CN110452998A - A Method for Genetic Analysis of Yellow Crucian Carp Using Mitochondrial DNA Control Region - Google Patents

Abstract

The invention provides a method for analyzing the genetics of crucian carp by using the mitochondrial DNA control region, which belongs to the technical field of genetic engineering and comprises the following steps: adding a lysate to the muscle tissue sample of the crucian carp for lysis, and then separating the supernatant to obtain mitochondria Genomic DNA; Mitochondrial Genomic DNA is screened for the target sequence; Carp crucian carp genetics analysis is carried out from the screening results; Wherein, the lysate is containing NaOH, Tris-HCl, EDTA, sodium glycocholate and dimercaptopropanol Aqueous solution; target sequences include all or part of the mitochondrial DNA control region. The present invention can easily and quickly obtain high-quality, high-purity mitochondrial genomic DNA that can be used as an amplification template, and has a high yield, and then uses the control region of the mitochondrial DNA of the yellow crucian carp to analyze the genetic information and genetic diversity of the yellow crucian carp. Fast, accurate, simple and practical.

Description

A Method for Genetic Analysis of Yellow Crucian Carp Using Mitochondrial DNA Control Region

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a method for genetically analyzing yellow crucian carp by using a mitochondrial DNA control region.

Background technique

Molecular markers generally refer to DNA markers, which are genetic markers based on nucleotide sequence variation in genetic material between individuals, and have been widely used in genetic research of marine organisms. Fish mitochondrial DNA (Mitochondrial DNA, mtDNA) has become an important molecular marker because of its simple structure, strict maternal inheritance, and lack of recombination. The mitochondrial DNA control region (Control region, CR, also known as the D-loop region) is located between the tRNA Pro and tRNA Phe genes in a non-coding region of mitochondrial DNA. The structure is slightly complex, the evolution rate is fast, and it is easy to accumulate more mutations (such as base substitution, insertion and deletion, etc.), are widely used in intraspecies molecular genetics research. Even though the mitochondrial DNA D-loop region is the most variable segment of mitochondrial DNA, there are also relatively conserved fragments. In terms of composition, the control region can be divided into three segments: the extended termination associated sequences (ETAS), the central conserved domain (CD) and the conserved sequence blocks (CSBs). Among them, the conserved sequence region and the central conserved region are relatively conservative, while mutations often occur in the terminator sequence region. There are multiple conserved fragments in the conserved sequence region, such as CSB-F, CSB-E, CSB-D, CSB-1, CSB-2, CSB-3, etc. These fragments are highly conserved in many mitochondrial DNA, and have not been found yet There are mutations, and there is currently no evidence on the function of the gene to verify its function. At present, the mitochondrial DNA control region structure of various fishes has been analyzed and studied, and the general form of each conserved segment in the control region has been summarized. Length polymorphisms are also found in the control region of mitochondrial DNA in some fishes, and the length polymorphism in the control region may be used in intra-species or inter-species population studies.

Yellow crucian carp (Setipinna tenuifilis) belongs to Clupeiformes, Engraulididae, and genus Crucian carp. It is widely distributed in the western Indian Ocean and Northwest Pacific Ocean, and is distributed in all coastal areas of China. Yellow crucian carp is an inshore and lower-level fish that likes to inhabit sea areas with sandy bottom and slow water flow. Yellow crucian carp is a small economic fish. Since the 1980s, with the decline of traditional economic fish resources such as small yellow croaker, blue-spotted mackerel and hairtail, the catch of yellow crucian carp has increased year by year. Due to excessive fishing pressure, the resources of yellow crucian carp have shown a downward trend. Therefore, how to rationally develop and utilize yellow crucian carp resources, and carry out scientific resource protection and restoration has become an urgent problem to be solved. The use of mitochondrial DNA control region sequences to study the genetic differences and differentiation degrees of different yellow crucian carp populations provides an important theoretical basis for the rational utilization and scientific protection of yellow crucian carp resources.

