CN108504743A - A kind of screening technique of Penaeus monodon A1426G single nucleotide polymorphism candidate sequences - Google Patents
A kind of screening technique of Penaeus monodon A1426G single nucleotide polymorphism candidate sequences Download PDFInfo
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- CN108504743A CN108504743A CN201810011659.5A CN201810011659A CN108504743A CN 108504743 A CN108504743 A CN 108504743A CN 201810011659 A CN201810011659 A CN 201810011659A CN 108504743 A CN108504743 A CN 108504743A
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Abstract
Disclosed by the invention is a kind of screening technique of Penaeus monodon single nucleotide polymorphism candidate sequence, is included the following steps：（1）Screen candidate sequence：（2）Screen candidate SNP locus：（3）The sites the A1426G_SNP primer sequence designed using PAMSA methods.The present invention can be under the information of not known Penaeus monodon genomic DNA mononucleotide polymorphism site, it is sequenced by transcript profile aggregate sample, filter out candidate sequence and candidate SNP locus, it lays the first stone for the screening and detection of Penaeus monodon A1426G single nucleotide polymorphisms, A1426G mononucleotide polymorphism sites are screened using bioinformatic analysis on the basis of this, and use PAMSA（PCR amplification of multiple specific alleles）Method designs specific primer, not only easily can complete the detection of Penaeus monodon A1426G single nucleotide polymorphism, verification and Genotyping by poly- propionamide gel electrophoresis, but also selective mechanisms expense is low, easy to operate quick.
Description
Technical field
The invention belongs to Penaeus monodon DNA molecular Genetic Markers, refer to a kind of Penaeus monodon A1426G monokaryons
The screening technique of nucleotide polymorphism mark candidate sequence.
Background technology
Penaeus monodon (Penaeus monodon) is the maximum species of Penaeus kind body, is commonly called as terrible shrimp, grass shrimp and nine sections
Shrimp is distributed widely in the littoral seas such as India, the Pacific Ocean, and southern Fujian Province, Guangdong Province, Guangxi Zhuang are mainly distributed in China
The littoral seas such as autonomous region of race and Hainan Province.Penaeus monodon growth is fast, individual is big, meat flavour is delicious and full of nutrition, and yield is high,
It is one of south China shrimps in culture principal item.Penaeus monodon Natural Population in China's is due to overfishing, disease infection at present
And habitat reasons, the germ plasm resource amount such as is destroyed and declines year by year, population quantity is fewer and fewer, can seldom form fishing season
.Also, the seed parent needed for breeding production is mainly derived from the wild female shrimp of Hainan marine site capture, further results in it certainly
Right resource gradually fails.It is extremely urgent that ecological protection is carried out to Penaeus monodon natural excellent germplasm.It carries out on this basis
Germplasm innovation, it is to restore its germ plasm resource to widely popularize fine-variety breeding, promotes the important measures of its industry sustainable health development.
Germ plasm resource is assessed and family tree identification needs a large amount of DNA molecular marker to improve reliability.Jerry et al. once carried out
Penaeus monodon pedigree appraisal because the factors such as microsatellite marker amplification efficiency, amorph and shadow band cause from
7 pairs of labels (30%) are only filtered out in 23 pairs of microsatellite markers to identify for pedigree[2].It can be seen that screening sufficient amount DNA molecular mark
Note is the premise guarantee that Penaeus monodon carries out Germplasm Identification.However, compared with the marine fish such as atlantic salmon, spot at present
The quality and quantity of section prawn molecular markers development cannot still meet the needs of researchs such as pedigree identification, genetic linkage maps.
As the DNA molecular marker of a new generation, single nucleotide polymorphism is the molecule that content is most abundant in genome
Label, and genetic stability is high, parting is accurate, it is easy to accomplish and high-throughput, automation detection, is current Penaeus monodon selection and breeding
The preferred DNA molecular mark of population genetic background check, individual selection and breeding, family tree identification and high density genetic linkage maps structure
One of note.But spot section DNA information is deficient, the rare report of research in relation to the exploitation of its single nucleotide polymorphism, application
Road.Matthew Baranski et al. report the SNP marker screening of Penaeus monodon (Penaeus monodon), used
Detection and genotyping method is chip typing, and this method label screening, verification and the cost of application are higher, and Penaeus monodon
During the detection and genotyping of the SNP marker screening of (Penaeus monodon), it is crucial that Penaeus monodon mononucleotide is more
The screening technique and candidate SNP locus screening technique of state property mark candidate sequence, filtered out polymorphism mark candidate sequence and
Candidate SNP locus, so that it may to realize screening and detection to Penaeus monodon single nucleotide polymorphism, to realize to spot section
Prawn single nucleotide polymorphism is verified and Genotyping, is conducive to the identification of spot section shrimp pedigree, genetic linkage maps structure etc.
