Genetic sex-specific multiple molecular marker of guppy, kit and application thereof

Abstract

The application discloses a court goldfish genetic sex-specific multiplex molecular marker, a kit and application thereof, wherein the multiplex molecular marker comprises the following A1) and A2), A1) is a DNA fragment with a length of 486 bp obtained by amplification with male chromosome-specific primers composed of single-stranded DNA molecules shown as SEQ ID NO. 1 and SEQ ID NO. 2 and taking genomic DNA of the court goldfish as a template; and A2) is a DNA fragment with a length of 204 bp obtained by amplification with universal primers composed of single-stranded DNA molecules shown as SEQ ID NO. 3 and SEQ ID NO. 4 and taking genomic DNA of the court goldfish as a template. The genetic sex-specific molecular marker is screened, a court goldfish genetic sex multiplex PCR identification method is established, and the method is applied to genetic sex identification in a court goldfish breeding process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular markers, and particularly relates to a genetic gender-specific multiplex molecular marker of palace goldfish, a kit and application thereof. BACKGROUND

[0002] The palace goldfish is a hard fish of the Cypriniformes Cyprinidae Carassius. China is the birthplace of the goldfish, and goldfish breeding has a history of more than 1700 years. The goldfish has experienced the development process of wildness, release, semi-domestication, domestication, artificial selection and crossbreeding. In particular, in the past ten years, with the growth of international market demand, the goldfish breeding and related industries in China have developed rapidly and become the backbone of the ornamental fishery industry. The goldfish has rich varieties, and the existing goldfish has four major strains of "grass species, literary species, dragon species and egg species", and more than 300 varieties. Raising the palace goldfish at home not only beautifies the home environment, but also cultivates the mind, and has the beautiful implication of "gold and jade filling the hall". Therefore, the palace goldfish has important ornamental value and economic value.

[0003] Correct gender identification is crucial for the breeding management and breeding plan of the palace goldfish. Only when the gender of the palace goldfish is known, the proportion of the population can be better grasped, and the problem of too many males or too many females can be avoided. At present, the gender identification of the palace goldfish mainly relies on external characteristics and gender judgment, and is observed and judged by body characteristics and fin morphology, which often leads to misjudgment. Especially in the early years, the morphological difference is very small and difficult to distinguish. After sexual maturity, the observation through gender characteristics has high accuracy, but the cycle is long. Therefore, it is particularly important to carry out screening of genetic gender-specific markers of the palace goldfish to quickly realize gender identification for the industrialized breeding management of the palace goldfish. SUMMARY

[0004] The purpose of the present application is to provide a multiplex molecular marker for identifying or assisting in identifying the genetic gender specificity of the palace goldfish, a kit and application thereof.

[0005] The present application provides a multiplex molecular marker for identifying or assisting in identifying the genetic gender of the palace goldfish, which comprises the following A1) and A2),

[0006] A1) using the genomic DNA of the palace goldfish as a template, using the male chromosome-specific primer composed of the single-stranded DNA molecule of SEQ ID NO. 1 and the single-stranded DNA molecule of SEQ ID NO. 2 to amplify a DNA fragment with a length of 486 bp;

[0007] A2) using the single-stranded DNA molecule represented by SEQ ID NO. 3 and the single-stranded DNA molecule represented by SEQ ID NO. 4 as the universal primers, genomic DNA of the Guppy as the template, and performing amplification to obtain a DNA fragment with a length of 204 bp.

[0008] The application provides a primer composition for identifying or assisting in identifying the genetic sex of the Guppy, which comprises the following B1) and B2),

[0009] B1) a male chromosome-specific primer pair composed of the single-stranded DNA molecule represented by SEQ ID NO. 1 and the single-stranded DNA molecule represented by SEQ ID NO. 2;

[0010] B2) a universal primer pair composed of the single-stranded DNA molecule represented by SEQ ID NO. 3 and the single-stranded DNA molecule represented by SEQ ID NO. 4.

[0011] The application also provides the use of the molecular marker or the primer pair composition in the preparation of a product for identifying or assisting in identifying the genetic sex of the Guppy.

[0012] The application further provides a kit comprising the primer composition.