Contents of the invention

An object of the present invention is to provide a fast, accurate, simple and practical method for genetic analysis of crucian crucian carp using the mitochondrial DNA control region. Mitochondrial genomic DNA with high yield.

The technical scheme that the present invention takes for realizing the above object is: utilize the mitochondrial DNA control region to carry out the method for the genetic analysis of yellow crucian carp, comprise the steps:

Adding lysate to the muscle tissue sample of yellow crucian carp for lysis, and then separating the supernatant to obtain mitochondrial genomic DNA;

Screening of mitochondrial genomic DNA for target sequences;

Carry out genetic analysis of yellow crucian carp from screening results;

Wherein, the lysate is an aqueous solution comprising NaOH, Tris-HCl, EDTA, sodium glycocholate and dimercaptopropanol; according to the feature that mitochondrial genomic DNA is independent of genomic DNA and exists in cells in a circular form, the The components in the lysate can play a positive role, quickly lyse and denature the cells of the crucian carp muscle tissue sample and the protein in the cells, release the mitochondrial genomic DNA, subtract the entire genomic DNA extraction process, and reduce DNA Therefore, high-quality, high-purity mitochondrial genomic DNA that can be used as a template for amplification can be easily and quickly obtained with a high yield;

Target sequences include all or part of the mitochondrial DNA control region. The full sequence of the amplified control region is used for structural analysis of the control region, and the hypervariable region of the amplified control region is used for length polymorphism analysis.

Preferably, the final concentration of NaOH in the lysate is 0.3-0.4M, the final concentration of Tris-HCl is 0.03-0.05M, the final concentration of EDTA is 4.0-5.0M, and the final concentration of sodium glycocholate is 6.0-10.0mM. The final concentration of propanol is 0.2-0.5 mM.

Preferably, the OD ₂₆₀ /OD ₂₈₀ of the mitochondrial genomic DNA is between 1.83-1.92, and the OD ₂₃₀ /OD ₂₆₀ is between 2.12-2.33.

Preferably, the concentration of mitochondrial genomic DNA is >73.5 ng/μL.

Preferably, screening is performed using PCR amplification of said target sequence.

More preferably, the hypervariable region primers in the front of the control region are: HJ primer-F: 5'-CACGCACTTAGCTCCAAGCTA-3'; HJ primer-R: 5'-GACGATACCAGAGTAGTGACG-3';

More preferably, the primers for the second half of the hypervariable region control region are: HJH primer-F: 5'-CAGTCTTCTCTATTCGTCTAG-3'; HJH primer-R: 5'-GTTAGCGAGCTTGATTGCTG-3'.

More preferably, the PCR reaction system: rTaq 0.25 μL, DNA template 2 μL, primer-F 1 μL, primer-R 1 μL, DNTP 2 μL, 10×PCR buffer 2 μL, deionized water 16.75 μL.

More preferably, the PCR amplification program is: denaturation at 95°C for 5 minutes; 40 cycles at 95°C for 30 s, 52°C for 405 s, and 72°C for 40 s; extension at 72°C for 5 min, and storage at 4°C.

Compared with prior art, the beneficial effect of the present invention is:

The components in the lysate for obtaining mitochondrial genomic DNA in the present invention can play a beneficial role, rapidly lyse and denature the protein in the muscle tissue sample cells and cells of the crucian crucian carp, release the mitochondrial genomic DNA, and subtract the extraction of the entire genomic DNA. The extraction process can easily and quickly obtain high-quality, high-purity mitochondrial genomic DNA that can be used as an amplification template, and the yield is high; the present invention uses the mitochondrial DNA control region of the yellow crucian carp to analyze the genetic information and genetic diversity of the yellow crucian carp, Fast, accurate, simple and practical.

The present invention adopts the above-mentioned technical scheme to provide a method for genetic analysis of crucian carp by utilizing the mitochondrial DNA control region, which makes up for the deficiencies in the prior art, and is reasonable in design and convenient in operation.