Research is carried out.
Invention content
The purpose of the present invention is to provide a kind of sieves of Penaeus monodon A1426G single nucleotide polymorphism candidate sequences
Choosing method is realized under the background of Penaeus monodon DNA information scarcity to Penaeus monodon single nucleotide polymorphism
Screening and detection are Penaeus monodon breeding to realize to the verification of Penaeus monodon single nucleotide polymorphism and Genotyping
Genetic background investigation in project and family tree Identification Service.
To achieve the above object, this invention takes following technical proposals, a kind of Penaeus monodon single nucleotide polymorphism marks
Remember that the screening technique of candidate sequence, the present invention include technical step in detail below：
(1) candidate sequence is screened：The carapace total serum IgE of 20 Penaeus monodon individuals is extracted, mixing sample carries out transcript profile
Sequencing, splicing Penaeus monodon carapace refer to transcript, and comparison positioning sequencing is read end (reads) and arrived with reference to transcript screening monokaryon
Nucleotide polymorphism site, and screen and compare mass fraction and be more than 40, minorAllele frequency 200 or more and secondary equipotential
Site of the gene frequency higher than 20% is candidate sequence；
(2) candidate SNP locus is screened：
The gene order of its candidate comp233997 is：
TTGGGCCAGTTGAAGGTACAACGCTGTATGATAAAAATAAATGCATTGAGGGGACGCAGTACAAGCAGGATGCATTC
CACCTTTTTTCTTCTCTCTTCAGAAAATATAACACTGAATCACACAAGACTCAATATCTAATATTCCAATTTTATTA
ATCTGTAACTTTATGATTTCAGGTTTTGGCCTAAAATGTTTACATAATATCACACAACAACACCAAAACACAACAAG
GCAATGACAATCTGTTAGATTTTGTAATTTGCAGTTTATTGAATGACAGAATAAATATATCCTATTAACTTGAGTTT
TTACCCCAAGTGACTAGGTATGGTTCACAGACCATGTAACATTTACAGATCTATATTTATCTTTTCTTCTTACTTGC
ATGGAATGACTGAGACATGTACATTCACCAAGCGACACTGTTTATCAAGTCCATATTTGACTGTTCATCTAAACATC
CTACTCCATTTTTGCTCAGCTCTATATACCTGTCCTACAACAAAACTTGGCAGCTTGTACTTGCAATAACATTGAAT
CAATCAAAAGAAAATTTACTAAGAGCAATACCCTATAAAAAGTAAAACTTAGTTTGGACTTGTCTGAAATTTTCCAA
ACATCTTAAACAATCTTATTGCAAGTCCCTGCTGTTTTAAGTGTAATCATATTCTCAAGACAAACTATATACAGGCA
ACAGGAATAGCAGCTTAAAACCATAACCCACATGATTAGAAGCCTTGTTAGCCAATCTTCAGCCAACTTTATATTTC
CTAGATGTGTGGATGTACAAGATTATCGAGTACCTTTCATATTATTAGTAATGCAGTGTACCCATCACAAAATGGAG
TTTAACTTTGATCTTAAGCATTTGTGACAGCTGTACAAGCACTCTGAACAGCAATAAAACAGAAGCTTTAAGGCTCC
CGATGAGTAACTTAACTTAACAAGCAATTAGTATCCCTTGTCAAGTCCATAACTGAGAAATTACTGTACGTACAGGA
CGCGCATGGTATTGGTAAAGCCAGCTCCCTTCTGCCAGCCAGTCACCACAACAACAGGGTCGCCGGGCTTGATGAAG
CCACACTCCTTGCCATACTGAACAGCATAGTCTACGCGAGCATTGACATCATTCATCCAGTCTTCAATACGTTCAGC
ATTCTGAGGCACAGTGTAGTGGATGGGAATAATACCACGGTAGAGATGGCACTGTCTGGCCACCTGTGGGAATCGAG
TTACAGCCACAATTGGGCAGCGAGGCCTGTACTTTGAAACCAAATGAGCAGAGCGACCAGTGGTGGTAATAACAATA
ATGGCTGTGGCCATTGCTTTGAATGAAGCTTCAACAGCAGCAATGGCTGTGGTGTGTGTTGAGTCAGTGGGCAGTTG
GACCTGCTGAGACAGCTCAGTGAAGAGTTGCTTGTGCCA
ATTGCAGCTTCAGCTTCACGTGCAATATTGGCCATGGTTCGCACACACACAAGAGGGTAGTCTCCCTTAGCAGTTTC