[0013] Further, the kit further comprises a PCR reaction system, wherein the PCR reaction system comprises Taq DNA polymerase, dNTPs, magnesium chloride, potassium chloride, betaine and glycerol, and the concentration of the Taq DNA polymerase is 0.3 U / μL, the concentration of the dNTPs is 200 μM, the concentration of the magnesium chloride is 3.5 mM, the concentration of the potassium chloride is 50 mM, the concentration of the betaine is 0.3 M, and the concentration of the glycerol is 5%.

[0014] The application also provides the use of the molecular marker, the primer pair composition or the kit in identifying or assisting in identifying the genetic sex of the Guppy.

[0015] The application further provides a method for identifying or assisting in identifying the genetic sex of the Guppy, which comprises the primer composition or the kit.

[0016] Further, the method comprises the following steps:

[0017] using the male chromosome-specific primer pair composed of the single-stranded DNA molecule represented by SEQ ID NO. 1 and the single-stranded DNA molecule represented by SEQ ID NO. 2, and the universal primer pair composed of the single-stranded DNA molecule represented by SEQ ID NO. 3 and the single-stranded DNA molecule represented by SEQ ID NO. 4 to amplify the genomic DNA of the sample.

[0018] When the genomic DNA of the sample to be tested is amplified to obtain the 204bp-long common chromosome-specific molecular marker of the palace goldfish, it is determined that the sample to be tested is a genetically female palace goldfish, and the sex chromosome genotype is XX;

[0019] When the genomic DNA of the sample to be tested is amplified to obtain both the 204bp-long common chromosome-specific molecular marker of the palace goldfish and the 486bp-long genetically male sex chromosome-specific molecular marker of the palace goldfish, it is determined that the sample to be tested is a genetically male palace goldfish, and the sex chromosome genotype is XY;

[0020] When the genomic DNA of the sample to be tested cannot be amplified to obtain the 204bp-long common chromosome-specific molecular marker of the palace goldfish, it is determined that the sample to be tested is not a palace goldfish.

[0021] Further, the method is:

[0022] The sample to be tested is subjected to multiplex PCR amplification by using the kit described above;

[0023] When the genomic DNA of the sample to be tested is amplified to obtain the 204bp-long common chromosome-specific molecular marker of the palace goldfish, it is determined that the sample to be tested is a genetically female palace goldfish, and the sex chromosome genotype is XX;

[0024] When the genomic DNA of the sample to be tested is amplified to obtain both the 204bp-long common chromosome-specific molecular marker of the palace goldfish and the 486bp-long genetically male sex chromosome-specific molecular marker of the palace goldfish, it is determined that the sample to be tested is a genetically male palace goldfish, and the sex chromosome genotype is XY;

[0025] When the genomic DNA of the sample to be tested cannot be amplified to obtain the 204bp-long common chromosome-specific molecular marker of the palace goldfish, it is determined that the sample to be tested is not a palace goldfish.

[0026] The application of the molecular marker described above, the primer pair composition described above or the kit described above in palace goldfish breeding should also be within the protection scope of the present application.

[0027] The present application has the following beneficial effects: 1) The present application firstly carries out genetic sex research on 19 representative varieties of domestic palace goldfish, screens genetic sex-specific molecular markers, establishes a multiplex PCR identification method for the genetic sex of palace goldfish, and applies the method to genetic sex identification in the palace goldfish breeding process. 2) The multiplex PCR identification method established by the present application can independently prepare a PCR reaction system, and the whole process has the characteristics of accuracy, high efficiency, stability, convenience and low cost, and has important application value in palace goldfish sex control and palace goldfish large-scale breeding. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1. DNA agarose gel electrophoresis quality control map of representative sample of Example 1 Goldfish;

[0029] Figure 2 Figure 2. Genetic sex GWAS analysis results map of Example 1 Goldfish;

[0030] Figure 3 Figure 3. Genetic sex specific multiplex molecular marker results map of Example 1 Goldfish;

[0031] Figure 4 Figure 4. Genetic sex specific multiplex molecular marker kit results map of Example 2 Goldfish;

[0032] Figure 5 Figure 5. Genetic sex specific multiplex molecular marker application results map of Example 3 Goldfish. DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the present application, not to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0034] The experimental methods in the following examples are all routine methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0035] The quantitative experiments in the following examples are all set up in triplicate, and the results are averaged, unless otherwise specified.

