Primer combination, kit and application for identifying genetic sex of sturgeon

Abstract

The present invention provides a primer combination, a kit and an application for identifying the genetic sex of sturgeons. Through two pairs of primers, not only can the genetic sex of sturgeons be identified simply and quickly, but also experimental errors can be effectively avoided, thereby improving the accuracy of sturgeon sex identification. At the same time, the present invention is not limited by the age of sturgeons, will not cause major damage to sturgeons, and has good universality, which can help sturgeon sex control breeding and germplasm resource protection and utilization, and provide technical support for biodiversity research and Yangtze River protection work.

Description

Technical Field

[0001] The invention relates to molecular biotechnology, and in particular to a primer combination, a kit and an application for identifying the genetic sex of sturgeons. Background Art

[0002] Sturgeon is the abbreviation of Acipenseriformes. Sturgeon is the only existing large cartilaginous fish in the class of bony fish. It matures at a long age and spawns in rivers. Most species have the habit of migrating between rivers and seas. There are 27 species in the world, which have great academic significance and economic value. However, the intensification of human activities and overfishing over the past 100 years have led to a sharp decline in the amount of all sturgeon resources. All species are currently endangered to varying degrees, which has attracted widespread attention from the society.

[0003] Sturgeons do not have obvious external secondary sexual characteristics such as pearl stars. Therefore, it is difficult to distinguish male sturgeons from immature female sturgeons in non-spawning areas. In spawning areas, semen can generally be squeezed out of the genital pores of mature male sturgeons, while female sturgeons generally have swollen abdomens and thicker waists. However, female sturgeons that are smaller and about to spawn for the first time are often confused with male sturgeons without semen and immature female sturgeons. The external morphology of the genital pores of sturgeons can be used to identify males and immature females. Observations of wild adults of North American sturgeons, middle-nosed sturgeons, Atlantic sturgeons and short-nosed sturgeons have found that the female genital pores are generally "O"-shaped, while the male genital pores are generally "Y"-shaped. The accuracy of this identification method is 82% in live specimens, but only 29% in dead specimens. Some scholars have identified the sex of Russian sturgeons by measuring measurable traits of the head, with an accuracy rate of more than 90%. For sturgeons whose gonads have developed to the second stage or above, the most direct method of sex identification is to distinguish the ovaries and testes through abdominal surgery. However, the above-mentioned sturgeon sex identification methods can only identify sturgeons that have developed to a certain age, and cannot identify the sex of young fish. In addition, some sex identification methods can cause great damage to sturgeons, such as abdominal surgery techniques. There are reports about female-specific DNA fragments in sturgeon sex identification methods. In the operation of this method, if DNA extraction fails, drugs are added incorrectly or omitted, the sturgeons to be sexed will be misjudged as male sturgeons, resulting in errors in sturgeon sex identification.

[0004] Exploring the regulatory mechanism of sexual differentiation in sturgeons and establishing new technologies for breeding all-female or high-proportion female sturgeons have important theoretical and practical significance for the protection and development of their germplasm resources. The development of sex-specific molecular markers helps to accurately and quickly obtain phenotypes and determine sex, thereby carrying out artificial breeding and breeding, and also helps to carry out research on sex-controlled breeding technology in commercial production, and then establish a single-sex system. Therefore, the development of a molecular marker for accurately identifying the sex of sturgeons has great production application value in artificial breeding of sturgeons. Summary of the invention

[0005] The present invention provides a primer combination, a kit and applications thereof for identifying the genetic sex of sturgeons, which improves the accuracy of sex identification of sturgeons and enables sex identification to be performed throughout the entire life cycle of sturgeons.

[0006] The present invention also provides a method for identifying the genetic sex of sturgeons, which uses the above-mentioned primer combination or kit, is simple to operate, has accurate results, and can be used for identification throughout the life cycle of sturgeons.

[0007] The invention provides a primer combination for identifying the genetic sex of sturgeon, comprising a first primer pair and a second primer pair; the first primer pair comprises a first primer and a second primer, the nucleotide sequence of the first primer is shown as SEQ ID NO:1, and the nucleotide sequence of the second primer is shown as SEQ ID NO:2; the second primer pair comprises a third primer and a fourth primer, the nucleotide sequence of the third primer is shown as SEQ ID NO:3, and the nucleotide sequence of the fourth primer is shown as SEQ ID NO:4.