ACCAGACAGCATGACACAGTCAGCACCATCAAGAATAGCATTACCAACATCAGACACTTCAGCACGTGTGGGACGGG
GTTTCTTCACCATGGATTCCAACATCTGGGTAGCACAAATGACTGGCTTTCCAACTTTGTTGCACTTGGCAATCATC
TGTTTCTGGGCCACAAAGACCTTCTCGGCTGGGATCTCAATACCCAAATCACCACGAGCAATCATGATACCATCTCC
CTCTTCAATGATGTCATCAATGTTCTTGCATCCCTGGTGATTCTCAATCTTGCTGATGATTTTAATGTTCTTGCCCT
TTTCACCAAGGACTTCCCTGATCTCACGGACTCCAGCTGCATCACGGATGAAGGATGCAAACACAATGTCTACTCCC
ATTTTGACACCAAGGAGGAGGTCACCACGGTCCTTCTCAGAGACTGCAGGAAGGTCTACTGGGACACCTGGGAGGTT
CATGCCCTTCTTGCTCCCAAGCATACCTCCATTTTCAACCTCACATTCAATGGAATCACTGCCCACATCCTTGGCAA
TAAGAGAGATAAGGCCATCATCAACAAAAATGCGGTTGCCAGGTTTCACAACTTTGGTGATGTTGACATAGTCCAAG
TAAAGCACCTCTTCTGAGCACTTTTCGTAGTAACTGCTATCTGTGGTCAACTTGATTGTGGCACCTTCCTTCAATTC
AATTTCAGCTGTTGGTCCACCTTCCAAAAGGCCAGTACGAATTTCAGGTCCCTTTGTATCAAGAGCAATGGCAACAG
GATAAGAATGCCCAATCTTCTCGGAATACTTCTGGGCTGCCTTACGGACATTCATCATTGTCTCACTGTGGTATTCA
TGGGTGCCGTGGGAGAAGTTCATACGTGCGATGTTCATGCCAGCCTCCATCATCTTCTCCAGCATCTCCACAGACCG
AGAGACAGGCCCAATGGTGCAGATGATGCCGGAGAGTCGCTTCGAGAAAGGTTTAGAGTCGATGTCCAGGGCCGCCA
TGTGGTCCACCTGAGTGTGGGCATCTGCGGCCCCAAGCTGCATGGGTGCTACCTTCGACATCTTGAGATCTTGTATT
TGAACAGAGAGCAGCTTTTTCTTTCTTCCTCTACCGTGGCTGACCGGCGACTGCACGGAAATCAATGTTGCAATCCT
TTCACCCTTGACTTTGGCAGTTGCGTCTTATCGTGTAGAAAAGACAAAATCGATCGATGTGCTTATATTATGGTATT
AGATAATCTTTCTTAATTTCTTTTTTCATTTTCTATTGCCAATATCAAGGTAAAAAGATATCCAGGTTCAGGAAAAT
AGCCATTTCCAAAAGAATTCAAGAAATCGGACGTCATGATCTTTACCGCAG, wherein overstriking, the base underlined are
The candidate sites A1426G_SNP；
(3) sites the A1426G_SNP primer sequence designed using PAMSA methods for：
Positive strand primer one：5’-GGTTTTGAAGAGTTGCTTGTGACAA-3’；
Positive strand primer two：5’-ATTTTTTTGGTTTTGAAGAGTTGCTTGTGCCCG-3’；
Negative strand primer：5’-GTGAAGAAACCCCGTCCCA-3’.
The present invention of above-mentioned measure is taken, it can be in not known Penaeus monodon genomic DNA single nucleotide polymorphism
It under the information in site, is sequenced by transcript profile aggregate sample, filters out candidate sequence and candidate SNP locus, be Penaeus monodon
The screening and detection of A1426G single nucleotide polymorphisms lay the first stone, and are screened using bioinformatic analysis on the basis of this
A1426G mononucleotide polymorphism sites, and using PAMSA (PCR amplification of multiple specific
Alleles) method designs specific primer, not only easily can complete Penaeus monodon A1426G by poly- propionamide gel electrophoresis
Single nucleotide polymorphism detection, verification and Genotyping, and selective mechanisms expense is low, easy to operate quick.
Description of the drawings
Fig. 1 is mononucleotide polymorphic site A1426G_SNP provided by the invention to 24 individual detection figures of Penaeus monodon
Spectrum.
Specific implementation mode
The present invention is described in detail below by embodiment.
As shown in Figure 1, number 1-24 is 24 individuals of Penaeus monodon, AA, AG, GG are the genotype of Different Individual, 200bp
It is the length of Marker different fragments.
