CN105349675A - Larimichthys crocea genome-wide SNP and InDel molecular marker method based on double enzyme digestion - Google Patents
Larimichthys crocea genome-wide SNP and InDel molecular marker method based on double enzyme digestion Download PDFInfo
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Abstract
The invention discloses a larimichthys crocea genome-wide SNP and InDel molecular marker method based on double enzyme digestion. The method comprises the following steps: 1) joint sequence design; 2) genome DNA enzyme digestion; 3) joint sequence connection; 4) sample mixing and PCR amplification; 5) high-throughput sequencing; 6) SNP locus excavation and analysis through sequencing data. According to the method, the modern molecular biology and the advanced high-throughput sequencing technology are combined, the method of combination of EcoRII and NlaIII double enzyme digestion is used, the genome-wide DNA molecules are subjected to enzyme digestion, DNA segments with certain lengths are obtained for carrying out library establishment sequencing and SNP molecular marker excavation, and the uniformly distributed genome-wide SNP locus information is obtained; the workload and cost of genome-wide marker analysis are greatly reduced, and the method is also applicable to the genome-wide marker analysis of other species; the SNP marker excavated through the method is applicable to the study fields such as animal and plant species identification, species genetic genealogy analysis, germplasm resource genetic diversity analysis and genetic breeding.
Description
Technical field
The present invention relates to large yellow croaker full-length genome molecular markers development technical field, specifically a kind of large yellow croaker full-length genome SNP based on double digestion and InDel molecule marking method.
Background technology
Or molecule marker refers to reflect between biont or the DNA fragmentation base of certain difference characteristic in genome between population, it directly reflects the difference between genomic dna, thus, molecule marker is the mark on gene level, is a kind of important tool of biological heredity molecule; Mainly comprise RFLP (RestrictionFragmentLengthPolymorphism), RAPD (RandomAmplifiedPolymorphicDNA), AFLP (AmplifiedFragmentLengthPolymorphism),
SSR (SimpleSequenceRepeat), SNP (SingleNucleotidePolymorphisms) and InDel (smallInsertandDeletion) etc.; At present, along with the development of genotyping technique, except SSR, SNP and InDel are marked at and widely use, the mark of other types has been used fewer and feweri, although SSR has easy to detect, polymorphism high, but in today that high-flux sequence and analytical technology constantly improve, SNP and InDel becomes more and more important molecule marker, it has following characteristics for SSR: first, widely distributed: SNP and InDel is distributed in genome region widely, comprises coding region and non-coding region; The second, site-specific: SNP and InDel mark can explicitly point out the reason producing polymorphism, but the polymorphism of SSR also needs further sequence verification; 3rd, high-throughput: SNP and InDel mark can utilize the method for large scale sequencing to carry out high-throughout exploitation, and the number of the molecule marker of general exploitation can arrive several ten thousand to several ten million; Along with the progress of high-flux sequence method and declining fast further of cost, SNP and InDel mark has become the main mark type of molecule marker and genotype identification now, at present, the success of SNP and InDel mark is also widely used in the genetic map construction of Important Economic species, and important character QTL locates and in whole-genome association; Large yellow croaker is a kind of important fish being mainly distributed in China and East Asia, the maximum marine economy fish of China's output and nursery amount, the research of large yellow croaker important economical trait, the such as speed of growth etc., need a large amount of full-length genome SNP and InDel molecule marker; In prior art, the exploitation of SNP and the InDel molecule marker of large yellow croaker mainly relies on the method for full-length genome and transcript profile high-flux sequence; Although transcript profile can obtain SNP and the InDel site that lots of genes expresses district fast, express district and only account for about 2% of full-length genome, the mark of full genome cannot be obtained; Although genome sequencing can carry out SNP and InDel exploitation in full genome level, due to its high cost, high data analysis intensity limits its widespread use marked at large yellow croaker SNP and InDel; For these reasons, need to improve the large yellow croaker molecule marking method of prior art.
Summary of the invention
The object of the present invention is to provide a kind of can overcome the heavy sequencing technologies of existing transcript profile and full-length genome large yellow croaker molecule marker mark excavate skewness and high cost problem, high-throughout SNP marker excavation can be carried out based on the large yellow croaker full-length genome SNP of the double digestion of EcoRI and NlaIII and InDel molecule marking method within the scope of full-length genome, to solve the problem proposed in above-mentioned background technology with lower cost.
