CN110484638A - The evaluation method of Yong fish population genetic diversity - Google Patents

The evaluation method of Yong fish population genetic diversity Download PDF

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CN110484638A
CN110484638A CN201910911579.XA CN201910911579A CN110484638A CN 110484638 A CN110484638 A CN 110484638A CN 201910911579 A CN201910911579 A CN 201910911579A CN 110484638 A CN110484638 A CN 110484638A
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dna
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pcr amplification
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徐胜勇
蔡珊珊
高天翔
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Zhejiang Ocean University ZJOU
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    • C12Q2600/172—Haplotypes

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Abstract

The evaluation method of present invention offer Yong fish population genetic diversity; belong to marine organisms protection field; including carrying out DNA extraction to different groups Yong fish; PCR amplification, sequencing carry out analysis of genetic diversity; wherein; the material source of DNA is that musculature adds succinyl chloride after phenol/chloroform/isoamyl alcohol is added when DNA is extracted.The step of present invention can reduce the generation of phenomenon of rupture when DNA is extracted, and the appearance of non-specificity band when being able to suppress PCR amplification improves PCR product specificity, improves the quality of PCR product, can save PCR product quality testing and purifying, improves efficiency.

Description

The evaluation method of Yong fish population genetic diversity
Technical field
The invention belongs to marine organisms to protect field, and in particular to the evaluation method of Yong fish population genetic diversity.
Background technique
Understand group's connectivity and it is structural be marine ecology, marine resources conservation and evolution biology a basic mesh Mark.All the time, mitochondrial DNA is considered as the onlooker to evolve, and chondriogen site is generally assumed to be in evolutionary process In remain neutral or almost neutral.In addition, as a whole, in conjunction with chondriogen site include encoding gene and non- The evaluation method area of encoding gene control Yong fish population genetic diversity, is often used in Population Genetics research.However, More and more researches show that mitochondrion-encoded genes participate in oxidative phosphorylation approach, plays and energy is provided and adjusts temperature Effect.Therefore, regardless of neutral evolution force, mitochondrion-encoded genes all should by the very big influence of selective strength, So as to cause the adaptability of part.That is, Intramitochondrial genetic diversity can pass through research coding and noncoding region Domain and know.
Yong fish (Platycephalus sp.1) is a kind of demersal, the marine fishes with important commercial value, It is widely distributed along northwest Pacific.In view of biological characteristic, such as something lost of extensive distribution, the feature of fixation and neutrality Homogeney is passed, Platycephalus sp.1 is considered as studying the hereditary connectivity influenced by geographic isolation and local adaptation A preferable selection.Platycephalus sp.1 produces powerful genetic group in a limited distribution Differentiation.And the low-level genetic variation and genetic differentiation in Platycephalus sp.1 group is by utilizing mitochondrial cytochrome b region Segment can identify.
The prior art such as Authorization Notice No. is the Chinese invention patent of 105200047 B of CN, which is related to a kind of north The amplimer and its screening technique of common razor clam chondrioid cox3 gene, amplimer cox3CF are 5 '- TATCATTTAGTHGATATAAGACC-3'；Amplimer cox3CR is 5 '-CCATGAAADCCAGTTATYACAA-3 '；Amplification The preparation method of primer includes download sequence, sequence alignment, design of primers and synthesis, DNA extraction, five step of primer amplification；The hair It is bright to filter out a pair of primer cox3CF and cox3CR for being used to expand northern common razor clam chondrioid part cox3 sequence, and determine Its reaction system and PCR amplification condition, the experiment proved that the primer has the characteristics that band is single, amplification efficiency is high, it can Meet the needs of razor clam chondrioid genome research and subsequent genetic diversity and phylogenetics research.
Summary of the invention
The purpose of the present invention is to provide the evaluation method of Yong fish population genetic diversity, this method can reduce DNA and mention The generation of phenomenon of rupture when taking, the appearance of non-specificity band when being able to suppress PCR amplification improve PCR product specificity, improve The quality of PCR product improves efficiency the step of capable of saving PCR product quality testing and purify.
The technical solution that the present invention is taken to achieve the above object are as follows:
A kind of evaluation method of Yong fish population genetic diversity is provided, it is special using mitochondrion DNA control area as molecular labeling Sign is, comprising:
DNA extraction is carried out to different groups Yong fish；
PCR amplification；
Sequencing；
Carry out analysis of genetic diversity；
Wherein, the material source of DNA is that musculature adds fourth two after phenol/chloroform/isoamyl alcohol is added when DNA is extracted Acyl chlorides.When DNA is extracted, after phenol/chloroform/isoamyl alcohol is added, violent mixing time is too long, be easy to cause part DNA break, is added Succinyl chloride can reduce the generation of DNA break, amplify the target gene fragment of a large amount of expected molecular sizes, improve PCR and produce Object concentration improves the signal-to-noise ratio of consecutive nucleotides curve.In musculature have less DNA enzymatic, the DNA mass extracted compared with It is good.Regulating and controlling effect is played to the transcription and replication of mt DNA in mitochondrion DNA control area, and coded sequence is few, is mt DNA genome In sequence and length variation it is maximum, evolutionary rate is most fast, in the horizontal highest region of polymorphism and population genetic research most Effectively and most sensitive region of DNA domain.Mitochondria control region can verily reflect the genetic variation and genetic differentiation relationship between different groups, very suitable It shares in the genetic diversity of analysis group.
In certain embodiments, above-mentioned PCR amplification the primer includes:
Forward primer: 5 '-GCACTAACCAGGAACAATGGCTTG-3 '；
Reverse primer: 5 '-AAGCTCGGGGATAATGAGAC-3 '.Complementation will not occur for the PCR amplification primer, and formation is drawn Object dimer, and combination can be matched well with specific fragment, effectively amplify specific aim sequence.
In certain embodiments, the reaction system of above-mentioned PCR amplification are as follows: the BSA of 10 × buffer 3 μ L, 0.5mg/mL The Taq of 0.3 μ L, the 2.5U/ μ L of reverse primer of forward primer 0.3 the μ L, 10pM of dNTPs 1.2 the μ L, 10pM of 1.2 μ L, 2mM Archaeal dna polymerase 0.2-0.4 μ L, 1 μ L of template DNA, adds water to be supplemented to 30 μ L.The reaction system of PCR amplification provided by the invention has There is higher reaction efficiency, the generation of mispairing and non-specific amplification phenomenon can be reduced, increases reaction product.
In certain embodiments, peroxide list potassium sulfonate is also added in above-mentioned pcr amplification reaction system.PCR product is not It is pure, it is of poor quality, when there is the non-specific amplifications band such as unintelligible band, miscellaneous band, hangover, it is easy to cause the mistake of sequencing reaction It loses.Peroxide list potassium sulfonate is added during PCR amplification, is able to suppress the appearance of non-specific band, and it is special to improve PCR product Property, the template quality of sequencing reaction is improved, the signal-to-noise ratio of consecutive nucleotides curve is improved, improves sequencing accuracy, so as to The step of saving PCR product quality testing and purifying, improves efficiency.
In certain embodiments, the reaction condition of above-mentioned PCR amplification are as follows: 95 DEG C of 5min, 95 DEG C of 30s, 55-58 DEG C of 45s, 72 DEG C of 1min, 35 circulations, 4 DEG C.The reaction condition of PCR amplification provided by the invention can be improved the spy of reaction efficiency and product It is anisotropic.
It in certain embodiments, is bidirectional sequencing to above-mentioned sequencing.Bidirectional sequencing method is high-efficient, high sensitivity and accurate Property is high.
In certain embodiments, the analysis content of above-mentioned genetic diversity specifically includes: detecting the different groups Yong The base composition of the aim sequence of fish counts haplotype, and calculates haplotype diversity, nucleotide diversity, average nucleotide Difference number.
In certain embodiments, the distinctive haplotype of above-mentioned haplotype, population, calculating variant sites number, haplotype are more Sample, nucleotide diversity, average nucleotide difference number are analyzed using 3.5 software of Arlequin.
The invention has the benefit that
1) for the present invention by optimizing to DNA extraction method, succinyl chloride is added in when extracting, reduces the hair of DNA break It is raw, the appearance of false negative reaction during PCR amplification is reduced, the yield of the target gene fragment of expected molecular size is improved, mentions High PCR product concentration；
2) present invention is able to suppress the appearance of non-specific band, mentions to peroxide list potassium sulfonate is added during PCR amplification High PCR product specificity, improves the template quality of sequencing reaction, improves the signal-to-noise ratio of consecutive nucleotides curve, improving sequencing just True rate improves efficiency so as to save the step of PCR product quality testing is with purifying.
Detailed description of the invention
Fig. 1 is the mitochondria control region sequence variations site figure of Yong fish of the invention；
Fig. 2 is DNA concentration and A of the invention260/280Between 1.80 or so ratio chart；
Fig. 3 is the embodiment of the present invention 1, comparative example 1, comparative example 2, comparative example 3, the PCR products electrophoresis map of comparative example 4；
Fig. 4 is the part sequencer map of the embodiment of the present invention 1, comparative example 1, comparative example 2, comparative example 3, comparative example 4.
Description of symbols: " * " represents base identical with haplotype Hap1 corresponding site.
Specific embodiment
Present invention is further described in detail with reference to embodiments:
Embodiment 1:
Test comes from four groups with 96 tail Yong fish samples, picks up from respectively: Chinese Dongying (31 tail), Chinese Qingdao (24 Tail), Chinese Zhoushan (19 tail), Japan's Tokyo Bay's (22 tail), each department Yu Yang group number is respectively DY, QD, ZS, TB.Respectively It cuts after live body muscle fixed with 95% ethyl alcohol, is placed in -80 DEG C of preservations.
DNA is extracted: sample being taken out under the conditions of -80 DEG C, is placed in ice chest, clip 0.05g sample is rushed with sterile water It washes, removes the ethyl alcohol of sample surfaces；Sample is put into 1.5mL centrifuge tube, is placed on ice, 500 μ L buffers is added (100mmol/L NaCl；10mmol/L Tris-Cl,pH8.0；1mmol/L EDTA, pH8.0), 50 μ L 10%SDS solution, The Proteinase K of 10 μ L 20mg/ μ L, is ground, 56 DEG C of water-baths with grinding rod, and during which every 10min, which turns upside down, mixes once, Become limpid to solution；After solution is cooled to room temperature, it is separately added into 250 μ L saturation phenol and 250 μ L chloroforms and isoamyl alcohol (v/v =24/1) 100 μ L succinyl chlorides, are added, softly shake 10min, room temperature 12000rpm is centrifuged 5min, takes supernatant, repeats to extract 1 It is secondary.Supernatant liquid is transferred in new 1.5mL centrifuge tube, 500 μ L chloroform-isoamyl alcohols are added, softly shake 10min, room temperature 12000rpm is centrifuged 5min, takes supernatant, repeats above-mentioned steps 1 time, supernatant is taken to be transferred in new 1.5mL centrifuge tube, is added 50 μ L 3mol/LNaAc and 1mL dehydrated alcohols place 10min at -20 DEG C, and 12000rpm is centrifuged 10min, obtain nucleic acid precipitating； It is rinsed twice with 75% alcoholic solution of pre-cooling, the dehydrated alcohol of pre-cooling washs precipitating, spontaneously dries, and 60 μ L TE are added and buffer Liquid adds the RNA enzyme of final concentration of 0.05mg/mL to remove RNA.Supernatant is abandoned after being centrifuged 10min under the conditions of 10000rpm 100 μ L deionized waters are added in liquid in centrifuge tube after drying, dissolving DNA extracts quality with agarose gel electrophoresis detection DNA, DNA concentration is measured using Qubit luminoscope, sample is diluted to 100ng/ μ L.
Using primer CR-F:5 '-GCACTAACCAGGAACAATGGCTTG-3 ' and CR-R:5 '- AAGCTCGGGGATAATGAGAC-3 ' expands the mitochondrion DNA control area of sample used.Pcr amplification reaction uses 30 μ L reaction system: dNTPs 1.2 the μ L, 10pM of bovine serum albumin 1.2 the μ L, 2mM of 10 × buffer 3 μ L, 0.5mg/mL are just To the peroxide list sulphur of Taq archaeal dna polymerase 0.4 the μ L, 2mg/mL of 0.3 μ L, the 2.5U/ μ L of reverse primer of primer 0.3 μ L, 10pM Sour 1.8 μ L of potassium solution, 1 μ L of template DNA add water to be supplemented to 30 μ L.Reaction condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 circulations, 4 DEG C keep the temperature.