(1) bioinformatic analysis is utilized, the splicing sequence containing candidate mononucleotide polymorphic site, detailed process are obtained
It is as follows：The transcript profile sequencing of the Penaeus monodon carapace of mixing sample is carried out first, and presses default parameters using trinity softwares
Splice carapace and refer to transcript, comparing positioning transcript profile sequencing by default parameters using BWA softwares reads end (reads) to reference
Transcript compares mass fraction using GATK software screening methods and is more than 40, minorAllele frequency 200 or more and secondary etc.
Site of the position gene frequency higher than 20% is candidate locus；
(2) screening comp233997 splicings sequence is used for design of primers, and sequence is：
TTGGGCCAGTTGAAGGTACAACGCTGTATGATAAAAATAAATGCATTGAGGGGACGCAGTACAAGCAGGATGCATTC
CACCTTTTTTCTTCTCTCTTCAGAAAATATAACACTGAATCACACAAGACTCAATATCTAATATTCCAATTTTATTA
ATCTGTAACTTTATGATTTCAGGTTTTGGCCTAAAATGTTTACATAATATCACACAACAACACCAAAACACAACAAG
GCAATGACAATCTGTTAGATTTTGTAATTTGCAGTTTATTGAATGACAGAATAAATATATCCTATTAACTTGAGTTT
TTACCCCAAGTGACTAGGTATGGTTCACAGACCATGTAACATTTACAGATCTATATTTATCTTTTCTTCTTACTTGC
ATGGAATGACTGAGACATGTACATTCACCAAGCGACACTGTTTATCAAGTCCATATTTGACTGTTCATCTAAACATC
CTACTCCATTTTTGCTCAGCTCTATATACCTGTCCTACAACAAAACTTGGCAGCTTGTACTTGCAATAACATTGAAT
CAATCAAAAGAAAATTTACTAAGAGCAATACCCTATAAAAAGTAAAACTTAGTTTGGACTTGTCTGAAATTTTCCAA
ACATCTTAAACAATCTTATTGCAAGTCCCTGCTGTTTTAAGTGTAATCATATTCTCAAGACAAACTATATACAGGCA
ACAGGAATAGCAGCTTAAAACCATAACCCACATGATTAGAAGCCTTGTTAGCCAATCTTCAGCCAACTTTATATTTC
CTAGATGTGTGGATGTACAAGATTATCGAGTACCTTTCATATTATTAGTAATGCAGTGTACCCATCACAAAATGGAG
TTTAACTTTGATCTTAAGCATTTGTGACAGCTGTACAAGCACTCTGAACAGCAATAAAACAGAAGCTTTAAGGCTCC
CGATGAGTAACTTAACTTAACAAGCAATTAGTATCCCTTGTCAAGTCCATAACTGAGAAATTACTGTACGTACAGGA
CGCGCATGGTATTGGTAAAGCCAGCTCCCTTCTGCCAGCCAGTCACCACAACAACAGGGTCGCCGGGCTTGATGAAG
CCACACTCCTTGCCATACTGAACAGCATAGTCTACGCGAGCATTGACATCATTCATCCAGTCTTCAATACGTTCAGC
ATTCTGAGGCACAGTGTAGTGGATGGGAATAATACCACGGTAGAGATGGCACTGTCTGGCCACCTGTGGGAATCGAG
TTACAGCCACAATTGGGCAGCGAGGCCTGTACTTTGAAACCAAATGAGCAGAGCGACCAGTGGTGGTAATAACAATA
ATGGCTGTGGCCATTGCTTTGAATGAAGCTTCAACAGCAGCAATGGCTGTGGTGTGTGTTGAGTCAGTGGGCAGTTG
GACCTGCTGAGACAGCTCAGTGAAGAGTTGCTTGTGCCA
ATTGCAGCTTCAGCTTCACGTGCAATATTGGCCATGGTTCGCACACACACAAGAGGGTAGTCTCCCTTAGCAGTTTC
ACCAGACAGCATGACACAGTCAGCACCATCAAGAATAGCATTACCAACATCAGACACTTCAGCACGTGTGGGACGGG
GTTTCTTCACCATGGATTCCAACATCTGGGTAGCACAAATGACTGGCTTTCCAACTTTGTTGCACTTGGCAATCATC
TGTTTCTGGGCCACAAAGACCTTCTCGGCTGGGATCTCAATACCCAAATCACCACGAGCAATCATGATACCATCTCC
CTCTTCAATGATGTCATCAATGTTCTTGCATCCCTGGTGATTCTCAATCTTGCTGATGATTTTAATGTTCTTGCCCT
TTTCACCAAGGACTTCCCTGATCTCACGGACTCCAGCTGCATCACGGATGAAGGATGCAAACACAATGTCTACTCCC