For achieving the above object, the invention provides following technical scheme:
Based on large yellow croaker full-length genome SNP and the InDel molecule marking method of double digestion, comprise the following steps:
1) joint sequence design;
2) genomic dna enzyme is cut;
3) joint sequence connects;
4) sample mix and pcr amplification;
5) high-flux sequence;
6) sequencing data analysis mining SNP site.
As the further scheme of the present invention: the described large yellow croaker full-length genome SNP based on double digestion and InDel molecule marking method, specifically comprise the following steps:
1) joint sequence design: the check order primer sequence of Platform Requirements of the design of joint sequence and two enzyme end of EcoRI and NlaIII and follow-up IlluminaHiseq is consistent, joint sequence is made up of check order platform joint sequence and sample label sequence two part; Sample label design is according to following principle:
A. must the rarest two distinguishing base between each sample label sequence;
B. sample label sequence can not contain the identical base of continuous print two;
C. sample label can not comprise and can not form restriction enzyme site distinguished sequence with joint sequence;
2) genomic dna enzyme is cut: the genomic dna of 200ng carries out two endonuclease reactions in the reaction system of 20ul simultaneously;
3) joint sequence connects: joint sequence is connected to genomic dna enzyme and cuts in same test tube and carry out, and is connected on the enzyme simple stage property end of DNA fragmentation by the joint sequence of design by ligase enzyme;
4) sample mix builds storehouse from pcr amplification: the DNA fragmentation of different individual of sample jointing sequence is carried out balanced mix, ensures the harmony of each sample DNA segment number, adds PCR primer sequence and DNA synthetic enzyme carries out PCR reaction, and amplification connects product;
5) high-flux sequence: utilized by the amplified production in step 4 IlluminaHiseq2000 order-checking platform to check order;
6) sequencing data analysis mining SNP site: first order-checking raw data carries out sorting by the special sequence label of sample, obtain the order-checking section of the reading sequence of each sample, the section of the reading sequence of each sample utilized short data records comparison software BWA comparison on the reference genome of large yellow croaker, and use GATK to carry out SNP excavation.
Compared with prior art, the invention has the beneficial effects as follows: the present invention adopts based on the large yellow croaker full-length genome SNP of double digestion and InDel molecule marking method, SNP marker excavation can be carried out in large yellow croaker full-length genome level, contain genes encoding and non-coding region; And owing to using enzyme to cut the method reclaimed with specific DNA fragments, greatly reduce the scope that sequence carries out checking order, thus effectively control marker development cost; Present invention employs the method for EcoRI and NlaIII enzymes combinations, the DNA fragmentation containing two kinds of restriction enzyme sites in amplification large yellow croaker full-length genome that can be special, thus reach the object simplifying gene establishment storehouse and order-checking; The present invention greatly reduces high-throughput in large yellow croaker full-length genome SNP excavation and builds storehouse, the complexity of order-checking and SNP exploitation and workload, shorten the work period of the research fields such as the cultivar identification, the kind Genetic lineages that utilize full-length genome scope SNP to carry out are analyzed, Genetic Diversity of Germplasm analysis and genetic breeding, SNP excavation can be carried out on large yellow croaker full-length genome with lower cost, for large yellow croaker cultivar identification, kind Genetic lineages are analyzed and Genetic Diversity of Germplasm analysis, the research fields such as genetic breeding provide effective molecule marking method.
Accompanying drawing explanation
Fig. 1 is molecule marker schema of the present invention.
Fig. 2 carries out the fragment length distribution plan that double digestion builds storehouse in application example of the present invention.
Fig. 3 utilizes the full-length genome SNP marker of excavation to carry out group clustering analysis in application example of the present invention.
Embodiment
Be described in more detail below in conjunction with the technical scheme of embodiment to this patent.
Embodiment 1
Refer to Fig. 1-3, a kind of large yellow croaker full-length genome SNP based on double digestion and InDel molecule marking method, comprise the following steps:
1) experiment material is chosen
Contemporaneity 1 age cultured large yellow croaker random population 96 tail is got in this experiment, weight range and the long scope of body similar; After the characteristic index that all large yellow croaker individual records are basic, stay fin ray, preserve with the alcohol that volume fraction is 70%, save backup under putting into-20 ° of C.