Description of drawings

Fig. 1 shows the purity and yield of mitochondrial DNA in Test Example 1 of the present invention.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the associated drawings. Some embodiments of the invention are shown in the drawings. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

One embodiment of the present invention provides a method for genetic analysis of crucian carp using the mitochondrial DNA control region, comprising the following steps:

S1: adding lysate to the crucian carp muscle tissue sample for lysis, and then separating the supernatant to obtain mitochondrial genomic DNA;

S2: Screen the mitochondrial genomic DNA for the target sequence;

S3: Carry out genetic analysis of yellow crucian carp from the screening results;

Wherein, the lysate is an aqueous solution comprising NaOH, Tris-HCl, EDTA, sodium glycocholate and dimercaptopropanol; according to the feature that mitochondrial genomic DNA is independent of genomic DNA and exists in cells in a circular form, the The components in the lysate can play a positive role, quickly lyse and denature the cells of the crucian carp muscle tissue sample and the protein in the cells, release the mitochondrial genomic DNA, subtract the entire genomic DNA extraction process, and reduce DNA Therefore, high-quality, high-purity mitochondrial genomic DNA that can be used as a template for amplification can be easily and quickly obtained with a high yield;

Target sequences include all or part of the mitochondrial DNA control region. The full sequence of the amplified control region is used for structural analysis of the control region, and the hypervariable region of the amplified control region is used for length polymorphism analysis.

In one of the embodiments, the final concentration of NaOH in the lysate is 0.3-0.4M, the final concentration of Tris-HCl is 0.03-0.05M, the final concentration of EDTA is 4.0-5.0M, and the final concentration of sodium glycocholate is 6.0-10.0 mM, the final concentration of dimercaptopropanol is 0.2-0.5mM.

In one embodiment, the OD ₂₆₀ /OD ₂₈₀ of the mitochondrial genomic DNA is between 1.83-1.92, and the OD ₂₃₀ /OD ₂₆₀ is between 2.12-2.33.

In one embodiment, the concentration of mitochondrial genomic DNA is >73.5 ng/μL.

In one of the embodiments, the specific steps of obtaining mitochondrial genomic DNA include:

S11: Mix the scorpionfish muscle tissue sample with the lysate at a volume ratio of 1:2-5, grind, transfer the liquid to a centrifuge tube, and incubate in a water bath at 90-95°C for 10-20 minutes, then ice-bath 10-30min, centrifuge at 10000-15000rpm/min for 5-10min, take the supernatant, and get the crude DNA product; among them, the final concentration of NaOH in the lysate is 0.3-0.4M, the final concentration of Tris-HCl is 0.03-0.05M, EDTA The final concentration of sodium glycocholate is 4.0-5.0M, the final concentration of sodium glycocholate is 6.0-10.0mM, and the final concentration of dimercaptopropanol is 0.2-0.5mM; further, the lysate also includes a DNaseI digestion step, which helps to eliminate nuclear DNA residues;

S12: Mix isopropanol with crude DNA, centrifuge to precipitate DNA to remove protein, then mix the precipitate containing DNA with washing solution I, and then centrifuge again to further precipitate protein to remove protein in DNA. Then take the supernatant, add washing solution II and absolute ethanol, and place it at -20°C for 6-12h to promote DNA precipitation to remove impurities such as proteins in the DNA. Then use 70-80% ethanol to remove the cations in the DNA to obtain purified mitochondrial genomic DNA; wherein, the washing solution I contains phosphate buffer with a final concentration of 0.08-0.10M and potassium acetate with a final concentration of 0.01-0.02M ; Wash II contains sodium acetate at a final concentration of 1.4-1.8M and sodium thioglycolate at a final concentration of 0.3-0.4mM. The washing solution can cooperate with the lysate, and can better remove impurities such as proteins, RNA, sugars, salt ions and polyphenols, improve the purity of the extracted mitochondrial genomic DNA, and protect the integrity of the mitochondrial genomic DNA. sex. Further, during washing DNA with washing solution I and washing solution II, it also includes adding RNase A digestion step, which helps to remove RNA residues, and the extracted mitochondrial DNA electrophoresis band is a clear, neat and uniform band, The background is clear, and there is no macromolecule DNA and front-end RNA tailing near the sample well.