ATTTTGACACCAAGGAGGAGGTCACCACGGTCCTTCTCAGAGACTGCAGGAAGGTCTACTGGGACACCTGGGAGGTT
CATGCCCTTCTTGCTCCCAAGCATACCTCCATTTTCAACCTCACATTCAATGGAATCACTGCCCACATCCTTGGCAA
TAAGAGAGATAAGGCCATCATCAACAAAAATGCGGTTGCCAGGTTTCACAACTTTGGTGATGTTGACATAGTCCAAG
TAAAGCACCTCTTCTGAGCACTTTTCGTAGTAACTGCTATCTGTGGTCAACTTGATTGTGGCACCTTCCTTCAATTC
AATTTCAGCTGTTGGTCCACCTTCCAAAAGGCCAGTACGAATTTCAGGTCCCTTTGTATCAAGAGCAATGGCAACAG
GATAAGAATGCCCAATCTTCTCGGAATACTTCTGGGCTGCCTTACGGACATTCATCATTGTCTCACTGTGGTATTCA
TGGGTGCCGTGGGAGAAGTTCATACGTGCGATGTTCATGCCAGCCTCCATCATCTTCTCCAGCATCTCCACAGACCG
AGAGACAGGCCCAATGGTGCAGATGATGCCGGAGAGTCGCTTCGAGAAAGGTTTAGAGTCGATGTCCAGGGCCGCCA
TGTGGTCCACCTGAGTGTGGGCATCTGCGGCCCCAAGCTGCATGGGTGCTACCTTCGACATCTTGAGATCTTGTATT
TGAACAGAGAGCAGCTTTTTCTTTCTTCCTCTACCGTGGCTGACCGGCGACTGCACGGAAATCAATGTTGCAATCCT
TTCACCCTTGACTTTGGCAGTTGCGTCTTATCGTGTAGAAAAGACAAAATCGATCGATGTGCTTATATTATGGTATT
AGATAATCTTTCTTAATTTCTTTTTTCATTTTCTATTGCCAATATCAAGGTAAAAAGATATCCAGGTTCAGGAAAAT
AGCCATTTCCAAAAGAATTCAAGAAATCGGACGTCATGATCTTTACCGCAG, wherein overstriking, the base underlined are
The candidate sites A1426G_SNP.
(3) it is set using PAMSA (PCR amplification of multiple specific alleles) methods
The sites the A1426G_SNP primer sequence of meter is：
Positive strand primer one：5’-GGTTTTGAAGAGTTGCTTGTGACAA-3’；
Positive strand primer two：5’-ATTTTTTTGGTTTTGAAGAGTTGCTTGTGCCCG-3’；
Negative strand primer：5’-GTGAAGAAACCCCGTCCCA-3’.
According to above-mentioned acquired candidate sequence, can also further carry out to Penaeus monodon A1426G mononucleotide polymorphics
Property label screening and detection, steps are as follows for particular technique：
(1) Penaeus monodon muscle of back genomic DNA to be extracted, 50ng/ul is diluted to, PCR reaction systems are 30ul,
Including 50ng/ul Penaeus monodon Genomic DNA solution 1ul, 10 × PCR buffer 3ul, 2.5mM dNTP Mixs 2.4ul,
25mM MgCl2The positive strand primers one of 4ul, 5U/ul Taq archaeal dna polymerases 0.15ul, 10uM, positive strand primer two and negative strand primer
Each 0.5ul, adds ddH2O to 30ul；PCR response procedures are：94 DEG C of denaturation 10min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C
30sec, 40 cycles；72 DEG C of extension 10min, 4 DEG C of preservations.
(2) the PCR product 10ul for taking the Different Individual of step 3 acquisition respectively carries out 6% and becomes under the firm power of 70W
Property poly- propionamide gel electrophoresis 2h, silver staining colour developing photographs to record that the results are shown in Figure 1 under gel imaging system, produced according to PCR
Genotype between the different instructions individual of object clip size counts the genotype of Different Individual, obtains Penaeus monodon mononucleotide
The mapping genetic variations of polymorphic site A1426G_SNP.