2) large yellow croaker extracting genome DNA and preservation
A. freezing fin is used mortar grinder powdered in liquid nitrogen, get the centrifuge tube that specification is 1.5ml, add 1ml and digest damping fluid, gently powder being added 1ml digests in damping fluid, powder is soaked at the surface uniform of digestion damping fluid, then shake makes sample submergence, and digests 5h at 37 DEG C, and per jolting half an hour once;
B. treat that solution is cooled to room temperature, be distributed into two pipes, often pipe absorption 0.5ml balance phenol mixes gently, until form emulsus, centrifugal 10min under 3000-5000 × g condition, shifts out aqueous phase with macropore suction pipe;
C. 0.5ml(same volume is used) phenol: chloroform: primary isoamyl alcohol (25:24:1) extracts twice again, aqueous phase is transferred in clean pipe, adds NaCl and add 50ul to concentration 0.3M(3MNaCl), the ethanol of 2.5 times of volumes is injected gently along tube wall, shake pipe gently, until solution mixes completely, very clearly can see that DNA rapid precipitation is got off, under 3000 × g condition after centrifugal 10min, with appropriate 70% ethanol rinse twice, centrifugal, abandon supernatant liquor, dry;
D. sample settling flux in 0.5mlTE, add NaCl and add 16.7ul to final concentration 100mM(3MNaCl), add (DNAsefree) RNAseA and add 5ul to concentration 100ug/ml(10mg/mlRNAseA), keep 3h at 37 DEG C after, add SDS and add 10ul to ultimate density 0.2%(10%SDS), and use same volume phenol: chloroform: primary isoamyl alcohol (25:24:1) extracts twice, aqueous phase is transferred in clean pipe, add NaCl and add 50ul to concentration 0.3M(3MNaCl), the ethanol of 2.5 times of volumes is injected gently along tube wall, shake pipe gently, until solution mixes completely, very clearly can see that DNA rapid precipitation is got off, DNA is placed on settling flux in 0.5ml10mMTE, last sample measures OD value in order to determine concentration under 260nm, save backup under the DNA sample extracted is placed in-20 ° of C.
3) joint sequence Design and synthesis
Refer to accompanying drawing 2, the joint sequence that the present embodiment has designed and synthesized accompanying drawing 2 carries out high-flux sequence and builds storehouse.
4) genomic dna enzyme is cut
Large yellow croaker genomic dna (about 200ng) joins in the reaction system of 20ul and carries out enzyme and cut, this reaction system comprises NEB4 damping fluid (NEBBuffer4), the PstI restriction endonuclease of 8U and the MspI restriction endonuclease of 8U, whole enzyme cuts through journey and continue 2h under 37 ° of C, reaction system is placed in the activity of 20min sex change PstI and MspI under 65 ° of C afterwards, suppresses endonuclease reaction.
5) genomic dna digestion products is connected with joint sequence
Joint sequence ligation is carried out in the same test tube of genomic dna endonuclease reaction, equally first adds NEB4 damping fluid (NEBBuffer4) and ATP, adds the corresponding forward joint of 0.1pmol and the reverse breeches joint sequence of 15pmol in test tube; Afterwards, add the T4 ligase enzyme of NEB4 damping fluid, ATP and 200U in the reaction system of each sample, whole reaction system remains under 22 ° of C and continues 2h, and reaction system is placed in 20min under 65 ° of C afterwards.
6) mixed sample and amplified library
Connect product by after Qbit accurate quantitative analysis, between sample, balanced mix is in a PCR test tube, and mixing sample is by 18 pcr amplification reactions of taking turns (95 ° of C, 30s; 62 ° of C, 30s; 68 ° of C, 30s), equivalent amplification is carried out to mixing sample, obtains treating sequencing library.
7) high-flux sequence and full-length genome SNP develop
Amplified production in step 6 builds storehouse according to Illumina and order-checking flow process uses Hiseq platform to carry out high-flux sequence, and order-checking adopts both-end 2X100bp pattern, and average each individuality order-checking 500M, obtains sequencing data altogether and be about 50G.
The primitive sequencer section of reading obtained is classified according to the sequence of the special label of sample, obtains 96 respectively and individual originally reads segment data; After the special label of its sample is deleted to each section of reading sequence, use BWA comparison on large yellow croaker reference genome sequence, obtain sequence alignment bam file; Use the bam file of GATK to gained to analyze, the polymorphism of each base level is judged, obtains the SNP marker of large yellow croaker full-length genome.
The present embodiment obtains 30194 SNP polymorphism marks altogether according to above-mentioned flow process, and wherein 12983 is that 128 large yellow croaker individualities are common; In order to obtain these 128 individual Genetic lineages information, utilize the full genome SNP marker genotype of above-mentioned acquisition, carry out genetic distance to these 128 fishes to analyze, found that there is obvious cluster between some of them individuality, show the offspring that these individualities may be same familys, as shown in Figure 3.
Above the better embodiment of this patent is explained in detail, but this patent is not limited to above-mentioned embodiment, in the ken that one skilled in the relevant art possesses, can also makes a variety of changes under the prerequisite not departing from this patent aim.
Claims (4)
1. the large yellow croaker full-length genome SNP based on double digestion and InDel molecule marking method, it is characterized in that: utilize EcoRI and NlaIII to combine and double digestion is carried out to genome, reach and simplify the object that gene sets up storehouse and order-checking, said method comprising the steps of:
1) joint sequence design: the check order primer sequence of Platform Requirements of the design of joint sequence and two enzyme end of EcoRI and NlaIII and follow-up IlluminaHiseq is consistent, joint sequence is made up of check order platform joint sequence and sample label sequence two part; Sample label design is according to following principle:
A. the rarest two distinguishing base between each sample label sequence;
B. sample label sequence is not containing the base that continuous print two is identical;
C. sample label does not comprise and does not form restriction enzyme site distinguished sequence with joint sequence;
2) genomic dna enzyme is cut: the genomic dna of 200ng carries out two endonuclease reactions in the reaction system of 20ul simultaneously;
3) joint sequence connects: joint sequence is connected to genomic dna enzyme and cuts in same test tube and carry out, and is connected on the enzyme simple stage property end of DNA fragmentation by the joint sequence of design by ligase enzyme;
4) sample mix builds storehouse from pcr amplification: the DNA fragmentation of different individual of sample jointing sequence is carried out balanced mix, ensures the harmony of each sample DNA segment number, adds PCR primer sequence and DNA synthetic enzyme carries out PCR reaction, and amplification connects product;
5) high-flux sequence: utilized by the amplified production in step 4 IlluminaHiseq2000 order-checking platform to check order;
6) sequencing data analysis mining SNP site: first order-checking raw data carries out sorting by the special sequence label of sample, obtain the order-checking section of the reading sequence of each sample, the section of the reading sequence of each sample utilized short data records comparison software BWA comparison on the reference genome of large yellow croaker, and use GATK to carry out SNP excavation.
2. the large yellow croaker full-length genome SNP based on double digestion according to right 1 and InDel molecule marking method, it is characterized in that, utilize two kinds of DNA restriction endonuclease EcoRI and NlaIII to combine, enzyme is carried out to all genes of individuals groups and cuts, obtain the DNA fragmentation of appropriate length, carry out building storehouse order-checking.
3. the large yellow croaker full-length genome SNP based on double digestion according to right 1 and InDel molecule marking method, it is characterized in that, for the enzyme simple stage property end of two kinds of DNA restriction endonuclease EcoRI and NlaIII and requirement, designed joint sequence and the sample label sequence of order-checking platform.
4. the large yellow croaker full-length genome SNP based on double digestion according to right 1 and InDel molecule marking method, is characterized in that, uses the genome digestion products of EcoRI and NlaIII to carry out high-flux sequence and build storehouse and order-checking, and carry out full-length genome SNP excavation.
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Cited By (9)
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| CN106086193A (en) * | 2016-06-27 | 2016-11-09 | 山西医科大学 | A kind of method analyzing mixing sample DNA based on INDEL SNP linkage relationship |
| CN106191246A (en) * | 2016-07-12 | 2016-12-07 | 集美大学 | A kind of method utilizing liquid phase capture to carry out Carnis Pseudosciaenae genomic gene typing |
| CN107841544A (en) * | 2017-12-14 | 2018-03-27 | 浙江海洋大学 | A kind of method for obtaining Japanese eel high density SNP marker |
| CN107937500A (en) * | 2017-11-17 | 2018-04-20 | 深圳华大生命科学研究院 | Batch obtains the method and kit of high-precision insect COI genetic barcodes |
| CN108998539A (en) * | 2018-08-15 | 2018-12-14 | 浙江海洋大学 | Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies |
| CN109022558A (en) * | 2018-08-15 | 2018-12-18 | 浙江海洋大学 | The flat Rockfish genome SNP marker method of Xu Shi based on enzymes combinations Genotyping sequencing technologies |
| CN109082461A (en) * | 2018-08-15 | 2018-12-25 | 浙江海洋大学 | Collichthys lucidus genome SNP marker method based on enzymes combinations Genotyping sequencing technologies |
| CN109182545A (en) * | 2018-10-18 | 2019-01-11 | 浙江海洋大学 | A kind of associated SNP marker of Larimichthys crocea Vibrio harveyi disease and its primer and application |
| CN113782103A (en) * | 2021-10-26 | 2021-12-10 | 大连大学 | DNA matrix processing method based on combined enzyme digestion mechanism |
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