[0008] The primer combination as described above, wherein the first primer pair is used to amplify a female sturgeon-specific DNA fragment, the nucleotide sequence of the female sturgeon-specific DNA fragment is shown in SEQ ID NO:5, and the fragment size is 473bp; the second primer pair is used to amplify a DNA fragment shared by female sturgeons and male sturgeons, the nucleotide sequence of the DNA fragment shared by female sturgeons and male sturgeons is shown in SEQ ID NO:6, and the fragment size is 717bp.

[0009] The primer combination as described above, wherein the sturgeon includes at least one of the Chinese sturgeon, the Yangtze sturgeon, the small-sterlet sturgeon, the Schrenckii sturgeon, the large-mixed sturgeon, the Siberian hybrid sturgeon, the Siberian sturgeon, the Dabryan sturgeon, the Russian sturgeon, the shining sturgeon, the European sturgeon, the lake sturgeon, the naked belly sturgeon, the Persian sturgeon, the high-headed sturgeon, and the snout sturgeon.

[0010] The invention provides a kit for genetic sex identification of sturgeons, comprising the above primer combination.

[0011] The kit as described above, wherein the kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, and loading buffer.

[0012] The present invention provides an application of the primer combination or the kit in identifying the genetic sex of sturgeons.

[0013] The present invention also provides a method for identifying the genetic sex of sturgeon, comprising the following steps:

[0014] Extract DNA from sturgeon samples to be tested;

[0015] Using the DNA of the sturgeon sample to be tested as a template, using the above primer combination or the above kit to perform PCR amplification to obtain a PCR amplification product;

[0016] The PCR amplification product was tested by agarose gel electrophoresis. When two bands of 473 bp and 717 bp appeared in the test results, the genetic sex of the sturgeon was identified as female; when one band of 717 bp appeared in the test results, the genetic sex of the sturgeon was identified as male.

[0017] In the method as described above, the sample of sturgeon to be tested includes at least one of fins and mucus of the sturgeon.

[0018] As described above, in the PCR amplification, the PCR reaction system includes 3-5 μL of DNA template, 0.5-1.5 μL of the first primer, 0.5-1.5 μL of the second primer, 0.25-0.75 μL of the third primer, 0.25-0.75 μL of the fourth primer, 10-15 μL of 2×Taq Master Mix and 4-6 μL of ddH 2 O.

[0019] As described above, in the PCR amplification, the PCR amplification program includes pre-denaturation at 93-95°C for 3 min; denaturation at 93-95°C for 10 s, annealing at 54-56°C for 10 s, and extension at 72°C for 19 s, for a total of 35 cycles; and finally extension at 72°C for 10 min.

[0020] The primer combination of the present invention provides two pairs of primers, which can avoid experimental errors caused by unsuccessful DNA extraction, wrong addition of drugs or omission of drugs, thereby improving the accuracy of sturgeon sex identification. At the same time, the primer combination of the present invention is not limited by the age of the sturgeon, and sex identification can be performed throughout the life cycle of the sturgeon. The present invention provides assistance for sex-controlled breeding of sturgeons and protection and utilization of germplasm resources, and provides technical support for biodiversity research and Yangtze River protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the electrophoresis diagram of genetic sex identification of Chinese sturgeon (Acipenser sinensis) in Example 1;

[0022] Figure 2 The electrophoresis diagram of genetic sex identification of Yangtze sturgeon (Acipenser dabryanus) in Example 2;

[0023] Figure 3 This is the electrophoresis diagram of genetic sex identification of Acipenser ruthenus in Example 3;

[0024] Figure 4This is the electrophoresis diagram of genetic sex identification of Acipenser schrencki in Example 4;

[0025] Figure 5 This is the electrophoresis diagram of genetic sex identification of large mixed sturgeon (Acipenser schrencki♂×Huso dauricus♀) in Example 5;

[0026] Figure 6 This is the electrophoresis diagram of genetic sex identification of Siberian hybrid sturgeon (Acipenser schrenckii♂×Acipenser baerii♀) in Example 6;

[0027] Figure 7 This is the electrophoresis diagram of genetic sex identification of Siberian sturgeon (Acipenser baerii) in Example 7;

[0028] Figure 8 This is the electrophoresis diagram of genetic sex identification of Huso dauricus in Example 8;

[0029] Fig. 9 The electrophoresis diagram of genetic sex identification of Russian sturgeon (Acipenser gueldenstaedti) in Example 9;

[0030] Fig.10 The electrophoresis diagram of genetic sex identification of Acipenser stellatus in Example 10;

[0031] Fig.11 This is the electrophoresis diagram of genetic sex identification of European snapper (Huso huso) in Example 11;

[0032] Fig.12 The electrophoresis diagram of genetic sex identification of lake sturgeon (Acipenser fulvescens) in Example 12;

[0033] Fig.13 The electrophoresis diagram of genetic sex identification of naked belly sturgeon (Acipenser nudiventris) in Example 13;

[0034] Fig.14 The electrophoresis diagram of genetic sex identification of Acipenser persicus in Example 14;

[0035] Fig.15 This is the electrophoresis diagram of genetic sex identification of Acipenser transmontanus in Example 15;

[0036] Fig.16This is the electrophoresis diagram for genetic sex identification of sturgeon (Acipenser medirostris) in Example 16. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific implementation methods listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0038] A first aspect of the present invention provides a primer combination for identifying the genetic sex of sturgeon, comprising a first primer pair and a second primer pair; the first primer pair comprises a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO:1, and the nucleotide sequence of the second primer is shown in SEQ ID NO:2; the second primer pair comprises a third primer and a fourth primer, the nucleotide sequence of the third primer is shown in SEQ ID NO:3, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO:4.

[0039] The primer combination for identifying the genetic sex of sturgeon includes a first primer pair and a second primer pair. In the first primer pair of the present invention, the first primer is an upstream primer and the second primer is a downstream primer; in the second primer pair of the present invention, the third primer is an upstream primer and the fourth primer is a downstream primer.

[0040] The present invention obtains a primer combination for identifying the genetic sex of sturgeons based on sturgeon genomic DNA. When designing the primer combination, the present invention not only considers how to simply and quickly identify the genetic sex of sturgeons, but also takes how to avoid experimental errors as a core factor, thereby obtaining a primer combination including a first primer pair and a second primer pair.

[0041] The first primer pair is used to simply and quickly identify whether the genetic sex of the sturgeon is female, and the second primer pair is used to avoid experimental errors caused by unsuccessful DNA extraction, wrong addition of drugs, or omission of drugs. When the second primer pair amplifies a DNA fragment shared by female and male sturgeons, it proves that the experimental operation is correct or the experimental materials are normal, and there are no situations such as unsuccessful DNA extraction, wrong addition of drugs, or omission of drugs. At this time, the results amplified by the first primer pair are more accurate.

[0042] Furthermore, in order to reduce the impact of DNA fragment loss caused by severe DNA degradation on the identification of the genetic sex of sturgeons, a primer pair as shown in SEQ ID NO: 1-SEQ ID NO: 4 was designed. The amplified fragments obtained based on the primer pair are not only located on the same chromosome, but also the distance between the two is less than 20k bp. Therefore, even if severe DNA degradation occurs, it will not affect the identification results.

[0043] Therefore, the primer combination of the present invention not only has the ability to simply and quickly identify the genetic sex of sturgeons, but also has the effect of avoiding experimental errors, thereby improving the accuracy of sturgeon sex identification.

[0044] Further, the first primer pair is used to amplify a female sturgeon-specific DNA fragment, the nucleotide sequence of which is shown in SEQ ID NO: 5, and the fragment size is 473 bp. The second primer pair is used to amplify a DNA fragment common to female and male sturgeons, the nucleotide sequence of which is shown in SEQ ID NO: 6, and the fragment size is 717 bp.

[0045] To ensure the accuracy of the amplification results, the present invention defines the fragments after amplification. When the results after amplification do not meet the above limitations, it means that the experimental operation is incorrect or there may be problems with the experimental materials. Therefore, the accuracy of the results can be guaranteed by re-experimentation.

[0046] The present invention does not limit the primer combination and can be used for genetic sex determination of various sturgeons, for example, it can be Chinese sturgeon, Yangtze sturgeon, small-body sturgeon, Acipenser schrenckii, large-mixed sturgeon, Siberian hybrid sturgeon, Siberian sturgeon, Dabryan sturgeon, Russian sturgeon, flash sturgeon, European sturgeon, lake sturgeon, naked belly sturgeon, Persian sturgeon, high-headed sturgeon, and snout sturgeon.