In order to adapt to the reading of computer, following sequences are generated by computer：
Sequence table
<110>Guangxi Zhuang Autonomous Region Institute Of Oceanology
<120>A kind of screening technique of Penaeus monodon A1426G single nucleotide polymorphism candidate sequences
<130> 2017
<140> 2018100116595
<141> 2018-01-05
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2863
<212> DNA
<213> Penaeus monodon
<400> 1
ttgggccagt tgaaggtaca acgctgtatg ataaaaataa atgcattgag gggacgcagt 60
acaagcagga tgcattccac cttttttctt ctctcttcag aaaatataac actgaatcac 120
acaagactca atatctaata ttccaatttt attaatctgt aactttatga tttcaggttt 180
tggcctaaaa tgtttacata atatcacaca acaacaccaa aacacaacaa ggcaatgaca 240
atctgttaga ttttgtaatt tgcagtttat tgaatgacag aataaatata tcctattaac 300
ttgagttttt accccaagtg actaggtatg gttcacagac catgtaacat ttacagatct 360
atatttatct tttcttctta cttgcatgga atgactgaga catgtacatt caccaagcga 420
cactgtttat caagtccata tttgactgtt catctaaaca tcctactcca tttttgctca 480
gctctatata cctgtcctac aacaaaactt ggcagcttgt acttgcaata acattgaatc 540
aatcaaaaga aaatttacta agagcaatac cctataaaaa gtaaaactta gtttggactt 600
gtctgaaatt ttccaaacat cttaaacaat cttattgcaa gtccctgctg ttttaagtgt 660
aatcatattc tcaagacaaa ctatatacag gcaacaggaa tagcagctta aaaccataac 720
ccacatgatt agaagccttg ttagccaatc ttcagccaac tttatatttc ctagatgtgt 780
ggatgtacaa gattatcgag tacctttcat attattagta atgcagtgta cccatcacaa 840
aatggagttt aactttgatc ttaagcattt gtgacagctg tacaagcact ctgaacagca 900
ataaaacaga agctttaagg ctcccgatga gtaacttaac ttaacaagca attagtatcc 960
cttgtcaagt ccataactga gaaattactg tacgtacagg acgcgcatgg tattggtaaa 1020
gccagctccc ttctgccagc cagtcaccac aacaacaggg tcgccgggct tgatgaagcc 1080
acactccttg ccatactgaa cagcatagtc tacgcgagca ttgacatcat tcatccagtc 1140
ttcaatacgt tcagcattct gaggcacagt gtagtggatg ggaataatac cacggtagag 1200
atggcactgt ctggccacct gtgggaatcg agttacagcc acaattgggc agcgaggcct 1260
gtactttgaa accaaatgag cagagcgacc agtggtggta ataacaataa tggctgtggc 1320
cattgctttg aatgaagctt caacagcagc aatggctgtg gtgtgtgttg agtcagtggg 1380
cagttggacc tgctgagaca gctcagtgaa gagttgcttg tgccaaattg cagcttcagc 1440
ttcacgtgca atattggcca tggttcgcac acacacaaga gggtagtctc ccttagcagt 1500
ttcaccagac agcatgacac agtcagcacc atcaagaata gcattaccaa catcagacac 1560
ttcagcacgt gtgggacggg gtttcttcac catggattcc aacatctggg tagcacaaat 1620
gactggcttt ccaactttgt tgcacttggc aatcatctgt ttctgggcca caaagacctt 1680
ctcggctggg atctcaatac ccaaatcacc acgagcaatc atgataccat ctccctcttc 1740
aatgatgtca tcaatgttct tgcatccctg gtgattctca atcttgctga tgattttaat 1800
gttcttgccc ttttcaccaa ggacttccct gatctcacgg actccagctg catcacggat 1860
gaaggatgca aacacaatgt ctactcccat tttgacacca aggaggaggt caccacggtc 1920
cttctcagag actgcaggaa ggtctactgg gacacctggg aggttcatgc ccttcttgct 1980
cccaagcata cctccatttt caacctcaca ttcaatggaa tcactgccca catccttggc 2040
aataagagag ataaggccat catcaacaaa aatgcggttg ccaggtttca caactttggt 2100
gatgttgaca tagtccaagt aaagcacctc ttctgagcac ttttcgtagt aactgctatc 2160
tgtggtcaac ttgattgtgg caccttcctt caattcaatt tcagctgttg gtccaccttc 2220