In one embodiment, screening is performed using PCR amplification of said target sequence.

In one of the embodiments, the primers for the front hypervariable region of the control region are: HJ primer-F: 5'-CACGCACTTAGCTCCAAGCTA-3'; HJ primer-R: 5'-GACGATACCAGAGTAGTGACG-3';

In one of the embodiments, the primers for the second half of the hypervariable region control region are: HJH primer-F: 5'-CAGTCTTCTCTATTCGTCTAG-3'; HJH primer-R: 5'-GTTAGCGAGCTTGATTGCTG-3'.

In one embodiment, the PCR reaction system: rTaq 0.25 μL, DNA template 2 μL, primer-F 1 μL, primer-R 1 μL, DNTP 2 μL, 10×PCR buffer 2 μL, deionized water 16.75 μL.

In one embodiment, the PCR amplification program is: denaturation at 95°C for 5 minutes; 40 cycles at 95°C for 30s, 52°C for 405s, and 72°C for 40s; extension at 72°C for 5 minutes, and storage at 4°C.

The following will be described in detail in conjunction with specific embodiments. The following examples, unless otherwise specified, do not include other components except unavoidable impurities. The drugs and instruments used in the examples are all routine choices in the art unless otherwise specified. The experimental methods for which specific conditions are not indicated in the examples are implemented according to conventional conditions, such as the conditions described in literature, books or the method recommended by the manufacturer.

Example 1:

A method for carrying out genetic analysis of yellow crucian carp by using the mitochondrial DNA control region, comprising the steps of:

S1: adding lysate to the crucian carp muscle tissue sample for lysis, and then separating the supernatant to obtain mitochondrial genomic DNA;

S11: Yellow crucian carp samples were collected from 7 locations along the coast of China (Dongying, Yantai, Qingdao, Nantong, Wenzhou, Xiamen, and Beibu Gulf) from 2005 to 2013. A total of 202 individuals (Table 1) were collected in the experimental samples through morphological identification. Finally, a small piece of muscle tissue was fixed in 95% alcohol solution and stored in a -20°C refrigerator for later use;

Table 1 Sample information of yellow crucian carp

group sampling location Sea area sampling time Number of samples Dy Dongying bohai sea 2007.05 45 YT Yantai yellow sea 2008.04 twenty three QD Qingdao yellow sea 2007.04 30 NT Nantong yellow sea 2013.01 29 WZ wenzhou East China Sea 2008.03 33 XM Xiamen East China Sea 2013.03 20 BB Beibu Gulf South China Sea 2013.01 twenty two

S12: Mix the scorpionfish muscle tissue sample with the lysate at a volume ratio of 1:4, grind, transfer the liquid to a centrifuge tube, and incubate in a water bath at 92°C for 15 minutes, then ice-bath for 20 minutes, at 12,000 rpm/min Centrifuge for 8 minutes, take the supernatant, and get the crude DNA product; among them, the final concentration of NaOH in the lysate is 0.35M, the final concentration of Tris-HCl is 0.04M, the final concentration of EDTA is 4.6M, and the final concentration of sodium glycocholate is 7.5mM , the final concentration of dimercaptopropanol is 0.3mM;