Sequencing: sequencing primer is amplimer, and sequencing reaction uses 10 μ L reaction systems: template 20ng, BigDye Mix 1.5 μ L, 0.5 μm of ol/L of sequencing primer, reaction condition are as follows: 96 DEG C of initial denaturation 2min, 96 DEG C of denaturation 10s, 55 DEG C of annealing 10s, 60 DEG C extend 4min, 35 circulation, 4 DEG C heat preservation.Sequencing reaction product is purified with 70% ethanol precipitation, in automatic sequencer Sequencing, all samples carry out bidirectional sequencing.
The processing of mitochondrion DNA control area sequence data: sequence assembly, and artificial school are carried out using 7.1 software of DNAStar It is right, it is ensured that the accuracy of sequence.Multiple Sequence Alignment is carried out using MAFFT method in Unipro UGENE 1.21.0.It uses Arlequin3.5 software analysis haplotype (Hap), distinctive haplotype (pHap), calculating variant sites number (S), haplotype are more Sample (h), nucleotide diversity (π), average nucleotide difference number (k).The nucleotide of sequence is found out using 5.0 software of MEGA Composition.
Sequencing obtains the mitochondria control region sequence of 96 tail Yong fishes altogether, is compared and correction is eventually used for population data analysis Mitochondria control region sequence length be 459bp.Fig. 1 is the mitochondria control region sequence variations site of Yong fish, can be seen by Fig. 1 Out, in 459 sites, polymorphic site 21, the 4.58% of mass point sum, including be distributed in the 56th, 88,165,183, 203,8 brief informative sites on 256,324,437 sites and be distributed in the 39th, 72,117,151,178,221,242, 291,13 single mutable sites on 315,343,357,382,448 sites.Through 5.0 software of MEGA analyze, obtain A, T, C, The average content of G base is respectively 32.03%, 32.24%, 21.57%, 14.16%, G base relatively lack, A+T content (64.27%) be apparently higher than G+C content (35.73%), show apparent AT preference and very strong anti-G bias occurred, this with The characteristic of most of fish is consistent.
The population genetic diversity obtained based on mitochondria control region is shown in Table 1, in 96 sequences of 4 geographical population altogether It was found that 29 kinds of haplotypes, wherein 23 haplotypes are the haplotype that single sampled point exclusively enjoys, remaining 5 haplotype is 2 or more The shared haplotype of a sampled point, haplotype Hap-5 are shared by 4 sampled points.In distinctive haplotype, Chinese Dongying (DY) 7 kinds, Chinese 3 kinds of Qingdao (QD), Chinese 9 kinds of Zhoushan (ZS), 4 kinds of Japan's Tokyo Bay (TB).From the angle of each Yong fingerling group Degree sees that level of genetic diversity is not consistent, and the haplotype diversity and nucleotide of Japan's Tokyo Bay (TB) Yong fingerling group is more Sample is all minimum (0.318 ± 0.016,0.0007 ± 0.0003), the haplotype multiplicity of Chinese Zhoushan (ZS) Yong fingerling group Property and nucleotide diversity are all highest (0.912 ± 0.046,0.0046 ± 0.0023), Chinese Dongying (DY) Yong fingerling group Haplotype diversity and nucleotide diversity take second place.
The genetic diversity parameter of 1 four groups of table
Comparative example 1:
When being stripped to DNA, succinyl chloride is not added, rest part and embodiment 1 are completely the same.
Comparative example 2:
Pcr amplification reaction system is not added with peroxide list potassium sulfonate, and rest part and embodiment 1 are completely the same.
Comparative example 3:
When being stripped to DNA, succinyl chloride is not added, pcr amplification reaction system is not added with peroxide list potassium sulfonate, remaining Part and embodiment 1 are completely the same.
Comparative example 4:
After PCR amplification, product is purified, rest part and comparative example 2 are completely the same.
Test example 1:
Ultraviolet spectrophotometry detects DNA concentration and purity:
It takes 10 μ L DNA samples to dilute 100 times to 1000 μ L, measures OD with ultraviolet specrophotometer260、OD280, calculate OD260/OD280Ratio, it is best with 1.80 or so.According to OD260Calculate amount of DNA=50 × OD260Value × extension rate.DNA is dense Degree, A260/280Ratio between 1.80 or so is shown in Fig. 2.μg/mL
As seen from Figure 2, the concentration of the DNA sample of embodiment 1, A260/280Sample accounting between 1.80 or so is obvious Higher than comparative example 1, comparative example 2, comparative example 3, this illustrates, succinyl chloride is added when being stripped to DNA, and it is disconnected can to reduce DNA The generation split improves DNA mass.
