CN116042796B - Molecular marker and primer pair for identifying sex of Amyda sinensis and application of molecular marker and primer pair - Google Patents
Molecular marker and primer pair for identifying sex of Amyda sinensis and application of molecular marker and primer pair Download PDFInfo
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Abstract
The invention belongs to the technical field of animal sex identification by a molecular biological method, and particularly relates to a molecular marker and primer pair for identifying sex of a shanrui turtle and application thereof. Genomic DNA comprises the sequence set forth in SEQ ID NO:1 and SEQ ID NO:2, identifying the mountain soft-shelled turtle with the sequence shown in the formula 2 as female; genomic DNA comprises the sequence set forth in SEQ ID NO:3, identifying the shanrui turtle as male. The method for identifying the sex of the shan Rui soft-shelled turtles based on the molecular markers for identifying the sex of the shan Rui soft-shelled turtles, which is provided by the invention, is not limited by age and samples, has the advantages of low cost, simplicity and convenience in operation, capability of carrying out mass identification, quick identification, visual and accurate result, capability of avoiding the defects of long time consumption, high cost, easiness in error and the like of the traditional method, capability of carrying out sex identification on the shan Rui soft-shelled turtles in each period quickly and accurately, capability of shortening breeding time, greatly improving yield and reducing raising cost; meanwhile, an identification means is provided for wild population rescue, conservation, recovery and the like.
Description
Technical Field
The invention belongs to the technical field of animal sex identification by a molecular biological method, and particularly relates to a molecular marker and primer pair for identifying sex of a shanrui turtle and application thereof.
Background
Turtles are the older and most morphologically specialized reptiles, the earliest record of appearance on earth being the late triassic, and about 2.2 hundred million years of history have been known to date as "activated stones". The Amyda sinensis (Palea steindachneri) belongs to the class Reptilia (repilia), the order of the tortoise (Chelonia), the family of the soft-shelled turtles (Trionychidae), and the genus Amyda (Palea). In recent decades, wild resources have severely declined due to changes in their habitat, excessive capture, blind introduction of competing organisms, etc. At present, the mountain soft-shelled turtle has been listed by the international natural protection consortium (IUCN 2021) as an extremely dangerous species (CR), and also by the international trade convention for endangered wild animal and plant species (CITES) as incorporated in appendix ii. The artificial breeding can meet the demands of people on the soft-shelled turtles, lighten the damage of people on wild resources and even can supplement wild groups.
The appearance of the shan Rui turtle is similar to that of the Chinese soft-shelled turtle, obvious sex difference exists in growth, the male growth is quick, the sexually mature individuals are large, and the female is opposite, namely the shan Rui turtle breeding industry has the requirement of high-efficiency all-male breeding. The adult mountain soft-shelled turtle has obvious type II appearance, can judge male and female by means of the appearance, but has long growth period and sexual maturity of 5-6 years, and has very similar male and female appearance in the larva stage, and sex identification cannot be carried out from the body type appearance. If the genetic sex of the soft-shelled turtle can be identified after the soft-shelled turtle is taken out of the shell, the unisexual pond culture can be realized in the early stage of the soft-shelled turtle seedlings, the culture cost can be correspondingly reduced, and the yield and benefit can be obviously improved.
Generally, vertebrate sex determination mechanisms can be broadly divided into Genetic Sex Determination (GSD) and Environmental Sex Determination (ESD). Unlike mammal, bird and other higher vertebrates, sex determination of the body depends on genetic material, and the turtle is in the transition link from environment determination type to genetic determination type in animal sex determination mode, which makes elucidation of sex determination mechanism of turtle animal difficult. With the development of biotechnology, the identification of sex-determining means in the middle Hua Bie has gradually progressed from the initial temperature-dependent (TSD) mechanism to the Genomics (GSD) mechanism (ZZ/ZW type). The scholars reveal that the copy number of the 18SrRNA of the W sex chromosome of the turtle animal is much larger than Z, and a method for detecting the repeated copy number of the 18SrRNA amplification by qPCR is designed to identify the male and female, the success rate is 90 percent, but the method has the advantages of high cost, complex operation and 10 percent error rate.
Therefore, a method which is simple in operation, low in cost, high in identification efficiency and accuracy and capable of identifying the sex of the shanrui turtle before sexual maturity is necessary.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a molecular marker for identifying sex of shan Rui turtle, which can accurately identify sex before sexual maturity of shan Rui turtle by identifying the condition of the molecular marker in whole genome DNA of shan Rui turtle.
In order to achieve the above purpose, the following technical scheme may be adopted:
in one aspect, the invention provides a molecular marker for identifying sex of the shan Rui turtle, which comprises a sequence shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, the genomic DNA comprises a sequence as set forth in SEQ ID NO:1 and SEQ ID NO:2, identifying the mountain soft-shelled turtle with the sequence shown in the formula 2 as female; genomic DNA comprises the sequence set forth in SEQ ID NO:3, identifying the shanrui turtle as male.
In another aspect, the invention provides a primer pair for identifying sex of the shan Rui turtle, which is specifically amplified and comprises a primer sequence shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, and a primer pair of the sequence shown in 3.
In still another aspect, the invention provides a detection reagent for identifying sex of the shan Rui turtle, which comprises the primer pair for identifying sex of the shan Rui turtle.
In still another aspect, the invention provides a kit for identifying sex of a mountain soft-shelled turtle, which comprises the detection reagent.
In yet another aspect, the present invention provides a method for identifying sex of a soft-shelled turtle, comprising: extracting genome DNA of the Amyda sinensis as a DNA template, performing PCR amplification by using the primer pair for identifying the sex of the Amyda sinensis to obtain an amplification product, and performing electrophoresis amplification to obtain the amplification product; the amplified product has 366bp and 187bp bands at the same time, so that the amplified product is female mountain soft-shelled turtle; the amplified product has no 366bp band and 187bp band, and is male Amyda sinensis.
The beneficial effects of the invention at least comprise:
(1) The method for identifying the sex of the shan Rui soft-shelled turtles based on the molecular markers for identifying the sex of the shan Rui soft-shelled turtles, which is provided by the invention, is not limited by age and samples, has the advantages of low cost, simplicity and convenience in operation, capability of carrying out mass identification, quick identification, visual and accurate result, capability of avoiding the defects of long time consumption, high cost, easiness in error and the like of the traditional method, capability of carrying out sex identification on the shan Rui soft-shelled turtles in each period quickly and accurately, capability of shortening breeding time, greatly improving yield and reducing raising cost;
(2) The primer pair for identifying the sex of the mountain soft-shelled turtles provided by the invention has strong specificity and high accuracy of the molecular marker for identifying the sex of the mountain soft-shelled turtles, and can identify the genetic sex of the mountain soft-shelled turtles in early stages.
Drawings
FIG. 1 is a diagram showing an example of DNA electrophoresis of male and female Amyda sinensis, wherein male Amyda sinensis is female Amyda sinensis;
FIG. 2 is a diagram showing the verification electrophoresis of specific primers in Female and Male mountain soft-shelled turtle populations, wherein Male is Male, femate is Female, M:2000bpmarker.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides a molecular marker for identifying sex of a mountain soft-shelled turtle, which comprises a sequence shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, the genomic DNA comprises a sequence as set forth in SEQ ID NO:1 and SEQ ID NO:2, identifying the mountain soft-shelled turtle with the sequence shown in the formula 2 as female; genomic DNA comprises the sequence set forth in SEQ ID NO:3, identifying the shanrui turtle as male. The sex of the soft-shelled turtles can be identified by sequencing or characterizing genome DNA of the soft-shelled turtles by the existing sequencing technology, so that the soft-shelled turtles before sexual maturity can be identified quickly, accurately and efficiently.
Another embodiment of the invention provides a primer pair for identifying sex of the shan Rui turtle, which is specifically amplified and comprises a primer sequence shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, and a primer pair of the sequence shown in 3. In order to identify whether the genomic DNA of the shan Rui turtle contains the nucleotide sequence shown in SEQ ID NO: 1. SEQ ID NO:2 or SEQ ID NO:3, can be according to SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, designing a primer, carrying out PCR amplification on the genome DNA of the garter soft-shelled turtle, and identifying the gender of the garter soft-shelled turtle according to the condition of PCR amplification products, so that sequencing is not needed, and the identification cost is reduced; for example, the sex of the Amyda sinensis can be identified according to the condition of gel electrophoresis bands of PCR amplification products. In some embodiments, the nucleic acid sequence as set forth in SEQ ID NO:1 is 366bp band, comprising the nucleotide sequence shown as SEQ ID NO:2 and SEQ ID NO:3, the corresponding band of the genome DNA amplified product of the Amyda sinensis is a 187bp band. Namely female mountain soft-shelled turtles with 366bp bands and 187bp bands, and male mountain soft-shelled turtles with 187bp bands.
In some embodiments, the primer pair for identifying sex of the shan Rui turtle comprises a primer set as shown in SEQ ID NO:4 and SEQ ID NO: 5. Specifically, the primer pair for identifying the sex of the shan Rui soft-shelled turtle can be designed according to the molecular marker for identifying the sex of the shan Rui soft-shelled turtle, and the designed primer needs to be identical to the primer pair shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, and the sequences shown in 3 are all specific. The invention designs a polypeptide shown in SEQ ID NO:4 and SEQ ID NO:5 can specifically amplify the sequence shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, the specificity is strong, and the identification accuracy is high.
In still another embodiment, the invention provides a detection reagent for identifying sex of the shan Rui turtle, which may include the primer pair for identifying sex of the shan Rui turtle. It should be noted that, the primer pair for identifying the sex of the mountain soft-shelled turtle can be combined with a conventional auxiliary reagent to form a detection reagent for identifying the sex of the mountain soft-shelled turtle; conventional auxiliary reagents are known in the art, such as PCR reaction buffers, DNA polymerase or dNTPs in a PCR amplification reaction system, and the like. The detection reagent comprising the primer pair for identifying the sex of the mountain soft-shelled turtle can enable the molecular marker for identifying the sex of the mountain soft-shelled turtle and the primer pair to be subjected to specific amplification, so that the sex of the mountain soft-shelled turtle is identified.
In still another aspect, the invention provides a kit for identifying sex of a mountain soft-shelled turtle, which comprises the detection reagent. It should be noted that the composition of the detection kit is in a conventional form known in the art, for example, the detection kit may be provided with a kit instruction, for example, a reagent bottle containing various detection reagents may be provided, for example, a plurality of small grids may be provided for placing the detection reagent bottle, for example, a dropper may be provided for removing the detection reagents, etc. Similarly, the primer pair for identifying the sex of the mountain softshell turtles based on the detection kit has relatively strong specificity to the molecular marker for identifying the sex of the mountain softshell turtles.
Still another embodiment of the present invention provides a method for identifying sex of a shan Rui turtle, which may include: extracting genome DNA of the Amyda sinensis as a DNA template, performing PCR amplification by using the primer pair for identifying the sex of the Amyda sinensis to obtain an amplification product, and performing electrophoresis amplification to obtain the amplification product; the amplified product has 366bp and 187bp bands at the same time, so that the amplified product is female mountain soft-shelled turtle; the amplified product has no 366bp band and 187bp band, and is male Amyda sinensis. The primer pair for identifying the sex of the shan Rui soft-shelled turtle is used for PCR amplification on the genome DNA of the shan Rui soft-shelled turtle, then gel electrophoresis is carried out on the PCR amplification product, and the sex of the shan Rui soft-shelled turtle is identified through electrophoresis strips, so that the cost is low, the result is visual and the accuracy is high.
In some embodiments, the above method for identifying sex of shan Rui turtle, the PCR amplification procedure may include: 94 ℃ for 30s;98 ℃ for 10s;63 ℃ for 30s;72 ℃,60s;35 cycles, 72℃for 2min.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
In the following examples, the experimental use of the mountain soft-shelled turtles all come from the market of the flower and bird worms in Guangzhou urban area of Guangdong province, 60 mountain soft-shelled turtles (30 male mountain soft-shelled turtles and 30 female mountain soft-shelled turtles) are randomly selected, after sex detection by gonad anatomy and histology, the muscle tissues of each individual are cut off, and the individual is soaked in absolute ethyl alcohol and stored at-20 ℃ for standby.
In the following examples, specific amplification (PCR amplification) was performed according to the following procedure: (1) Extracting the whole genome DNA of male and female mountain Ruiye turtles by using a DNA extraction kit, detecting the DNA quality by using gel electrophoresis, and storing the DNA at the temperature of-20 ℃ for standby after the DNA detection is qualified; (2) use of primer F: SEQ ID NO:3, a step of; r: SEQ ID NO:4 (sex-specific molecular database constructed according to the deep whole genome sequencing comparative analysis of the obtained male and female turtles, designing and verifying the obtained sex-labeled primers on the sex-specific DNA sequence fragments), carrying out PCR amplification on the extracted male and female turtles DNA, wherein the PCR amplification reaction volume is 20uL, and the method comprises the following steps: PCRMix 10. Mu.L, 10. Mu. Mol/L forward and reverse primers each 1. Mu.L, 50. Mu.g/ml to 100. Mu.g/ml template DNA 1. Mu.L, ultrapure water 7. Mu.L; the PCR reaction procedure was: 94 ℃ for 30s;98 ℃ for 10s;63 ℃ for 30s;72 ℃,60s;35 cycles, 72 ℃,2min; (3) After the completion of the reaction, the reaction mixture was subjected to 1.2% agarose gel electrophoresis.
In the following examples, specific primer amplified fragment sequencing was performed according to the following steps: the PCR amplified product was purified and recovered by E.Z.N.A.TMGelExactionkit (omega Bio-Tek, guangzhou, china), and the recovered DNA was ligated to PMDTM19-T (TaKaRaBiotechnology, guangzhou, china) vector, and positive clone detection was selected for sequencing.
Example 1 validation of specific primer pairs in populations
(1) Selecting 30 samples of male and female mountain soft-shelled turtles, respectively carrying out whole genome DNA according to the specific amplification (PCR amplification) method, and detecting the DNA extraction quality by using gel electrophoresis, wherein the result is shown in figure 1, and the result shows that the DNA extraction quality is good;
(2) According to the specific amplification (PCR amplification) method, the whole genome DNA extracted in the step (1) is used as a DNA template to carry out PCR amplification respectively to obtain amplification products, then gel electrophoresis is carried out, the result is shown in figure 2, in 30 samples, the female Shanzhong DNA amplification products have PCR specific bands at 187bp and 366bp, and the male Shanzhong DNA amplification products have PCR specific bands at 187bp, so that the primer can accurately identify the sex of the Shanzhong;
(3) Sequencing the amplified product in the step (2), and combining the sequencing result with 366bp and 187bp bands to obtain a female garter turtle strip sequencing sequence as shown in SEQ ID NO:1, the sequencing sequence of the female short stripe of the mountain soft-shelled turtle is shown as SEQ ID NO:2 is shown in the figure; the sequencing sequence of the male mountain soft-shelled turtle strip is shown as SEQ ID NO: 3.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (6)
1. The molecular marker for identifying the sex of the mountain soft-shelled turtle is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1. SEQ ID NO:2 and seq id no:3, the genomic DNA comprises a sequence as set forth in seq id no:1 and seq id no:2, identifying the mountain soft-shelled turtle with the sequence shown in the formula 2 as female; genomic DNA does not comprise as set forth in seq id no:1, comprising a sequence as set forth in seq id no:3, identifying the shanrui turtle as male.
2. Primer pair for identifying sex of the shan Rui turtle, which is characterized in that the primer pair is specifically amplified and comprises the primer pair as shown in SEQ ID NO: 1. SEQ ID NO:2 and seq id no:3, a primer pair of the sequence shown in 3; nucleotide sequences of the primer pair are as shown in SEQ ID NO:4 and seq id no: 5.
3. The detection reagent for identifying the sex of the mountain soft-shelled turtle is characterized by comprising the primer pair for identifying the sex of the mountain soft-shelled turtle according to claim 2.
4. A kit for identifying sex of Amyda sinensis, which is characterized by comprising the detection reagent according to claim 3.
5. A method for identifying sex of a shan Rui turtle, comprising: extracting genome DNA of the Amyda sinensis as a DNA template, performing PCR amplification by using the primer pair of claim 2 to obtain an amplification product, and performing electrophoresis amplification to obtain the amplification product; the amplified product has 366bp and 187bp bands at the same time, so that the amplified product is female mountain soft-shelled turtle; the amplified product has no 366bp band and 187bp band, and is male Amyda sinensis.