S13: Mix isopropanol with the crude DNA product at a volume ratio of 1:2, centrifuge at 10,000 rpm/min for 5 min, and centrifuge to precipitate DNA; then add washing solution I to the precipitate containing DNA at a volume ratio of 1:1.5, and mix. Fully dissolve the precipitate, incubate on ice for 60 minutes, centrifuge at 10,000 rpm/min for 5 minutes, and take the supernatant; then add washing solution II and absolute ethanol to the supernatant at a volume ratio of 1:2, place at -20°C for 12 hours, and then dissolve at 10,000 rpm Centrifuge at 10,000rpm/min for 5min, discard ethanol, then add 75% ethanol, flick the centrifuge tube several times to suspend DNA in 75% ethanol, then centrifuge at 10000rpm/min for 5min, discard 75% ethanol to obtain purified DNA ; Wherein, washing liquid I contains the potassium acetate that the phosphate buffer solution that final concentration is 0.08M and the final concentration is 0.015M; Washing liquid II contains the sodium acetate of final concentration 1.5M and the sodium thioglycolate of final concentration 0.37mM;

S2: Amplify the entire sequence of the mitochondrial genomic DNA control region into two sections for PCR amplification. The primer sequences for PCR amplification are shown in Table 2. The PCR amplification system is as follows:

The PCR amplification program was: denaturation at 95°C for 5 min; 40 cycles at 95°C for 30 s, 52°C for 405 s, and 72°C for 40 s; extension at 72°C for 5 min, and storage at 4°C; 2 μL of the PCR product was subjected to 1.5% agarose gel electrophoresis (U =300V), use the recovery kit to recover and purify the target band, and use the ABI Prism 3730 DNA sequencer to carry out positive and negative strand sequencing on the recovered and purified PCR product;

Table 2 adapter sequence, PCR amplification primer sequence

Primer name Primer name HJ primer-F 5'-CACGCACTTAGCTCCAAGCTA-3' HJ primer-R 5'-GACGATACCAGAGTAGTGACG-3' HJH primer-F 5'-CAGTCTTTCTCTATTCGTCTAG-3' HJH primer-R 5'-GTTAGCGAGCTTGATTGCTG-3'

S3: Carry out genetic analysis of yellow crucian carp from the screening results;

The DNASTAR software package (DNASTAR, Inc., Madison, USA) was used to compare, edit and analyze the sequences, and the results were supplemented by manual correction; the Microsoft Excel 2010 software was used to perform statistical analysis on the repeat sequence frequency; the RNAstructure 4.3 software was used to analyze The secondary structure of the repeat sequence was calculated and the released free energy was calculated; the chi-square test (Chi-square test) was performed on the repeat sequence frequency using SPSS18.0 software to calculate the significance of the difference in the repeat sequence frequency between any two groups.

According to the theory of Birky et al., calculate the genetic diversity at different levels. Since there is no intra-individual heterogeneity in the DNA in the control area of crucian carp, the intra-individual genetic diversity K _b =0; the intra-individual genetic diversity K _c =1– _{ΣX i} ² , where Xi refers to a certain DNA type (length Difference) in the population frequency; overall genetic diversity K _t = 1-ΣY _i ² , Yi refers to the frequency of a certain DNA type (length difference) in all individuals. According to the formula C _ip = (mean K _c -K _b )/K _t and C _pt = (K _t -mean K _c )/K _t to calculate the proportion of genetic differences within and between populations.

After editing and manually correcting the sequence of the control region of the yellow crucian carp, the length of the complete sequence of the yellow crucian carp was 1192-1271bp, and the termination sequence region, the central conserved region and the conserved sequence region were identified. The extended termination associated sequence ETAS (TACAT and ATGCA) and six conserved fragments (CSB-D, E, F and CSB-1, 2, 3) were analyzed and the characteristic sequences of each conserved fragment were given. The statistics of the sequence length of the hypervariable region in the control region of 202 yellow crucian carp individuals showed that no length heterogeneity was found in the yellow crucian carp individuals, and the length polymorphism only appeared among individuals. Statistical analysis of the repetition frequency of yellow crucian carp individuals showed that the frequency of 7 repetitions was the highest in the Beibu Gulf population, and the frequency of 6 repetitions was the highest in the other 6 populations; 79.77% of the genetic differences caused by the repetition sequence came from among individuals within the population; the chi-square test results It shows that there are extremely significant differences in the frequency of repeat sequences between the Beibu Gulf population and the other 6 populations. Repeat sequence analysis may be used as an auxiliary method in the population genetics study of crucian carp.