[0047] Among them, the large mixed sturgeon refers to the hybrid of Acipenser schrenckii and Acipenser dabryanus. In one embodiment of the present invention, the large mixed sturgeon used is the hybrid of Acipenser schrenckii, the male parent is Acipenser dabryanus, and the female parent is Acipenser dabryanus. The Siberian hybrid sturgeon is the hybrid of Acipenser siberiensis and Acipenser schrenckii. In one embodiment of the present invention, the Siberian hybrid sturgeon used is the hybrid of Acipenser schrenckii, the male parent is Acipenser siberiensis, and the female parent is Acipenser siberiensis.

[0048] The second aspect of the present invention provides a kit for genetic sex identification of sturgeon, comprising the primer combination provided by the first aspect of the present invention.

[0049] Since the kit includes the primer combination provided by the first aspect of the present invention, the kit also has the ability to simply and quickly identify the genetic sex of sturgeons and avoid experimental errors, thereby improving the accuracy of sturgeon sex identification.

[0050] In a specific embodiment, the kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, and loading buffer.

[0051] Among them, water can provide the ionic environment and water molecule medium required for PCR reaction, so that the interaction between various molecules in the reaction can proceed smoothly, and it can also help maintain the temperature stability of the reaction system to ensure the reliability and accuracy of the reaction. PCR buffer can adjust the pH value of the reaction system to keep the working environment of TaqDNA polymerase alkaline. 2+ It can also affect the specificity of the reaction and the yield of the amplified fragment. dNTPs refers to four free deoxyribonucleoside triphosphates, which are the raw materials necessary for PCR amplification. DNA polymerase refers to a type of enzyme that uses parent DNA as a template to catalyze the polymerization of substrate dNTPs molecules to form daughter DNA. The DNA polymerase in PCR technology is generally thermally stable. The loading buffer can increase the sample density and prevent the sample from floating out of the spotting hole. The indicator contained in it can also show the progress of electrophoresis. Some loading buffers also contain sodium dodecyl sulfate, which can promote the denaturation of DNA polymerase and prevent residual DNA polymerase from binding to the DNA double strand and affecting the electrophoretic migration rate.

[0052] The third aspect of the present invention provides a use of the above-mentioned primer combination or the above-mentioned kit in identifying the genetic sex of sturgeon.

[0053] In a specific embodiment, Figure 1 The electrophoresis test results of genetic sex identification of Chinese sturgeon provided by one embodiment of the present invention are as follows: Figure 1 As shown, when the test results show two bands of 473bp and 717bp, the genetic sex of the sturgeon is female; when the test results show a band of 717bp, the genetic sex of the sturgeon is male.

[0054] A fourth aspect of the present invention provides a method for identifying the genetic sex of sturgeon, comprising the following steps:

[0055] Extract DNA from sturgeon samples to be tested;

[0056] Using the DNA of the sturgeon sample to be tested as a template, using the above primer combination or the above kit to perform PCR amplification to obtain a PCR amplification product;

[0057] The PCR amplification product was tested by agarose gel electrophoresis. When two bands of 473 bp and 717 bp appeared in the test results, the genetic sex of the sturgeon was identified as female; when one band of 717 bp appeared in the test results, the genetic sex of the sturgeon was identified as male.

[0058] According to the primer combination provided in the first aspect of the present invention, the DNA of the sturgeon sample to be tested is subjected to PCR detection and agarose gel electrophoresis detection, and the genetic sex of the sturgeon sample to be tested can be identified according to the number and size of the bands that appear in the detection, that is, the sex of the sturgeon sample to be tested is identified as female or male. The identification method provided by the present invention only needs to extract a trace amount of DNA from the sturgeon to be tested, which will not cause significant impact and damage to the sturgeon, and will not be limited by the age of the sturgeon. The genetic sex identification can be performed throughout the life cycle of the sturgeon.

[0059] In detail, when the identification is performed according to the above steps, the sample of the sturgeon to be tested includes at least one of the fins and mucus of the sturgeon, wherein the mucus can specifically be mucus on the surface of the sturgeon. The genomic DNA of the sample of the sturgeon to be tested can be extracted by conventional technical means in the art, such as the CTAB method.

[0060] Further, in PCR amplification, the PCR reaction system includes 3-5 μL of DNA template, 0.5-1.5 μL of the first primer, 0.5-1.5 μL of the second primer, 0.25-0.75 μL of the third primer, 0.25-0.75 μL of the fourth primer, 10-15 μL of 2×Taq Master Mix and 4-6 μL of ddH 2 O.

[0061] In addition, when the PCR amplification program includes 93-95℃ pre-denaturation for 3min; 93-95℃ denaturation for 10s, 54-56℃ annealing for 10s, 72℃ extension for 19s, for a total of 35 cycles; and finally 72℃ extension for 10min, it helps to improve the specificity of the PCR reaction and thus improve the accuracy of the identification of the genetic sex of sturgeon.

[0062] Finally, the PCR amplification product is detected according to the conventional agarose gel electrophoresis detection method in the art, and the genetic sex of the sturgeon sample to be detected can be identified based on the number and size of the bands displayed by agarose gel electrophoresis.

[0063] The technical solution of the present application will be further explained below in conjunction with specific embodiments.

[0064] In the following examples, agarose gel electrophoresis uniformly uses DL5000 DNA molecular weight standard (DNA Marker, referred to as M), and its electrophoresis bands are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom. The remaining experimental methods without specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The reagents used, unless otherwise specified, are commercially available or publicly available.

[0065] The primer sequences involved in the following examples are shown in Table 1:

[0066] Table 1

[0067]

[0068]

[0069] Example 1: Identification of genetic sex of Chinese sturgeon

[0070] After the genetic sex of Chinese sturgeon was determined by spawning and squeezing, the fin rays of 12 female Chinese sturgeons and 12 male Chinese sturgeons were collected, among which the Chinese sturgeons were from the Chinese Sturgeon Research Institute of China Three Gorges Corporation. DNA of the above 24 Chinese sturgeons was extracted from the fin rays of Chinese sturgeon using a DNA extraction kit to prepare DNA samples. The integrity of the DNA samples was identified by 1.5% agarose gel electrophoresis, and the OD value of the DNA samples was determined by ultraviolet spectrophotometer and diluted to 50 ng / μL.

[0071] Primers XYJD15F and XYJD15R were designed based on the female sturgeon-specific DNA fragment, wherein the nucleotide sequence of the female sturgeon-specific DNA fragment is shown in SEQ ID NO: 5, and the fragment size is 473 bp. Primers XYXY38F and XYXY38R were designed based on the common DNA fragment of female and male sturgeons, wherein the nucleotide sequence of the common DNA fragment of female and male sturgeons is shown in SEQ ID NO: 6, and the fragment size is 717 bp.

[0072] The above DNA samples were used as templates and PCR technology was used for amplification to obtain PCR products. The PCR amplification system was 25 μL, including 4 μL DNA template, 1 μL XYJD15F, 1 μL XYJD15R, 0.5 μL XYXY38F, 0.5 μL XYXY38R, 12.5 μL 2× TaqMaster Mix (With Dye) and 5.5 μL ddH 2 O. The PCR amplification procedure included 3 min of pre-denaturation at 94°C, 35 cycles of denaturation at 94°C for 10 s, annealing at 56°C for 10 s, and extension at 72°C for 19 s, and finally extension at 72°C for 10 min and storage at 4°C. The PCR product was added to a 3% agarose gel, and the voltage was set to 120 V for 25 min for electrophoresis. The agarose gel after electrophoresis was analyzed using a gel imaging system.

[0073] like Figure 1As shown, all 12 female Chinese sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male Chinese sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Chinese sturgeons obtained through spawning and squeezing.

[0074] Example 2: Identification of genetic sex of Yangtze sturgeon

[0075] The steps of Example 1 can be referred to in Example 2, except that: after the genetic sex of Yangtze sturgeon is determined by spawning and squeezing, fin rays of 12 female Yangtze sturgeon and 12 male Yangtze sturgeon are taken, wherein the Yangtze sturgeon comes from the Chinese Acipenser Research Institute of China Three Gorges Corporation.

[0076] like Figure 2 As shown, all 12 female Yangtze sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male Yangtze sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the genetic sex identification results of Yangtze sturgeons obtained through spawning and sperm squeezing.

[0077] Example 3: Identification of genetic sex of sterlet

[0078] The steps of Example 1 can be referred to in Example 3, except that: after the genetic sex of the sterlet sturgeons is determined by dissection, fin rays of 12 female sterlet sturgeons and 12 male sterlet sturgeons are obtained, wherein the sterlet sturgeons are from a sturgeon breeding farm in Yichang, Hubei, China.

[0079] like Figure 3 As shown, all 12 female sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of sturgeons obtained through dissection.

[0080] Example 4: Identification of genetic sex of Acipenser schrenckii

[0081] The steps of Example 1 can be referred to in Example 4, except that: after the genetic sex of Acipenser schrenckii is determined by dissection, fin rays of 12 female Acipenser schrenckii and 12 male Acipenser schrenckii are obtained, wherein the Acipenser schrenckii is from a sturgeon breeding farm in Yichang, Hubei, China.

[0082] like Figure 4 As shown, all 12 female Acipenser schrenckii had two bands, 473 bp and 717 bp in size, respectively; all 12 male Acipenser schrenckii had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Acipenser schrenckii obtained through dissection.

[0083] Example 5: Identification of genetic sex of large sturgeon

[0084] The steps of Example 1 can be referred to in Example 5, except that: the large mixed sturgeons used in this experiment are the first generation hybrids of Acipenser schrenckii, the male parent, and Acipenser dabryanus, the female parent, and the genetic sex of the large mixed sturgeons is determined by dissection, and the fin rays of 12 female large mixed sturgeons and 12 male large mixed sturgeons are obtained, wherein the large mixed sturgeons are from the sturgeon breeding farm in Yichang, Hubei, China.

[0085] like Figure 5 As shown, all 12 female sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of sturgeons obtained through dissection.

[0086] Example 6: Identification of genetic sex of Siberian hybrid sturgeon

[0087] The steps of Example 1 can be referred to in Example 6, except that: the Siberian hybrid sturgeon used in this experiment is the first generation hybrid of a female Siberian sturgeon and a male Acipenser schrenckii. After the genetic sex of the Siberian hybrid sturgeon is determined by dissection, fin rays of 12 female Siberian hybrid sturgeons and 12 male Siberian hybrid sturgeons are obtained, wherein the Siberian hybrid sturgeon comes from a sturgeon breeding farm in Yichang, Hubei, China.

[0088] like Figure 6 As shown, all 12 female Siberian hybrid sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male Siberian hybrid sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Siberian hybrid sturgeons obtained by dissection.

[0089] Example 7: Identification of genetic sex of Siberian sturgeon

[0090] The steps of Example 1 can be referred to in Example 7, except that: after the genetic sex of the Siberian sturgeon is determined by dissection, fin rays of 12 female Siberian sturgeons and 12 male Siberian sturgeons are obtained, wherein the Siberian sturgeons are from a sturgeon breeding farm in Yichang, Hubei, China.

[0091] like Figure 7 As shown, all 12 female Siberian sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male Siberian sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Siberian sturgeons obtained by dissection.

[0092] Example 8: Identification of genetic sex of Pseudoalterus dabryanus

[0093] The steps of Example 1 can be referred to in Example 8, except that: after the genetic sex of the sturgeon is determined by dissection, fin rays of 12 female sturgeons and 12 male sturgeons are taken, wherein the sturgeons are from the sturgeon farm in Beijing, China.

[0094] like Figure 8 As shown, all 12 female Dabryanus had two bands, 473 bp and 717 bp in size, respectively; all 12 male Dabryanus had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Dabryanus obtained by dissection.

[0095] Example 9: Identification of genetic sex of Russian sturgeon

[0096] The steps of Example 1 can be referred to in Example 9, except that: after the genetic sex of the Russian sturgeon is determined by dissection, fin rays of 12 female Russian sturgeons and 12 male Russian sturgeons are obtained, wherein the Russian sturgeon comes from a sturgeon breeding farm in Beijing, China.

[0097] like Fig. 9 As shown, all 12 female Russian sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male Russian sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Russian sturgeons obtained by dissection.

[0098] Example 10: Identification of genetic sex of Sturgeon radiata

[0099] The steps of Example 1 can be referred to in Example 1, except that: after the genetic sex of the glistening sturgeon is determined by dissection, fin rays of 12 female glistening sturgeons and 12 male glistening sturgeons are obtained, wherein the glistening sturgeons are from a sturgeon breeding farm in Beijing, China.

[0100] like Fig.10 As shown, all 12 female sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of sturgeons obtained by dissection.

[0101] Example 11: Identification of genetic sex of European snapper

[0102] The steps of Example 1 can refer to those of Example 1, except that: after the genetic sex of the European sturgeon is determined by endoscopic technology, fin rays of 12 female European sturgeons and 12 male European sturgeons are taken, wherein the European sturgeons come from a sturgeon farm in Zhejiang, China.

[0103] like Fig.11As shown, all 12 female European guppies had two bands, 473 bp and 717 bp in size, respectively; all 12 male European guppies had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of European guppies obtained by endoscopic technology.

[0104] Example 12: Identification of genetic sex of lake sturgeon

[0105] The steps of Example 1 can be referred to in Example 1, except that: after the genetic sex of the lake sturgeon is determined by dissection, fin rays of 12 female lake sturgeons and 12 male lake sturgeons are taken, wherein the lake sturgeons come from a sturgeon farm in Xinjiang, China.

[0106] like Fig.12 As shown, all 12 female lake sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male lake sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of lake sturgeons obtained by dissection.

[0107] Example 13: Identification of genetic sex of naked sturgeon

[0108] The steps of Example 1 can be referred to in this Example 1, except that: after the genetic sex of naked sturgeons is determined by dissection, fin rays of 12 female naked sturgeons and 12 male naked sturgeons are taken, wherein the naked sturgeons are from a sturgeon farm in Xinjiang, China.

[0109] like Fig.13 As shown, all 12 female naked belly sturgeons had two bands, 473bp and 717bp in size, respectively; all 12 male naked belly sturgeons had one band, 717bp in size. The results of this experiment are consistent with the results of genetic sex identification of naked belly sturgeons obtained by dissection.

[0110] Example 14: Identification of genetic sex of Persian sturgeon

[0111] The steps of Example 1 can be referred to in Example 1, except that: after the genetic sex of the Persian sturgeon is determined by dissection, fin rays of 12 female Persian sturgeons and 12 male Persian sturgeons are obtained, wherein the Persian sturgeons are from a sturgeon breeding farm in Xinjiang, China.

[0112] like Fig.14 As shown, all 12 female Persian sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male Persian sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of Persian sturgeons obtained by dissection.

[0113] Example 15: Identification of genetic sex of Acipenser chinensis

[0114] The steps of Example 1 can be referred to in Example 1, except that: after the genetic sex of the sturgeon is determined by dissection, fin rays of 12 female sturgeons and 12 male sturgeons are taken, wherein the sturgeons are from a sturgeon breeding farm in Xinjiang, China.

[0115] like Fig.15 As shown, all 12 female sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of sturgeons obtained by dissection.

[0116] Example 16: Identification of genetic sex of Sturgeon

[0117] The steps of Example 1 can be referred to in Example 1, except that: after the genetic sex of the Chinese sturgeon is determined by dissection, fin rays of 12 female Chinese sturgeons and 12 male Chinese sturgeons are taken, wherein the Chinese sturgeons are from a sturgeon farm in Xinjiang, China.

[0118] like Fig.16 As shown, all 12 female sturgeons had two bands, 473 bp and 717 bp in size, respectively; all 12 male sturgeons had one band, 717 bp in size. The results of this experiment are consistent with the results of genetic sex identification of sturgeons obtained by dissection.

[0119] In summary, the present invention is applicable to a variety of sturgeons and has good universality. The genetic sex of the sturgeon identified by the present invention is consistent with the genetic sex of the sturgeon obtained by the dissection method, the spawning and squeezing method or the endoscopic technique, indicating that the present invention can correctly identify the genetic sex of the sturgeon; at the same time, compared with the dissection method, the present invention only needs to extract a trace amount of DNA from the sturgeon to be detected, which will not cause significant impact and damage to the sturgeon; compared with the spawning and squeezing method and the endoscopic technique, the present invention is not limited by the age of the sturgeon, and the genetic sex can be identified throughout the life cycle of the sturgeon. Since the primer combination of the present invention provides two pairs of primers, the present invention can also avoid experimental errors caused by unsuccessful DNA extraction, wrong or missed addition of drugs, and ensure the accuracy of sturgeon sex identification; and since the two DNA fragments amplified by the two pairs of primers are located on the same chromosome, the distance is not greater than 20k bp, so the experimental error caused by severe DNA degradation can be effectively reduced. The present invention can provide assistance for the sex control breeding of sturgeons and the protection and utilization of germplasm resources, and provide technical support for biodiversity research and the Yangtze River protection work.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer combination for identifying the genetic sex of sturgeon, characterized in that: It includes a first primer pair and a second primer pair; the first primer pair includes a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO:1, and the nucleotide sequence of the second primer is shown in SEQ ID NO:2; the second primer pair includes a third primer and a fourth primer, the nucleotide sequence of the third primer is shown in SEQ ID NO:3, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO:

4.

