cephalopods and methods for their qualitativeand quantitative analysis | |
| CN101967519A (en) | Method for identifying fx151 genetic variation map by using litopenaeus vannamei boone fx151 microsatellite DNA marker | |
| CN106282379B (en) | Hybridize the CH of Pelteobagrus fulvidraco4Digestion identification method | |
| Kathiravan et al. | Short tandem repeat based analysis of genetic variability in Kanarese buffalo of South India | |
| Liu et al. | P1009 Sheep reference genome sequence updates: Texel improvements and Rambouillet progress |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |