CN111662965A - Method for noninvasive identification of pseudo-female soft-shelled turtles of Chinese soft-shelled turtles by fluorescent quantitative PCR (polymerase chain reaction) - Google Patents
Method for noninvasive identification of pseudo-female soft-shelled turtles of Chinese soft-shelled turtles by fluorescent quantitative PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention relates to the technical field of biological information, and discloses a method for noninvasive identification of a pseudo-female soft-shelled turtle by fluorescent quantitative PCR. The method specifically comprises the step of setting a pair of primer groups for identifying the sex of the pseudofemale soft-shelled turtle of the Chinese soft-shelled turtle, wherein the primer groups comprise primer pairs designed by DNA sequences of target genes 18S rRNA and Gapdh. Fluorescent quantitative PCR data processing Using 2–ΔΔCtMethod, using Gapdh as reference gene, the relative copy number of 18S rRNA in genome is calculated. And (3) screening the pseudofemale trionyx sinensis with the gonad being ovary and the genotype being ZZ by utilizing the fact that the copy number of the DNA sequence of 18S rRNA on the W chromosome of the trionyx sinensis is higher than that on the Z chromosome. The identification method of the invention enables the fluorescent quantitative PCR technology to be used for identifying the sex of the Chinese soft-shelled turtle, can detect the sex by only trace blood, has no influence on the health of the Chinese soft-shelled turtle by non-invasively collecting the tail blood of the Chinese soft-shelled turtle, and has the advantages of high efficiency, high sensitivity,The method has the characteristics of good stability and strong specificity, solves the blank of identifying the pseudo-female soft-shelled turtles of the Chinese soft-shelled turtles, is beneficial to the breeding of all-male soft-shelled turtle seedlings, and improves the culture benefit.
Description
Technical Field
The invention relates to the technical field of biological information, in particular to a method for noninvasive identification of a pseudo-female soft-shelled turtle by fluorescent quantitative PCR.
Background
Chinese soft-shelled turtle (Pelodiscussinensis) is commonly called soft-shelled turtle and is an important and rare aquaculture variety in China. The male soft-shelled turtle has higher growth speed, wide and thick skirt edge, less fat and high price than the female soft-shelled turtle, so that the development of the unisexual culture of the full male seedlings of the soft-shelled turtles can obviously improve the economic benefit and has great significance for the development of the breeding industry of the soft-shelled turtles.
The sex of normally developed Chinese soft-shelled turtle belongs to female heterozygosis type, i.e. female soft-shelled turtle sex chromosome is ZW type, male soft-shelled turtle sex chromosome is ZZ type. The 17 beta-estradiol drug treatment of the early Chinese soft-shelled turtle egg can induce the undifferentiated gonad of the Chinese soft-shelled turtle to develop to the female gonad, and produce the pseudo-female soft-shelled turtle with the ovary genotype of ZZ. The pseudonymous Chinese soft-shelled turtle (ZZ) and the original male Chinese soft-shelled turtle (ZZ) are screened out for reproduction, and the reproduction of the full male soft-shelled turtle can be realized. However, the appearance of the pseudo-female soft-shelled turtle is not greatly different from that of a normally developed Chinese soft-shelled turtle, and the pseudo-female soft-shelled turtle is difficult to identify from the apparent form.
Patent publication No. CN108588237B discloses a method for identifying the sex of trionyx sinensis by using the difference between genome sequences on chromosomes Z and W, which extracts liver tissues of trionyx sinensis to cause death of trionyx sinensis. Moreover, the sex-linked gene primer is detected by common PCR, has relatively low specificity, is easy to generate dimer and has certain interference on result judgment. In addition, the common PCR amplification reaction time is long, and the PCR product needs to be subjected to electrophoresis detection, so that the detection efficiency is reduced.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a method for non-invasively identifying the false female soft-shelled turtle of the Chinese soft-shelled turtle by fluorescent quantitative PCR;
in order to solve the technical problem, the technical scheme adopted by the invention is a method for non-invasively identifying the pseudo-female soft-shelled turtles by fluorescent quantitative PCR, a group of primer pairs for identifying the sex of the pseudo-female soft-shelled turtles of the Chinese soft-shelled turtles is set, the primer pairs comprise a pair of target gene 18S rRNA and an internal reference gene Gapdh, the fluorescent quantitative PCR reaction is adopted, the pseudo-female soft-shelled turtles are screened out by utilizing the difference of the DNA copy number of the target gene 18S rRNA on W chromosomes and Z chromosomes, the target gene 18S rRNA and the internal reference gene Gapdh primers comprise F and R primers, and the primer sequences of the target gene 18S rRNA and the internal reference gene Gapd:
the method for non-invasively identifying the pseudo-female soft-shelled turtle by the fluorescent quantitative PCR comprises the following further technical scheme:
preferably, the specific steps for identifying the false female soft-shelled turtle of the Chinese soft-shelled turtle are as follows:
s1, taking more than 50 Chinese soft-shelled turtle blood DNA with male sex determination, arranging a group of reaction tubes in each individual by adopting a 20-microliter PCR reaction solution system, adding 18SrRNA of a target gene and a Gapdh primer pair of an internal reference gene into each group of reaction tubes respectively, making 3 parallel repeated holes for 18S rRNA of the target gene and the Gapdh of the internal reference gene, carrying out fluorescent quantitative PCR amplification, and calculating the relative copy number of the 18S rRNA of the target gene;
s2, selecting one of the male Chinese soft-shelled turtles in the step S1 as a control sample A, setting the relative copy number of 18SrRNA of the control sample A to be 1, calculating the relative value of the relative copy number of 18S rRNA of other male Chinese soft-shelled turtles to A to obtain the relative value of the relative copy number of 18S rRNA of all male Chinese soft-shelled turtles, and calculating the average value of the relative values of all male soft-shelled turtles
And standard deviation SD, to
As a threshold value;
s3, processing the trionyx sinensis eggs in the gonad differentiation period by using an estrogen drug, then normally culturing to 2 years, screening individuals of the phenotype female trionyx sinensis, extracting blood DNA, carrying out fluorescence quantitative PCR reaction in the step S1 to obtain the relative copy number of the target gene 18S rRNA of each phenotype female trionyx sinensis, and calculating the relative value Y of the relative copy number of each phenotype female trionyx sinensis relative to the relative copy number of the control sample A in the step S2;
s4, comparing the relative value Y calculated in step S3 with the threshold value set in step S2, e.g.
The male soft-shelled turtle is the female soft-shelled turtle,
the soft-shelled turtle is a false female soft-shelled turtle.
Preferably, the 20 μ L PCR reaction liquid system in step S1 is:
preferably, the reaction conditions for the fluorescent quantitative PCR amplification described in step S1 are:
preferably, the storage concentration of the target gene 18S rRNA and the internal reference gene Gapdh primer pair in step S1 is 10. mu. mol/L.
Preferably, the calculation manner of the relative copy number of the target gene 18S rRNA in the step S1 is as follows: setting Gapdh as reference gene, substituting the Ct values of target gene 18S rRNA and Gapdh amplified reference gene into relative quantitative calculation formula 2-ΔΔCtThereby obtaining the relative copy number of 18S rRNA per individual.
Preferably, the estrogen drug in step S3 is 17 β -estradiol solution prepared with 95 wt% ethanol as solvent, and the concentration is 50 μ g/μ L.
Preferably, the gonad differentiation period in step S3 is a period from embryo hatching to 15-18.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention establishes a method for non-invasively identifying the pseudo-female soft-shelled turtle of the Chinese soft-shelled turtle by fluorescent quantitative PCR, adopts a method for taking blood from the tail of the Chinese soft-shelled turtle to take trace blood, overcomes the defect of death or wound of the pseudo-female soft-shelled turtle in the prior art, and has no adverse effect on the growth and development of the Chinese soft-shelled turtle.
