CN109536620B - Method and kit for paternity test of xenocypris davidi - Google Patents
Method and kit for paternity test of xenocypris davidi Download PDFInfo
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- CN109536620B CN109536620B CN201811588161.1A CN201811588161A CN109536620B CN 109536620 B CN109536620 B CN 109536620B CN 201811588161 A CN201811588161 A CN 201811588161A CN 109536620 B CN109536620 B CN 109536620B
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention relates to a xenocypris davidi paternity test method and a kit, the method comprises the following steps: (1) extracting DNA of xenocypris davidi; (2) performing PCR amplification on the obtained 59 microsatellite markers by high-throughput sequencing, screening and separating primers with high polymorphism, and further performing temperature gradient screening analysis; (3) screening to obtain 16 pairs of microsatellite markers with high polymorphism and stable amplification, respectively labeling two fluorescent primers, and reading allele values after PCR amplification; (4) selecting a group of xenocypris davidi individual for fertilization, hatching and breeding, extracting 30 individual DNAs as a filial generation group, and selecting 33 breeding parents for establishing a paternity test system for the candidate parents to perform paternity test analysis. The invention establishes the xenocypris davidi bleeker paternity test technology by using the microsatellite, has high accuracy, can be used for the later-stage individual identification of xenocypris davidi bleekers, and carries out the effect evaluation of proliferation and releasing; and population genetics analysis can also be carried out, so that the close-relative propagation and the decline of germplasm resources can be effectively prevented.
Description
Technical Field
The invention belongs to the field of aquaculture, and relates to a xenocypris davidi parent-child identification method and a kit.
Background
Xenocypris davidi (Xenocypris davidi) Belongs to Cyprinidae (Cyprinidae), Xenocyprinae (Xenocyprinae) and Xenocypris (Xenocypris), is a small and medium-sized fresh water economic fish, and is widely distributed in various large water systems and streams in China. Feeding nature is omnivorous fish, mainly ingests organic debris, filamentous algae, diatom and green algae; the secondary food is zooplankton, called "scavenger" in water, and is widely used for breeding and releasing natural water areas such as reservoirs and lakes.
The microsatellite DNA is also called Simple Sequence Repeats (SSR) or Short Tandem Repeat (STR), generally consists of two parts of a core repeat unit of 1-6 bp and conserved sequences at two ends of a repeat region, different repeat numbers and repeat positions are the basis for forming the polymorphism of the microsatellite loci, and the microsatellite molecular marker has many advantages and huge application prospects, is widely applied to various biological researches, and is mainly applied to aspects of population genetics, genetic relationship identification and individual identification, genetic map construction, molecular marker assisted breeding and the like in aquatic animals.
The effect evaluation of the later proliferation and releasing of xenocypris davidi as the main releasing fish in the country is the most interesting problem in the proliferation and releasing process of fishery resources, and the evaluation of the effect evaluation of the later proliferation and releasing of xenocypris davidi determines the 'what is released (releasing type)' the 'release amount' and the 'how to release (releasing mode)', and the key for solving the problems needs to be evaluated by reliable means such as molecular markers and the like.
Disclosure of Invention
The invention provides a xenocypris davidi paternity test method, which is characterized in that a xenocypris davidi paternity test technology is established by utilizing a microsatellite, so that the accuracy is high, and the xenocypris davidi can be used for later-stage individual identification of xenocypris davidi and effect evaluation of proliferation and releasing is carried out; and population genetics analysis can also be carried out, so that the close-relative propagation and the decline of germplasm resources can be effectively prevented.
A method for paternity test of xenocypris davidi bleeker, comprising the following steps:
(1) extracting individual DNA of xenocypris davidi: extracting DNA of a xenocypris davidi parent and filial generation for later use;
(2) screening of xenocypris davidi bleekers microsatellite markers: performing PCR amplification on the obtained 59 microsatellite markers by high-throughput sequencing, screening and separating primers with high polymorphism, and further performing temperature gradient screening analysis;
(3) synthesizing a fluorescence label of a xenocypris davidi microsatellite primer and carrying out PCR amplification: screening to obtain 16 pairs of microsatellite markers with high polymorphism and stable amplification, respectively labeling two fluorescent primers, and reading allele values after PCR amplification;
(4) the establishment of xenocypris davidi paternity test technology: selecting a group of xenocypris davidi individual for fertilization, hatching and breeding, extracting 30 individual DNAs as a filial generation group, and selecting 33 breeding parents for establishing a paternity test system for the candidate parents to perform paternity test analysis.
TABLE 116 microsatellite marker characteristics of xenocypris davidi
Preferably, the paternity test PCR reaction system of xenocypris davidi in the step (3) is as follows:
preferably, the paternity test PCR reaction conditions of xenocypris davidi are as follows:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at the annealing temperature of the respective primers for 45s, extension at 72 ℃ for 90s, 35 cycles; the PCR product was re-extended at 72 ℃ for 10 min and stored at 4 ℃.
The invention also provides a xenocypris davidi paternity test kit which can realize paternity test of xenocypris davidi.
Kit (100-fold specification), consisting of:
the invention has the beneficial effects that: the invention establishes the xenocypris davidi bleeker paternity test technology by using the microsatellite, has high accuracy, can be used for evaluating the proliferation and releasing effect of xenocypris davidi bleekers in the later period, and can also effectively prevent the inbreeding and the decline of germ plasm resources.