2. The primer combination according to claim 1, characterized in that The first primer pair is used to amplify a female sturgeon-specific DNA fragment, the nucleotide sequence of the female sturgeon-specific DNA fragment is shown in SEQ ID NO:5, and the fragment size is 473 bp; the second primer pair is used to amplify a DNA fragment shared by female and male sturgeons, the nucleotide sequence of the DNA fragment shared by female and male sturgeons is shown in SEQ ID NO:6, and the fragment size is 717 bp.

3. The primer combination according to claim 1 or 2, characterized in that: The sturgeon is at least one of the Chinese sturgeon, the Yangtze sturgeon, the small-bodied sturgeon, the Schrenckii sturgeon, the large-bodied sturgeon, the Siberian hybrid sturgeon, the Siberian sturgeon, the Dabryan sturgeon, the Russian sturgeon, the flash sturgeon, the European sturgeon, the lake sturgeon, the naked belly sturgeon, the Persian sturgeon, the high-headed sturgeon, and the snout sturgeon.

4. A kit for genetic sex identification of sturgeon, characterized in that: The invention comprises the primer combination according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, and loading buffer.

6. Use of the primer combination according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 in identifying the genetic sex of sturgeon; in, The identification of the genetic sex of sturgeon comprises the following steps: Extract DNA from sturgeon samples to be tested; Using the DNA of the sturgeon sample to be tested as a template, PCR amplification is performed using the primer combination described in any one of claims 1 to 3 or the kit described in any one of claims 4 to 5 to obtain a PCR amplification product; The PCR amplification product is subjected to agarose gel electrophoresis detection. When the detection result shows two bands of 473 bp and 717 bp, the genetic sex of the sturgeon is identified as female; when the detection result shows a band of 717 bp, the genetic sex of the sturgeon is identified as male; The sturgeon is at least one of the Chinese sturgeon, the Yangtze sturgeon, the small-bodied sturgeon, the Schrenckii sturgeon, the large-bodied sturgeon, the Siberian hybrid sturgeon, the Siberian sturgeon, the Dabryan sturgeon, the Russian sturgeon, the flash sturgeon, the European sturgeon, the lake sturgeon, the naked belly sturgeon, the Persian sturgeon, the high-headed sturgeon, and the snout sturgeon.

7. A method for identifying the genetic sex of sturgeon, characterized in that: The steps include: Extract DNA from sturgeon samples to be tested; Using the DNA of the sturgeon sample to be tested as a template, PCR amplification is performed using the primer combination described in any one of claims 1 to 3 or the kit described in any one of claims 4 to 5 to obtain a PCR amplification product; The PCR amplification product is subjected to agarose gel electrophoresis detection. When the detection result shows two bands of 473 bp and 717 bp, the genetic sex of the sturgeon is identified as female; when the detection result shows a band of 717 bp, the genetic sex of the sturgeon is identified as male; The sturgeon is at least one of the Chinese sturgeon, the Yangtze sturgeon, the small-bodied sturgeon, the Schrenckii sturgeon, the large-bodied sturgeon, the Siberian hybrid sturgeon, the Siberian sturgeon, the Dabryan sturgeon, the Russian sturgeon, the flash sturgeon, the European sturgeon, the lake sturgeon, the naked belly sturgeon, the Persian sturgeon, the high-headed sturgeon, and the snout sturgeon.

8. The method according to claim 7, characterized in that The sturgeon sample to be tested includes at least one of sturgeon fins and mucus.

9. The method according to claim 7 or 8, characterized in that: In the PCR amplification, the PCR reaction system includes 3-5 µL of DNA template, 0.5-1.5 µL of the first primer, 0.5-1.5 µL of the second primer, 0.25-0.75 µL of the third primer, 0.25-0.75 µL of the fourth primer, 10-15 µL of 2×Taq Master Mix and 4-6 µL of ddH2O.

10. The method according to claim 9, characterized in that In the PCR amplification, the PCR amplification program includes 93-95°C pre-denaturation for 3 min; 93-95°C denaturation for 10 s, 54-56°C annealing for 10 s, 72°C extension for 19 s, for a total of 35 cycles; and finally 72°C extension for 10 min.

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