2) The invention firstly applies a fluorescent quantitative PCR method to identify the pseudo-female soft-shelled turtle of the Chinese soft-shelled turtle, designs fluorescent quantitative PCR primer sequences with extremely high specificity on a target gene and an internal reference gene, and identifies the pseudo-female soft-shelled turtle of the Chinese soft-shelled turtle by detecting the difference of the copy numbers of DNA sequences of 18S rRNA of the target gene on Z chromosomes and W chromosomes through the fluorescent quantitative PCR. The method overcomes the defects of long period and high error rate in the prior art, and has the characteristics of high sensitivity, good stability and strong specificity. The primer designed by the fluorescent quantitative PCR has extremely high specificity, and the specificity of an amplification product can be judged through a melting curve peak diagram. The sex linked gene primer is detected by common PCR, has relatively low specificity, is easy to generate dimer and has certain interference on result judgment. In addition, the fluorescent quantitative PCR is real-time detection, the PCR time is shorter, the PCR end result can be interpreted, and the method is more suitable for screening high-throughput samples in production; the common PCR amplification reaction time is longer, and the PCR product needs to be subjected to electrophoresis detection, so that the detection efficiency is reduced. The DNA extracted by only collecting trace blood can meet the requirement of fluorescent quantitative PCR detection, the detected DNA can be as low as pg level, and the method is suitable for large-scale detection.
3) The identification method provides a basis for further cultivating a new variety of the Chinese soft-shelled turtles in full-male mode, and the Chinese pseudo-female soft-shelled turtle (ZZ) and the original line Chinese soft-shelled turtle in male mode (ZZ) are bred, so that the breeding of the Chinese soft-shelled turtles in full-male mode can be achieved. Therefore, the method for identifying the pseudo-female soft-shelled turtles of the Chinese soft-shelled turtles is the key for cultivating the new varieties of the full-male soft-shelled turtles of the Chinese soft-shelled turtles. The invention is not only beneficial to the aquaculture seed selection and fine breed cultivation of the Chinese softshell turtles, but also reduces the cultivation cost, improves the cultivation benefit and establishes an all-male breeding mode. The method can also be used for further understanding the sex reversal process of the Chinese softshell turtle, provides a basis for researching the molecular mechanism of the sex reversal of the Chinese softshell turtle and the sex gene regulation of the Chinese softshell turtle, and even has important significance for the research of the Chinese softshell turtle and the large-scale whole reptile animals.
Drawings
Fig. 1 is a bar graph showing the relative values of the gene copy numbers of 50 male turtles, wherein the abscissa is the sample number, the ordinate is the relative value of the gene copy number, the gene copy number of the male turtles of the sample number 1 is set to be 1, and the relative values of the gene copy numbers of the other 49 male turtles and the gene copy number of the male turtles are calculated.
Detailed Description
The present invention will be described in detail with reference to the following examples.
The invention is further illustrated by the following examples, without restricting it.
The experimental methods in the present invention are conventional methods unless otherwise specified.
The percentages in the present invention are all by mass unless otherwise specified.
The proportions in the present invention are volume proportions unless otherwise specified.
Example 1 cultivation of pseudo-female Soft-shelled turtles induced by estrogens
1.1 incubation of Chinese Soft shelled turtle egg and estrogen drug treatment
Taking 60 soft-shelled turtle eggs as a treatment group, beginning to treat the soft-shelled turtle eggs with a 17 beta-estradiol solution of 50 mu g/mu L of sex hormone of the soft-shelled turtle when the soft-shelled turtle eggs are incubated to 15 periods, wherein the 17 beta-estradiol solution is prepared by using 95 wt% ethanol as a solvent, determining the distribution position of the embryo of the soft-shelled turtle in the soft-shelled turtle eggs by using an egg candler, and dripping 5 mu L of medicament on the shell of the embryo surface of the soft-shelled turtle eggs. Carrying out drug treatment once in each period of turtle eggs of the embryo of the Chinese softshell turtle incubated to the 15-18 period, wherein the position and the dosage of each dripping mode are unchanged, and carrying out sex hormone treatment for four times in total, wherein the embryo development morphological change characteristics in the 15-18 period are as follows:
simultaneously taking 60 turtle eggs as a control group, dripping 5 mu L of 95 wt% ethanol on the surface of the egg shell closest to the embryo during the 15-18 period of incubation of each turtle egg, wherein other incubation conditions are completely the same as those of the treatment group;
hatching the turtle eggs of the treated group and the control group until the turtle eggs are out of the shell, and transferring the young turtles to a greenhouse for cultivation to 1 year after the turtle eggs are out of the shell for one month.