caaaaggcca gtacgaattt caggtccctt tgtatcaaga gcaatggcaa caggataaga 2280
atgcccaatc ttctcggaat acttctgggc tgccttacgg acattcatca ttgtctcact 2340
gtggtattca tgggtgccgt gggagaagtt catacgtgcg atgttcatgc cagcctccat 2400
catcttctcc agcatctcca cagaccgaga gacaggccca atggtgcaga tgatgccgga 2460
gagtcgcttc gagaaaggtt tagagtcgat gtccagggcc gccatgtggt ccacctgagt 2520
gtgggcatct gcggccccaa gctgcatggg tgctaccttc gacatcttga gatcttgtat 2580
ttgaacagag agcagctttt tctttcttcc tctaccgtgg ctgaccggcg actgcacgga 2640
aatcaatgtt gcaatccttt cacccttgac tttggcagtt gcgtcttatc gtgtagaaaa 2700
gacaaaatcg atcgatgtgc ttatattatg gtattagata atctttctta atttcttttt 2760
tcattttcta ttgccaatat caaggtaaaa agatatccag gttcaggaaa atagccattt 2820
ccaaaagaat tcaagaaatc ggacgtcatg atctttaccg cag 2957
<210> 2
<211> 25
<212> DNA
<213> rengongxulie
<400> 2
ggttttgaag agttgcttgt gacaa 25
<210> 3
<211> 33
<212> DNA
<213> rengongxulie
<400> 3
atttttttgg ttttgaagag ttgcttgtgc ccg 33
<210> 4
<211> 19
<212> DNA
<213> rengongxulie
<400> 4
gtgaagaaac cccgtccca 19
Claims (1)
1. a kind of screening technique of Penaeus monodon single nucleotide polymorphism candidate sequence, it is characterised in that the method includes
Following particular technique steps：
(1) candidate sequence is screened：
The carapace total serum IgE of 20 tail Penaeus monodons is extracted, mixing sample carries out transcript profile sequencing, splicing Penaeus monodon carapace ginseng
Transcript is examined, comparison positioning sequencing is read end (reads) and arrived with reference to transcript screening mononucleotide pleomorphism site, and screens comparison
Mass fraction is more than 40, minorAllele frequency
Candidate sequence；
(2) candidate SNP locus is screened
The gene order of its candidate comp233997 is：
TTGGGCCAGTTGAAGGTACAACGCTGTATGATAAAAATAAATGCATTGAGGGGACGCAGTACAAGCAGGATGC
ATTCCACCTTTTTTCTTCTCTCTTCAGAAAATATAACACTGAATCACACAAGACTCAATATCTAATATTCCAATTTT
ATTAATCTGTAACTTTATGATTTCAGGTTTTGGCCTAAAATGTTTACATAATATCACACAACAACACCAAAACACAA
CAAGGCAATGACAATCTGTTAGATTTTGTAATTTGCAGTTTATTGAATGACAGAATAAATATATCCTATTAACTTGA
GTTTTTACCCCAAGTGACTAGGTATGGTTCACAGACCATGTAACATTTACAGATCTATATTTATCTTTTCTTCTTAC
TTGCATGGAATGACTGAGACATGTACATTCACCAAGCGACACTGTTTATCAAGTCCATATTTGACTGTTCATCTAAA
CATCCTACTCCATTTTTGCTCAGCTCTATATACCTGTCCTACAACAAAACTTGGCAGCTTGTACTTGCAATAACATT
GAATCAATCAAAAGAAAATTTACTAAGAGCAATACCCTATAAAAAGTAAAACTTAGTTTGGACTTGTCTGAAATTTT
CCAAACATCTTAAACAATCTTATTGCAAGTCCCTGCTGTTTTAAGTGTAATCATATTCTCAAGACAAACTATATACA
GGCAACAGGAATAGCAGCTTAAAACCATAACCCACATGATTAGAAGCCTTGTTAGCCAATCTTCAGCCAACTTTATA
TTTCCTAGATGTGTGGATGTACAAGATTATCGAGTACCTTTCATATTATTAGTAATGCAGTGTACCCATCACAAAAT
GGAGTTTAACTTTGATCTTAAGCATTTGTGACAGCTGTACAAGCACTCTGAACAGCAATAAAACAGAAGCTTTAAGG
CTCCCGATGAGTAACTTAACTTAACAAGCAATTAGTATCCCTTGTCAAGTCCATAACTGAGAAATTACTGTACGTAC
AGGACGCGCATGGTATTGGTAAAGCCAGCTCCCTTCTGCCAGCCAGTCACCACAACAACAGGGTCGCCGGGCTTGAT
GAAGCCACACTCCTTGCCATACTGAACAGCATAGTCTACGCGAGCATTGACATCATTCATCCAGTCTTCAATACGTT
CAGCATTCTGAGGCACAGTGTAGTGGATGGGAATAATACCACGGTAGAGATGGCACTGTCTGGCCACCTGTGGGAAT
CGAGTTACAGCCACAATTGGGCAGCGAGGCCTGTACTTTGAAACCAAATGAGCAGAGCGACCAGTGGTGGTAATAAC