Test example 2:
Agarose gel electrophoresis detects pcr amplification product:
Pcr amplification product is detected with 1% agarose gel electrophoresis, voltage 110V, electrophoresis time is about 30min.Electricity Gel is placed in EB solution after having swum and dyes 20min, distilled water rinsing, then result is observed with gel imaging system.PCR product Electrophoretogram is shown in Fig. 3.
Embodiment 1, comparative example 1, comparative example 4 only have a unique specific amplification band, and meet as seen from Figure 3 The size of target sequence, but the band of comparative example 1 is obviously darker, and comparative example 2, comparative example 3 have non-specific amplification item to take out of Existing, succinyl chloride is added when being stripped to DNA in this explanation, can reduce the generation of DNA break, improves DNA mass, amplifies The target gene fragment of a large amount of expected molecular sizes, improves PCR product concentration；Peroxide list potassium sulfonate is added during PCR amplification, It is able to suppress the appearance of non-specific band, improves PCR product specificity.
Fig. 4 is the part Sequencing chromatogram of embodiment 1, comparative example 1, comparative example 2, comparative example 3, comparative example 4.It can be with by Fig. 4 Find out that the background of 1 nucleotide curve peak of embodiment is noiseless, each small peak corresponds to particular bases, 4 nucleotide curve peak of comparative example Background only have few noise, but each peak corresponds to particular bases, the background noise of comparative example 1, comparative example 2, comparative example 3 Miscellaneous, signal-to-noise ratio is low, and sequence curve is made an uproar peak appearance, and base is not easy interpretation, and signal is weak, and it is unknown that many small peaks correspond to base.This says It is bright, succinyl chloride is added when being stripped to DNA, can be improved DNA mass, improves pcr amplification product concentration, improves Sequence kernel The signal-to-noise ratio of thuja acid curve can be improved the specificity of PCR product, mention after pcr amplification reaction system adds peroxide list potassium sulfonate Height sequencing accuracy, it is convenient to omit the purification step of pcr amplification product is sequenced.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Sequence table
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aagctcgggg ataatgagac 20

Claims (8)

1. a kind of evaluation method of Yong fish population genetic diversity, using mitochondrion DNA control area as molecular labeling, feature exists In, comprising:
1) DNA extraction is carried out to different groups Yong fish；
2) PCR amplification；
3) it is sequenced；
4) analysis of genetic diversity is carried out；
Wherein, the material source of the DNA is that musculature adds after phenol/chloroform/isoamyl alcohol is added when the DNA is extracted Succinyl chloride.
2. evaluation method according to claim 1, it is characterised in that: PCR amplification the primer includes: in the step 2)
Forward primer: 5 '-GCACTAACCAGGAACAATGGCTTG-3 '；
Reverse primer: 5 '-AAGCTCGGGGATAATGAGAC-3 '.
3. evaluation method according to claim 2, it is characterised in that: the reaction system of PCR amplification in the step 2) are as follows: Forward primer 0.3 the μ L, 10pM of dNTPs 1.2 the μ L, 10pM of BSA 1.2 the μ L, 2mM of 10 × buffer 3 μ L, 0.5mg/mL Taq the archaeal dna polymerase 0.2-0.4 μ L, 1 μ L of template DNA of 0.3 μ L, 2.5U/ μ L of reverse primer add water to be supplemented to 30 μ L.
4. evaluation method according to claim 3, it is characterised in that: in the step 2) in pcr amplification reaction system also Added with peroxide list potassium sulfonate.
5. evaluation method according to claim 3, it is characterised in that: the reaction condition of PCR amplification in the step 2) are as follows: 95 DEG C of 5min, 95 DEG C of 30s, 55-58 DEG C of 45s, 72 DEG C of 1min, 35 circulations, 4 DEG C.
6. evaluation method according to claim 1, it is characterised in that: the sequencing in the step 3) is bidirectional sequencing.
7. evaluation method according to claim 1, it is characterised in that: the analysis content of genetic diversity in the step 4) It specifically includes: detecting the base composition of the aim sequence of the different groups Yong fish, count haplotype, and calculate haplotype multiplicity Property, nucleotide diversity, average nucleotide difference number.
8. evaluation method according to claim 7, it is characterised in that: the haplotype diversity, nucleotide diversity are put down Equal nucleotide difference number is calculated using 3.5 software of Arlequin.
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