1.2 statistics of the number of the phenotype males and females of the Chinese soft-shelled turtles in the control group and the treatment group
Observing the development condition of the gonads of the Chinese softshell turtles, recording the number of phenotype males and females of the Chinese softshell turtles of the treatment group and the control group, and performing statistical analysis, wherein the results are shown in the following table 1:
TABLE 1 number of phenotypic males and females of Chinese softshell turtle in control group and treatment group
As can be seen from table 1, the ratio of the phenotype female soft-shelled turtles in the treatment group is significantly higher than that in the control group, which indicates that a part of the male soft-shelled turtles with ZZ chromosomes in the treatment group develop into pseudo-female soft-shelled turtles with female appearance; the estrogen treatment is carried out in the period of hatching the embryo of the Chinese softshell turtle to 15-18, namely the key period of gonad differentiation of the Chinese softshell turtle, so that a good sex reversion effect can be achieved, and the treatment mode is simple and convenient to operate, low in cost and suitable for production.
Example 2 fluorescent quantitative PCR assay
The fluorescence quantitative PCR test is carried out on the Chinese softshell turtle, and the specific steps are as follows:
1.1 blood drawing of Chinese softshell turtle
1) Placing the screened female Chinese soft-shelled turtle with the phenotype on a clean table, controlling the soft-shelled turtle and straightening the tail.
2) The needle is penetrated into the positions at the two sides of the tail part, the needle is slightly stirred, and after blood seeps out, the syringe is slowly pulled to take out the blood.
1.2 extraction of DNA from blood of Trionyx sinensis Wiegmann
Extracting total DNA of each group of trionyx sinensis blood, and extracting the extracted DNA with ddH2O was diluted 50-fold. Fluorescent quantitative PCR was performed using 18SrRNA as the target gene primer, Gapdh as the internal control, and diluted DNA as the template. The genomic DNA extraction process was as follows:
1) taking 5-10 mu L of fresh or frozen blood sample of the Chinese softshell turtle, adding Buffer GTL to complement to 200 mu L;
2) adding 20 mu L of protease K;
3) adding 200 mu L of Buffer GL, whirling, shaking, fully mixing, and carrying out water bath at 56 ℃ for 10 minutes;
4) centrifuging for a short time to remove water drops on the inner wall of the tube cover, adding 200 mu L of absolute ethyl alcohol, performing vortex oscillation, fully mixing, and centrifuging for a short time;
5) adding all the solution obtained in the previous step into an adsorption column (Spin Columns DM) filled in a collecting tube, if the solution cannot be added at one time, transferring for many times, centrifuging at 12,000rpm for 1 minute, pouring off the waste liquid in the collecting tube, and replacing the adsorption column into the collecting tube again.
6) Adding 500 μ L Buffer GW1 into the adsorption column, centrifuging at 12,000rpm for 1 min, pouring off waste liquid in the collection tube, and replacing the adsorption column into the collection tube;
7) mu.L of Buffer GW2 was added to the adsorption column, centrifuged at 12,000rpm for 1 minute, the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube.
8) And 7, repeating the step.
9) Centrifugation was carried out at 12,000rpm for 2 minutes, the waste liquid in the collection tube was discarded, and the adsorption column was left at room temperature for several minutes to completely dry.
10) Placing the adsorption column in a new centrifuge tube, suspending 50-200 mu LBuffer GE or sterilized water in the middle of the adsorption column, placing at room temperature for 2-5 minutes, centrifuging at 12,000rpm for 1 minute, collecting DNA solution, and storing DNA at-20 ℃ for subsequent fluorescent quantitative PCR experiment.
1.3 fluorescence quantitative PCR of Chinese softshell turtle blood DNA
Primers F and R were designed by primer analysis using Oligo6.0 based on the target gene sequence already recorded in the Gen-Bank of the National Center for Biotechnology Information (NCBI).