[0036] The experimental methods in the following examples that are not specified for specific conditions are generally carried out according to routine conditions, for example, the conditions described in the Molecular Cloning Experiments Guide (Third Edition, J. Sambrook et al., Huang Peitang et al. Translated, Science Press, 2002) or according to the conditions recommended by the reagent manufacturers.

[0037] Example 1: Genetic sex specific multiplex molecular marker of Goldfish

[0038] 1. Genome sequencing of representative varieties of Goldfish

[0039] Select 19 representative varieties of palace goldfish, namely black butterfly, red dragon eye, five-flower dragon eye, tiger head, blue lion head, red lion head, black dragon eye, purple body red ball, purple tiger head, crane top red, black tiger, red and white high head, red and white butterfly tail, black large eye, pearl, blue water bubble, goose head red, king tiger head, egg type velvet ball (all test varieties are from Beijing Aquaculture Technology Promotion Station Tongzhou Base), 2-3 purebred samples are selected from each variety, a total of 48, including 1-2 male and female samples. DNA extraction was performed on the above samples, and the kit was purchased from Beijing Tongnong Detection Technology Co., Ltd., with the order number DE302-MB-100, and the experimental process is as follows:

[0040] ① Cut about 50 mg of goldfish tail in a 2.0 mL centrifuge tube, add 500 μL of tissue lysis solution and 20 μL (20 mg / mL) of proteinase K, shake well, and incubate at 65°C for 60 min.

[0041] ② Add 350 μL of isopropanol and 10 μL of magnetic beads to the centrifuge tube, shake well, and stand for 10 min; then place the centrifuge tube on the magnetic stand and magnetize for 1 min, remove the waste liquid.

[0042] ③ Add 500 μL of washing solution I, shake well to suspend the magnetic beads, and wash for 3 min; then place the centrifuge tube on the magnetic stand and magnetize for 1 min, remove the waste liquid.

[0043] ④ Add 500 μL of washing solution II, shake well to suspend the magnetic beads, and wash for 3 min; then place the centrifuge tube on the magnetic stand and magnetize for 1 min, remove the waste liquid; dry for 5 min.

[0044] ⑤ Add 100 μL of double distilled water, shake well to suspend the magnetic beads, and elute at 56°C for 10 min; then place the centrifuge tube on the magnetic stand and magnetize for 1 min, take the DNA solution and store for later use.

[0045] ⑥ The DNA solution was detected by Nanodrop for the concentration and purity of DNA, and 1.5% agarose gel electrophoresis was used to detect the integrity of DNA.

[0046] After quality inspection, the average concentration of the sample DNA was higher than 100 ng / μL, OD260 / OD280=1.80-1.90, OD2360 / OD280=1.70-1.90, and the electrophoretic band was clear without tailing, as shown in Figure 1 . The extracted DNA was entrusted to Beijing Tongnong Detection Technology Co., Ltd. for library construction and whole genome sequencing, with a sequencing depth of 5x and a sequencing amount of 8G / sample.

[0047] 2. Development of genetic sex-linked molecular markers by GWAS analysis

[0048] After obtaining the above sample sequencing data, raw data was filtered using Fastp software to obtain clean data, with a ratio of 98.71%. The clean data was then aligned to the reference genome GCF_003368295.1 of the *Goldfish simonii* using BWA software. Samtools and GATK software were used to obtain the whole genome INDEL and SNP mutation sites of the *Goldfish simonii*. GWAS analysis was used to statistically analyze the distribution of sex-linked SNPs and locate the genetic sex-linked region of *Goldfish simonii* at chromosome CHR22:20-30M (see...). Figure 2 Then, the BAM files after comparison are sorted, indexed, and opened for viewing using the visualization software IGV. Genetic sex-linked primers are designed where there are obvious structural variations in male and female individuals, and universal primers are designed where there are no obvious mutations in either male or female individuals. PCR amplification is performed on male and female fish respectively to screen for sex-specific molecular markers.

[0049] Twenty-two goldfish samples from the aforementioned 48 were selected for primer testing. One to two goldfish of each variety were selected, with koi, carp, and sturgeon (all tested varieties were from the Tongzhou Base of the Beijing Aquatic Technology Extension Station) samples serving as controls. PCR detection was performed using the aforementioned universal primers for goldfish and genetic sex-linked primers. The PCR products were then mixed together and detected by 1.5% agarose gel electrophoresis. The PCR system consisted of: 2 μL DNA (30 ng / μL), 0.4 μL primer mixture (10 mM), 5 μL 2×PCRMix, and 2.6 μL double-distilled water. The PCR reaction program was as follows: S1, 95℃ for 10 min, 1 cycle; S2, 95℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 35 cycles; S3, 72℃ for 5 min. The PCR reagent used was a kit from Tiangen Biotech (Beijing) Co., Ltd., catalog number KT109.

[0050] The results showed that the primer sequences shown in SEQ ID NO.3 and SEQ ID NO.4 could amplify a specific band of 204 bp in all goldfish samples, but could not amplify in koi, carp, or sturgeon. Figure 3 The primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2 exhibit significant sex specificity, amplifying a specific 486 bp band only in XY individuals, but not in XX individuals, and not in koi, carp, or sturgeon. Figure 3 The test results were consistent with the sex results of the goldfish samples, indicating that the primers of this invention have a promising application prospect in the genetic sex identification of goldfish.

[0051]

[0052]

[0053] Example 2: Obtainment of the genetic sex-specific multiplex molecular marker kit of G. regalis

[0054] In order to improve the detection efficiency and reduce the detection cost, the PCR reaction reagent and the multiplex PCR reaction system are self-developed due to the high cost and low detection efficiency of using single PCR reaction and commercial PCR reagent. The 2x PCR Mix is prepared as follows: the concentration of Taq DNA polymerase is 0.6 U / μL, the concentration of dNTPs is 400 μM, the concentration of magnesium chloride is 8 mM, the concentration of potassium chloride is 100 mM, the concentration of betaine is 0.6 M, and the concentration of glycerol is 10%. In order to adjust the optimal multiplex PCR effect, the addition amount of the genetic sex-specific molecular marker mixture and the universal chromosome-specific molecular marker mixture of the PCR system is adjusted and tested, and the optimal PCR reaction system is finally determined as follows: 2 μL of DNA (30 ng / μL), 0.3 μL of genetic sex-specific molecular marker mixture (10 mM), 0.25 μL of universal chromosome-specific molecular marker mixture (10 mM), 5 μL of 2x PCR Mix, and 2.45 μL of double-distilled water. The Taq DNA polymerase is purchased from Tian Gen Bio-chemical Technology (Beijing) Co., Ltd., and the item number is ET106. Other general chemical reagents can meet the experimental conditions.

[0055] The 25 samples in Table 1 are detected according to the self-developed PCR reaction system preparation and multiplex PCR reaction, and the results are shown in Table 2. Figure 4 The gender detection is consistent with the results of the commercial reagent, and the specificity and uniformity of the two pairs of primers are better, which shows that the multiplex molecular marker kit of the application has strong universality in the genetic sex identification of different varieties of G. regalis and has good application prospect.

[0056] Example 3: Application of the genetic sex-specific multiplex molecular marker method of G. regalis

[0057] The genetic sex-specific multiplex molecular marker kit of G. regalis developed based on Examples 1 and 2 is used for genetic sex identification of the to-be-tested G. regalis. A total of 48 G. regalis are collected from Beijing Xianglong Technology Co., Ltd., Kunshan Dianshanhu Town Hengfeng Ornamental Fish Farm, and Jingxiguanzhong Pan's Ornamental Fish Farm in Minhou County as to-be-tested samples, and the gender identification is performed by using the method of the application, and the results are shown in Table 3. Figure 5and Table 2. A total of 46 samples were detected to be able to amplify 204bp band, and 2 samples were unable to amplify 204bp band; among the 46 samples, 9 samples were detected to be able to amplify 486bp band, and 37 samples were unable to amplify 486bp band, as shown in the following table. According to the comparison of the amplified bands in the table and the actual gender, it can be seen that the amplification results are completely consistent with the actual gender. The identification conclusion of the method of the present application is completely consistent with the actual situation.