Example 2:

The difference from Example 1 is that the lysate for extracting mitochondrial genomic DNA also includes the addition of 5 mg/mL DNaseI.

Example 3:

The difference from Example 1 is that when extracting mitochondrial genomic DNA, during washing the DNA with Washing Solution I and Washing Solution II, RNase A with a final concentration of 20ug/mL was added and digested at 37°C for 60min.

Example 3:

The difference from Example 2 is that: during washing the DNA with Washing Solution I and Washing Solution II, RNase A with a final concentration of 20ug/mL was added and digested at 37°C for 60min.

Comparative example 1:

The difference from Example 1 is that the lysate for extracting mitochondrial genomic DNA does not contain dimercaptopropanol.

Comparative example 2:

The difference from Example 1 is that the washing solution II for extracting mitochondrial genomic DNA does not contain sodium thioglycolate.

Test example 1:

1. Extract the Purity and Concentration of Mitochondrial Genomic DNA

0.8% agarose gel was electrophoresed in 1×TAE electrophoresis buffer at 135V for 20min, stained with EB, and photographed with an ultraviolet gel imaging system. The purity (OD ₂₆₀ /OD ₂₈₀ and OD ₂₃₀ /OD ₂₆₀ ) and concentration of the extracted DNA were measured with a NANODROP 1000TM UV/VIS Spectrophotometer. Among them, OD ₂₆₀ /OD ₂₈₀ is between 1.8-2.0, OD ₂₃₀ /OD ₂₆₀ is between 2.0-2.5, indicating that the extracted DNA is of high purity; OD ₂₆₀ /OD ₂₈₀ is between 1.8-2.0, indicating that the DNA sample has no protein And RNA pollution, good purity; OD ₂₆₀ /OD ₂₈₀ less than 1.8 indicates that the DNA sample contains protein contamination, OD ₂₆₀ /OD ₂₈₀ greater than 2.0 indicates that the DNA sample contains RNA contamination or DNA fragment fragmentation; OD ₂₃₀ /OD ₂₆₀ is 2.0-2.5 It shows that there is no pollution of impurities such as polysaccharides, phenols and salt ions in the DNA sample, and the purity is good. The purity and concentration of the DNA extracted in Examples 1-4 and Comparative Example 1-2 are shown in Figure 1 (individuals in the DY population). It can be seen that the OD ₂₆₀ /OD ₂₈₀ of the DNA extracted in Example 1 is 1.85, OD ₂₃₀ /OD ₂₆₀ is 2.18, OD ₂₆₀ /OD ₂₈₀ of the DNA extracted in Example 2 is 1.86, OD ₂₃₀ /OD ₂₆₀ is 2.25, OD ₂₆₀ /OD ₂₈₀ of the DNA extracted in Example 3 is 1.89, OD ₂₃₀ /OD ₂₆₀ is 2.31, and the OD ₂₆₀ /OD ₂₈₀ of the DNA that embodiment 4 extracts is 1.83, OD ₂₃₀ /OD ₂₆₀ is 2.14, and this illustrates that the DNA purity that embodiment 1-4 extracts is higher; The OD ₂₆₀ /OD ₂₈₀ of DNA is greater than 2.0, and the OD ₂₃₀ /OD ₂₆₀ is greater than 2.5, indicating that the DNA contains impurities such as protein, RNA or DNA fragment fragmentation, polysaccharides, phenols, and salt ions. The DNA extracted in Comparative Example 1-2 The OD ₂₆₀ /OD ₂₈₀ is less than 1.8, and the OD ₂₃₀ /OD ₂₆₀ is greater than 2.5, indicating that the DNA contains pollution from impurities such as protein, RNA, polysaccharides, phenols and salt ions; Scale 1-2. The above results show that the purity and yield of the extracted mitochondrial genomic DNA extracted in Example 1-4 are better than those in Comparative Example 1-2. It is further illustrated that the various components in the lysate used in the embodiment of the present invention can play a beneficial role, and the cells of the crucian carp muscle tissue sample and the protein in the cells are rapidly lysed and denatured to release the mitochondrial genomic DNA and obtain high-quality, high-quality samples. Purity of mitochondrial genomic DNA, and the yield is high; the embodiment of the present invention uses the washing solution to play a cooperative role with the lysate, and can better remove impurities such as proteins, RNA, sugars, salt ions and polyphenols, and improve The purity of the extracted mitochondrial genomic DNA.