The primer design sequence is as follows:
the 20 μ L PCR reaction liquid system optimization protocol was configured according to the following parameters:
the gene copy number analysis is carried out by adopting SYBR Green I method real-time fluorescence quantitative PCR (relative quantification) according to the following parameters, and the reaction condition optimization scheme is as follows:
1.4 statistics of the relative copy number of the Gene of interest
After PCR amplification is finished, Gapdh is set as an internal reference gene, and Ct values of 18S rRNA and Gapdh amplification are substituted into a relative quantitative calculation formula 2-ΔΔCtThus, the relative copy number of 18S rRNA was obtained for each individual.
Example 3 determination of threshold value
Taking blood of 50 trionyx sinensis whose sex is determined as male, performing fluorescence quantitative PCR according to the procedure of example 2, measuring gene copy number, setting the gene copy number of the male trionyx sinensis of sample number 1 as reference to 1, calculating the relative value Y of the gene copy number of other 49 male trionyx sinensis and the gene copy number of the male trionyx sinensis, wherein the relative value of the fluorescence quantitative result of 50 male trionyx sinensis is shown in FIG. 1, and calculating the average value by using the relative value of the fluorescence quantitative result of 50 male trionyx sinensis
And standard deviation SD, to
As the threshold, the threshold was obtained to be 2.57.
Example 4 screening of false female turtle
Observing the Chinese soft-shelled turtles of the treatment group in the example 1, distinguishing male soft-shelled turtles from phenotype female soft-shelled turtles according to differences of appearance and sex, screening the male soft-shelled turtles for continuous culture, carrying out fluorescence quantitative PCR in the example 2 on the phenotype female soft-shelled turtles, determining the gene copy number, comparing the gene copy number of the phenotype female soft-shelled turtles with the gene copy number of the male soft-shelled turtles of the sample number 1 in the example 3, and calculating to obtain the relative copy number Y of the genes;
comparing the relative copy number Y of the phenotypic female turtle gene to the threshold obtained in example 3, e.g.
The male soft-shelled turtle is the female soft-shelled turtle,
the false female soft-shelled turtle is selected, and the false female soft-shelled turtle is screened out.
It should be understood by those skilled in the art that the foregoing is only illustrative of several embodiments of the invention, and not of all embodiments. It should be noted that many variations and modifications are possible to those skilled in the art, and all variations and modifications that do not depart from the gist of the invention are intended to be within the scope of the invention as defined in the appended claims.
Claims (8)
1. A method for noninvasive identification of pseudo-female turtles of Chinese soft-shelled turtles by fluorescent quantitative PCR (polymerase chain reaction) is characterized in that a group of primer pairs for sex identification of the pseudo-female turtles of the Chinese soft-shelled turtles is set, the primer pairs comprise a pair of target gene 18S rRNA and an internal reference gene Gapdh, the pseudo-female turtles are screened out by using the difference of DNA copy numbers of the target gene 18S rRNA on W chromosomes and Z chromosomes by adopting the fluorescent quantitative PCR reaction, the target gene 18S rRNA and the internal reference gene Gapdh primers comprise F and R primers, and the primer sequences of the target gene 18S rRNA and the internal reference gene Gapdh are as follows:
2. the method for non-invasive identification of the pseudo-female trionyx sinensis by fluorescent quantitative PCR according to claim 1, which is characterized in that the specific steps of identifying the pseudo-female trionyx sinensis are as follows:
s1, taking more than 50 Chinese soft-shelled turtle blood DNA with male sex determination, arranging a group of reaction tubes in each individual by adopting a 20-microliter PCR reaction solution system, adding target gene 18S rRNA and reference gene Gapdh primer pairs into each group of reaction tubes respectively, making 3 parallel repeated holes for each target gene 18S rRNA and reference gene Gapdh, carrying out fluorescent quantitative PCR amplification, and calculating the relative copy number of the target gene 18S rRNA;
s2, selecting one of the male Chinese soft-shelled turtles in the step S1 as a control sample A, setting the relative copy number of 18S rRNA of the control sample A to be 1, calculating the relative value of the relative copy number of 18S rRNA of other male Chinese soft-shelled turtles to A, obtaining the relative value of the relative copy number of 18S rRNA of all male Chinese soft-shelled turtles, and calculating the average value of the relative values of all male soft-shelled turtles
And standard deviation SD, to
As a threshold value;
s3, processing the trionyx sinensis eggs in the gonad differentiation period by using an estrogen drug, then normally culturing to 2 years, screening individuals of the phenotype female trionyx sinensis, extracting blood DNA, carrying out fluorescence quantitative PCR reaction in the step S1 to obtain the relative copy number of the target gene 18S rRNA of each phenotype female trionyx sinensis, and calculating the relative value Y of the relative copy number of each phenotype female trionyx sinensis relative to the relative copy number of the control sample A in the step S2;
s4, comparing the relative value Y calculated in step S3 with the threshold value set in step S2, e.g.