Detailed Description
For further understanding of the contents and features of the present invention, the following detailed description is given:
a method for paternity test of xenocypris davidi bleeker, comprising the following steps:
extraction of individual DNA of xenocypris davidi
Collecting 33 tail of a tail fin sample of a parent fish of xenocypris davidi, collecting 30 offspring, and storing anhydrous alcohol at room temperature for later use. DNA is extracted by using a DNA extraction kit, then the DNA concentration is measured by NanoDrop2000, and then the DNA is diluted to 100ng/ul for storage and standby.
II) screening of xenocypris davidi microsatellite markers:
performing PCR amplification on the obtained 59 microsatellite markers by high-throughput sequencing, performing primary screening separation by using 12% polyacrylamide gel electrophoresis to obtain 16 microsatellite markers which have high polymorphism and can be stably amplified, and further performing temperature gradient screening and population analysis;
thirdly), synthesizing fluorescence labeling of xenocypris davidi microsatellite primers and PCR amplification:
screening to obtain 16 pairs of microsatellite markers (table 1) with high polymorphism and stable amplification, respectively labeling two fluorescent primers (5-HEX and 5-FAM), amplifying according to a PCR reaction system in table 2, primarily detecting products by 1% agarose gel electrophoresis, and reading allele values.
Fourth) establishing parent-child identification technology of xenocypris davidi
A group of individuals are selected from the full-artificial breeding offspring of xenocypris davidi for fertilization, hatching and breeding, 30 offspring are selected for DNA extraction, and meanwhile, 31 offspring of other breeding parents are used as candidate parents for establishing a paternity test system. Using cervus 3.0 software to count allelomorphs and heterozygosity: (H 0 ) Desired heterozygosity: (He) Polymorphic Information Content (PIC), average exclusion rate, and cumulative exclusion rate. See table 3 below.
Table 3: a figure of 16 microsatellite marker alleles, observed heterozygosity (Ho), expected heterozygosity (He), Polymorphic Information Content (PIC), cumulative exclusion probability of the first parent, cumulative exclusion probability of the second parent, and cumulative exclusion probability of the parents.
The method comprises the steps of taking 30 individuals as a progeny population, assuming that 33 candidate parents are all parents of the 30 progeny individuals, performing simulated paternity test on xenocypris microsatellites by using the screened 16, sampling 10000 times, performing modeling analysis by using the parents and the parents, selecting 33 parents of other propagation parent individuals as candidate parents to establish a paternity test system, performing paternity test analysis by using the parents or the parents which are known, the parents which are known and the parents which are unknown, and displaying a primary result, wherein the cumulative exclusion probability (CE-1P) of the first parent, the cumulative exclusion probability (CE-2P) of the second parent and the cumulative exclusion probability (CE-PP) of the parents are 39 0.99807593, 0.999983 and 0.99999999 respectively. The parents of all 30 offspring individuals can be accurately found, and the paternity test results are shown in table 4.
The above paternity test results for 30 individuals showed that the LOD value is the logarithmic value of the paternity index (paternity index), and the meaning that the LOD is greater than 0 is that the candidate parent is most likely to be the true parent compared to any parent. The results show that 30 individuals accurately find out real male parents and female parents of the xenocypris YM1 and YF1 respectively, and the analysis results prove that the 16 xenocypris markers for paternity test of xenocypris davidi.
In conclusion, the paternity test of the xenocypris davidi can be realized by the method, and the kit for paternity test of the xenocypris davidi is formed according to the reagents used in the method. Kit (100-fold format), table 5 below.
Sequence listing
<110> research institute for fresh water aquatic products in Zhejiang province
<120> xenocypris davidi paternity test method and kit
<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 (2)
1. The primer pair for paternity test of xenocypris davidi bleeker, which is characterized by comprising 16 pairs of primers with the following sequences: 5 primer of LOC: as shown in SEQ ID NO.17-18, primer of 8 LOC: as shown in SEQ ID NO.19-20, 13LOC primer: as shown in SEQ ID NO.21-22, primer of 14 LOC: as shown in SEQ ID NO.23-24, 18LOC primer: as shown in SEQ ID NO.25-26, 20LOC primer: primers for 21LOC shown in SEQ ID NO. 27-28: as shown in SEQ ID NO.29-30, primer of 24 LOC: as shown in SEQ ID NO.31-32, 28LOC primer: primers of 29LOC as shown in SEQ ID NO. 33-34: as shown in SEQ ID NO.35-36, primer of 30 LOC: primers of 37LOC as shown in SEQ ID NO. 37-38: as shown in SEQ ID NO.39-40, primer of 39 LOC: shown as SEQ ID NO.41-42, primer of 41 LOC: primers for 51LOC shown in SEQ ID NO. 43-44: as shown in SEQ ID NO.45-46, primer of 52 LOC: as shown in SEQ ID NO. 47-48.
2. A xenocypris davidi kiss-identification kit, characterized by comprising the primer pair for xenocypris davidi kiss-identification according to claim 1.
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| CN112877447A (en) * | 2021-04-21 | 2021-06-01 | 贵州大学 | Parent-child identification method for Qinling mountain brachymystax lenok |
| CN113774152B (en) * | 2021-10-21 | 2023-07-14 | 中国水产科学研究院南海水产研究所 | Primer and method for paternity test of lateolabrax japonicus based on microsatellite markers |
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 |
|---|
| "Isolation and characterization of 21 polymorphic microsatellite loci for the rockpool shrimp Palaemon elegans using Illumina MiSeq sequencing";Inés González-Castellano et al.;《SCIENNTIFIC REPORTS》;20181121;第8卷;第1-6页 * |
| "基于RNA-seq的黄尾鲴肝脏转录组测序与分析";张燕萍等;《水生态学杂志》;20181130;第39卷(第6期);第87-94页 * |
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