6. The method for identifying sex of mountain soft-shelled turtle as claimed in claim 5, wherein the PCR amplification procedure includes: 94 ℃ for 30s;98 ℃ for 10s;63 ℃ for 30s;72 ℃,60s;35 cycles, 72℃for 2min.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037128A (en) * | 1997-03-31 | 2000-03-14 | Council Of Scientific & Industrial Research | Process for the preparation of semisynthetic amplicon useful for sex determination of the papaya plant |
| CN108048579A (en) * | 2018-01-25 | 2018-05-18 | 湖南生物机电职业技术学院 | PCR amplification primer, method and the kit of Rapid identification Shelled Turtle Trionyx Sinensis genetic sex |
| CN109628569A (en) * | 2019-01-24 | 2019-04-16 | 中国水产科学研究院长江水产研究所 | A kind of Shelled Turtle Trionyx Sinensis female sex specific molecular marker and genetic sex identification method |
| CN109680076A (en) * | 2019-01-24 | 2019-04-26 | 中国水产科学研究院长江水产研究所 | A kind of molecular labeling and method for Shelled Turtle Trionyx Sinensis genetic sex identification |
| CN111662965A (en) * | 2020-07-17 | 2020-09-15 | 安徽农业大学 | Method for noninvasive identification of pseudo-female soft-shelled turtles of Chinese soft-shelled turtles by fluorescent quantitative PCR (polymerase chain reaction) |
| CN113755566A (en) * | 2021-09-26 | 2021-12-07 | 浙江省农业科学院 | PCR primer set, site, method and kit for rapidly identifying sex of wattle-necked softshell turtle |
| WO2022134451A1 (en) * | 2020-12-25 | 2022-06-30 | 中国水产科学研究院珠江水产研究所 | Snp molecular marker for identification of sex of osteoglossum bicirrhosum, and use thereof |
-
2022
- 2022-11-11 CN CN202211411732.0A patent/CN116042796B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037128A (en) * | 1997-03-31 | 2000-03-14 | Council Of Scientific & Industrial Research | Process for the preparation of semisynthetic amplicon useful for sex determination of the papaya plant |
| CN108048579A (en) * | 2018-01-25 | 2018-05-18 | 湖南生物机电职业技术学院 | PCR amplification primer, method and the kit of Rapid identification Shelled Turtle Trionyx Sinensis genetic sex |
| CN109628569A (en) * | 2019-01-24 | 2019-04-16 | 中国水产科学研究院长江水产研究所 | A kind of Shelled Turtle Trionyx Sinensis female sex specific molecular marker and genetic sex identification method |
| CN109680076A (en) * | 2019-01-24 | 2019-04-26 | 中国水产科学研究院长江水产研究所 | A kind of molecular labeling and method for Shelled Turtle Trionyx Sinensis genetic sex identification |
| CN111662965A (en) * | 2020-07-17 | 2020-09-15 | 安徽农业大学 | Method for noninvasive identification of pseudo-female soft-shelled turtles of Chinese soft-shelled turtles by fluorescent quantitative PCR (polymerase chain reaction) |
| WO2022134451A1 (en) * | 2020-12-25 | 2022-06-30 | 中国水产科学研究院珠江水产研究所 | Snp molecular marker for identification of sex of osteoglossum bicirrhosum, and use thereof |
| CN113755566A (en) * | 2021-09-26 | 2021-12-07 | 浙江省农业科学院 | PCR primer set, site, method and kit for rapidly identifying sex of wattle-necked softshell turtle |
Non-Patent Citations (5)
| Title |
|---|
| Farming Characteristics and the Ecology of Palea steindachneri(Trionychidae) in Vietnam;Olivier Le Duc等;Russian Journal of Herpetology;第27卷(第2期);第97-108页 * |
| 三种鳖线粒体DNA细胞色素b基因序列的比较分析;陈合格等;水生生物学报;第30卷(第4期);第380-385页 * |
| 三种鳖线粒体DNA部分基因序列的比较分析和分子鉴定标记;陈合格;中国优秀博硕士学位论文全文数据库（硕士）基础科学辑(第07期);第A006-49页 * |
| 两爪鳖和山瑞鳖线粒体全基因组分析及其在龟鳖目中的系统学地位;李小三;中国优秀硕士学位论文全文数据库 基础科学辑(第02期);第A006-259页 * |
| 山瑞鳖的研究进展;赵忠添等;南方农业学报;第45卷(第12期);第2280-2283页 * |
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