The conventional technologies in the above embodiments are known to those skilled in the art, so they will not be described in detail here.

The above embodiments are only used to illustrate the present invention, not to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the category of the present invention, and the scope of patent protection of the present invention should be defined by the claims.

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Claims (9)

1. utilize the method for the genetic analysis of yellow crucian carp in mitochondrial DNA control area, it is characterized in that: comprise the steps: Adding lysate to the muscle tissue sample of yellow crucian carp for lysis, and then separating the supernatant to obtain mitochondrial genomic DNA; The mitochondrial genomic DNA is subjected to target sequence screening; Carry out genetic analysis of yellow crucian carp from screening results; Wherein, the lysate is an aqueous solution comprising NaOH, Tris-HCl, EDTA, sodium glycocholate and dimercaptopropanol; The target sequence includes the whole or part of the mitochondrial DNA control region. 2. the method for utilizing the mitochondrial DNA control region to carry out the genetic analysis of yellow crucian carp according to claim 1, is characterized in that: the final concentration 0.3-0.4M of NaOH, the final concentration 0.03-0.05 of Tris-HCl in the described lysate M. The final concentration of EDTA is 4.0-5.0M, the final concentration of sodium glycocholate is 6.0-10.0mM, and the final concentration of dimercaptopropanol is 0.2-0.5mM. 3. The method of using the mitochondrial DNA control region to carry out genetic analysis of yellow crucian carp according to claim 1 or 2, characterized in that: the OD ₂₆₀ /OD ₂₈₀ of the mitochondrial genomic DNA is between 1.83-1.92, and the OD ₂₃₀ / OD260 is between _2.12-2.33 . 4. The method for genetic analysis of crucian carp using the mitochondrial DNA control region according to claim 1 or 2, characterized in that: the concentration of the mitochondrial genomic DNA is >73.5ng/μL. 5. The method for genetic analysis of yellow crucian carp using mitochondrial DNA control region according to claim 1, characterized in that: said screening is carried out by amplifying said target sequence by PCR. 6. the method for utilizing the mitochondrial DNA control region to carry out the genetic analysis of yellow crucian carp according to claim 5, is characterized in that: the hypervariable region primer in front of the control region is: HJ primer-F: 5'-CACGCACTTAGCTCCAAGCTA-3' ; HJprimer-R: 5'-GACGATACCAGAGTAGTGACG-3'. 7. the method for utilizing the mitochondrial DNA control region to carry out the genetic analysis of yellow crucian carp according to claim 5, is characterized in that: the second half of the primer of the hypervariable region control region is: HJH primer-F: 5'-CAGTCTTCTCTATTCGTCTAG- 3'; HJHprimer-R: 5'-GTTAGCGAGCTTGATTGCTG-3'. 8. The method for genetic analysis of crucian carp using mitochondrial DNA control region according to claim 5, characterized in that: said PCR reaction system: rTaq 0.25 μL, DNA template 2 μL, primer-F 1 μL, primer-R 1 μL , 2 μL of DNTP, 2 μL of 10×PCR buffer, and 16.75 μL of deionized water. 9. The method for genetic analysis of yellow crucian carp using the mitochondrial DNA control region according to claim 5, characterized in that: the PCR amplification program is: denaturation at 95°C for 5 minutes; 95°C for 30s, 52°C for 405s, and 72°C 40s, 40 cycles; 72°C extension for 5min, 4°C storage.