AATAATGGCTGTGGCCATTGCTTTGAATGAAGCTTCAACAGCAGCAATGGCTGTGGTGTGTGTTGAGTCAGTGGGCA
GTTGGACCTGCTGAGACAGCTCAGTGAAGAGTTGCTTGTGCCAAATTGCAGCTTCAGCTTCACGTGCAATATTGGCC
ATGGTTCGCACACACACAAGAGGGTAGTCTCCCTTAGCAGTTTCACCAGACAGCATGACACAGTCAGCACCATCAAG
AATAGCATTACCAACATCAGACACTTCAGCACGTGTGGGACGGGGTTTCTTCACCATGGATTCCAACATCTGGGTAG
CACAAATGACTGGCTTTCCAACTTTGTTGCACTTGGCAATCATCTGTTTCTGGGCCACAAAGACCTTCTCGGCTGGG
ATCTCAATACCCAAATCACCACGAGCAATCATGATACCATCTCCCTCTTCAATGATGTCATCAATGTTCTTGCATCC
CTGGTGATTCTCAATCTTGCTGATGATTTTAATGTTCTTGCCCTTTTCACCAAGGACTTCCCTGATCTCACGGACTC
CAGCTGCATCACGGATGAAGGATGCAAACACAATGTCTACTCCCATTTTGACACCAAGGAGGAGGTCACCACGGTCC
TTCTCAGAGACTGCAGGAAGGTCTACTGGGACACCTGGGAGGTTCATGCCCTTCTTGCTCCCAAGCATACCTCCATT
TTCAACCTCACATTCAATGGAATCACTGCCCACATCCTTGGCAATAAGAGAGATAAGGCCATCATCAACAAAAATGC
GGTTGCCAGGTTTCACAACTTTGGTGATGTTGACATAGTCCAAGTAAAGCACCTCTTCTGAGCACTTTTCGTAGTAA
CTGCTATCTGTGGTCAACTTGATTGTGGCACCTTCCTTCAATTCAATTTCAGCTGTTGGTCCACCTTCCAAAAGGCC
AGTACGAATTTCAGGTCCCTTTGTATCAAGAGCAATGGCAACAGGATAAGAATGCCCAATCTTCTCGGAATACTTCT
GGGCTGCCTTACGGACATTCATCATTGTCTCACTGTGGTATTCATGGGTGCCGTGGGAGAAGTTCATACGTGCGATG
TTCATGCCAGCCTCCATCATCTTCTCCAGCATCTCCACAGACCGAGAGACAGGCCCAATGGTGCAGATGATGCCGGA
GAGTCGCTTCGAGAAAGGTTTAGAGTCGATGTCCAGGGCCGCCATGTGGTCCACCTGAGTGTGGGCATCTGCGGCCC
CAAGCTGCATGGGTGCTACCTTCGACATCTTGAGATCTTGTATTTGAACAGAGAGCAGCTTTTTCTTTCTTCCTCTA
CCGTGGCTGACCGGCGACTGCACGGAAATCAATGTTGCAATCCTTTCACCCTTGACTTTGGCAGTTGCGTCTTATCG
TGTAGAAAAGACAAAATCGATCGATGTGCTTATATTATGGTATTAGATAATCTTTCTTAATTTCTTTTTTCATTTTC
TATTGCCAATATCAAGGTAAAAAGATATCCAGGTTCAGGAAAATAGCCATTTCCAAAAGAATTCAAGAAATCGGACG
TCATGATCTTTACCGCAG, wherein overstriking, the base underlined are the candidate sites A1426G_SNP；
3) specific primer, is designed
Specific primer, positive strand primer one are designed using PAMSA methods：5’-GGTTTTGAAGAGTTGCTTGTGACAA-3’；
Positive strand primer two：5 '-ATTTTTTTGGTTTTGAAGAGTTGCTTGTGCCCG-3 ', negative strand primer：5’-
GTGAAGAAACCCCGTCCCA-3’。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337578A (en) * | 2021-06-17 | 2021-09-03 | 集美大学 | Method for efficiently screening positive SNP of aquatic animals based on transcriptome data |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611460A (en) * | 2015-03-05 | 2015-05-13 | 厦门大学 | Method for screening and detecting single-nucleotide polymorphic site G642A of marsupenaeus japonicus |
-
2018
- 2018-01-05 CN CN201810011659.5A patent/CN108504743A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611460A (en) * | 2015-03-05 | 2015-05-13 | 厦门大学 | Method for screening and detecting single-nucleotide polymorphic site G642A of marsupenaeus japonicus |
Non-Patent Citations (2)
| Title |
|---|
| GLENN KL等: "SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp", 《ANIM GENET.》 * |
| YANG YU等: "SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing", 《PLOS ONE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337578A (en) * | 2021-06-17 | 2021-09-03 | 集美大学 | Method for efficiently screening positive SNP of aquatic animals based on transcriptome data |
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