[0058]

[0059]

[0060]

[0061] The present application has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. Use of a primer composition for identifying or aiding in the identification of the genetic sex of a guppy, characterized in that, The primer composition comprises B1) and B2), wherein B1) a male chromosome-specific primer pair consisting of a single-stranded DNA molecule shown as SEQ ID NO. 1 and a single-stranded DNA molecule shown as SEQ ID NO. 2; B2) a universal primer pair consisting of a single-stranded DNA molecule shown as SEQ ID NO. 3 and a single-stranded DNA molecule shown as SEQ ID NO. 4; A DNA fragment with a length of 486 bp is amplified from genomic DNA of the palace goldfish using the male chromosome-specific primer consisting of a single-stranded DNA molecule shown as SEQ ID NO. 1 and a single-stranded DNA molecule shown as SEQ ID NO. 2; A DNA fragment with a length of 204 bp is amplified from genomic DNA of the palace goldfish using the universal primer consisting of a single-stranded DNA molecule shown as SEQ ID NO. 3 and a single-stranded DNA molecule shown as SEQ ID NO. 4; The palace goldfish is a black butterfly, a red dragon eye, a five-color dragon eye, a tiger head, a blue lion head, a red lion head, a black dragon eye, a purple body red ball, a purple tiger head, a crane top red, a black tiger, a red and white high head, a red and white butterfly tail, a black large eye, a pearl, a blue water bubble, a goose head red, a king tiger head or an egg species velvet ball.

2. Use of a kit for identifying or aiding in the identification of the genetic sex of a guppy, characterized in that, The kit comprises the primer composition of claim 1; The palace goldfish is a black butterfly, a red dragon eye, a five-color dragon eye, a tiger head, a blue lion head, a red lion head, a black dragon eye, a purple body red ball, a purple tiger head, a crane top red, a black tiger, a red and white high head, a red and white butterfly tail, a black large eye, a pearl, a blue water bubble, a goose head red, a king tiger head or an egg species velvet ball.

3. Use according to claim 2, characterized in that, The kit further comprises a PCR reaction system, wherein the PCR reaction system comprises Taq DNA polymerase, dNTPs, magnesium chloride, potassium chloride, betaine and glycerol, wherein the concentration of the Taq DNA polymerase is 0.3 U / μL, the concentration of the dNTPs is 200 μM, the concentration of the magnesium chloride is 3.5 mM, the concentration of the potassium chloride is 50 mM, the concentration of the betaine is 0.3 M and the concentration of the glycerol is 5%.

4. A method for identifying or aiding in the identification of the genetic sex of a guppy, characterized in that, The method is: The genomic DNA of the sample to be tested is used as a template, and a male chromosome-specific primer pair consisting of a single-stranded DNA molecule shown as SEQ ID NO. 1 and a single-stranded DNA molecule shown as SEQ ID NO. 2, and a universal primer pair consisting of a single-stranded DNA molecule shown as SEQ ID NO. 3 and a single-stranded DNA molecule shown as SEQ ID NO. 4 are used for amplification; When only a palace goldfish universal chromosome-specific molecular marker with a length of 204 bp is amplified from the genomic DNA of the sample to be tested, it is determined that the sample to be tested is a palace goldfish female in genetics, and the sex chromosome genotype is XX. When the 204 bp common chromosome specific molecular marker of G. auratus and the 486 bp genetic male specific molecular marker of G. auratus are amplified from the genomic DNA of the sample to be tested at the same time, the sample to be tested is judged as a genetic male G. auratus with XY sex chromosome genotype; The G. auratus is G. auratus albino, G. auratus red dragon, G. auratus five-color dragon, G. auratus tiger head, G. auratus blue lion head, G. auratus red lion head, G. auratus black dragon, G. auratus purple body red ball, G. auratus purple tiger head, G. auratus crane top red, G. auratus black tiger, G. auratus red and white high head, G. auratus red and white butterfly tail, G. auratus black big eye, G. auratus pearl, G. auratus blue water bubble, G. auratus goose head red, G. auratus king tiger head or G. auratus egg type velvet ball.

5. The primer pair composition of claim 1 or the kit of claim 2 is used in the breeding of G. auratus; the G. auratus is G. auratus albino, G. auratus red dragon, G. auratus five-color dragon, G. auratus tiger head, G. auratus blue lion head, G. auratus red lion head, G. auratus black dragon, G. auratus purple body red ball, G. auratus purple tiger head, G. auratus crane top red, G. auratus black tiger, G. auratus red and white high head, G. auratus red and white butterfly tail, G. auratus black big eye, G. auratus pearl, G. auratus blue water bubble, G. auratus goose head red, G. auratus king tiger head or G. auratus egg type velvet ball; and the breeding is the genetic sex selection of G. auratus.

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