The male soft-shelled turtle is the female soft-shelled turtle,
the soft-shelled turtle is a false female soft-shelled turtle.
3. The method for non-invasive identification of the pseudo-female trionyx sinensis by fluorescent quantitative PCR according to claim 2, wherein the 20 μ LPCR reaction liquid system in step S1 is:
4. the method for noninvasive identification of pseudo-female trionyx sinensis through fluorescent quantitative PCR according to claim 2 or 3, wherein the reaction conditions of the fluorescent quantitative PCR amplification in the step S1 are as follows:
5. the method for non-invasive identification of the pseudo-female trionyx sinensis by fluorescent quantitative PCR according to claim 2 or 3, which is characterized in that: the storage concentration of the target gene 18S rRNA and the internal reference gene Gapdh primer pair in step S1 is 10. mu. mol/L.
6. The method for noninvasive identification of pseudo-female trionyx sinensis through fluorescence quantitative PCR according to claim 2, wherein the calculation manner of the relative copy number of the target gene 18S rRNA in the step S1 is as follows: setting Gapdh as reference gene, substituting the Ct values of target gene 18S rRNA and Gapdh amplified reference gene into relative quantitative calculation formula 2-ΔΔCtThereby obtaining the relative copy number of 18S rRNA per individual.
7. The method for non-invasive identification of the pseudo-female trionyx sinensis through the fluorescent quantitative PCR according to claim 2, wherein the estrogen drug in the step S3 is a 17 β -estradiol solution prepared by using 95 wt% ethanol as a solvent, and the concentration is 50 μ g/μ L.
8. The method for non-invasive identification of the pseudo-female trionyx sinensis through the fluorescent quantitative PCR according to claim 2, wherein the gonad differentiation period in the step S3 is a period from embryo incubation to 15-18.
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| CN116042796A (en) * | 2022-11-11 | 2023-05-02 | 中国水产科学研究院珠江水产研究所 | Molecular marker and primer pair for identifying sex of Amyda sinensis and application of molecular marker and primer pair |
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| CN109652563A (en) * | 2019-01-30 | 2019-04-19 | 浙江省水产技术推广总站 | The minimally invasive Rapid identification PCR primer group of Shelled Turtle Trionyx Sinensis ZW sex chromosome type, site, method and kit |
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| CN106811514A (en) * | 2015-12-01 | 2017-06-09 | 中华人民共和国上海出入境检验检疫局 | Soft-shelled turtle subfamily biotic component specificity real-time fluorescence detection method and its kit |
| CN108841945A (en) * | 2018-06-28 | 2018-11-20 | 中国水产科学研究院珠江水产研究所 | A kind of PCR amplification primer, method and the kit of Rapid identification Shelled Turtle Trionyx Sinensis genetic sex |
| CN109652563A (en) * | 2019-01-30 | 2019-04-19 | 浙江省水产技术推广总站 | The minimally invasive Rapid identification PCR primer group of Shelled Turtle Trionyx Sinensis ZW sex chromosome type, site, method and kit |
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| CN116042796A (en) * | 2022-11-11 | 2023-05-02 | 中国水产科学研究院珠江水产研究所 | Molecular marker and primer pair for identifying sex of Amyda sinensis and application of molecular marker and primer pair |
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