Genetic sex specific multi-molecular marker for court goldfish, kit and application of genetic sex specific multi-molecular marker

Abstract

The invention discloses a court goldfish genetic sex specific multiple molecular marker, a kit and application of the court goldfish genetic sex specific multiple molecular marker, and the multiple molecular marker comprises the following A1) and A2): A1) a DNA fragment with the length of 486bp is obtained by amplifying a male chromosome specific primer consisting of single-stranded DNA molecules as shown in SEQ ID NO.1 and SEQ ID NO.2 by taking genomic DNA of court goldfish as a template, and A2) a DNA fragment with the length of 486bp is obtained by amplifying a male chromosome specific primer consisting of single-stranded DNA molecules as shown in SEQ ID NO.1 and SEQ ID NO.2; a2) a DNA fragment with the length of 204bp is obtained by amplifying a universal primer consisting of single-stranded DNA molecules as shown in SEQ ID NO.3 and SEQ ID NO.4 by taking the genome DNA of the palace goldfish as a template. The genetic sex specific molecular marker is screened, the court goldfish genetic sex multiplex PCR identification method is established, and the method is applied to genetic sex identification in the court goldfish breeding process.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a palace goldfish genetic sex-specific multiple molecular marker, a kit and applications thereof. Background Art

[0002] The palace goldfish is a bony fish of the genus Carassius in the family Cyprinidae of the order Cypriniformes. my country is the hometown of goldfish. Goldfish breeding has a history of more than 1,700 years. It has experienced the development process of wild-release-semi-domestication-domestication-artificial selection and hybridization. Especially in the past decade, with the growth of international market demand, my country's goldfish breeding and related industries have developed rapidly and become the pillar industry of ornamental fisheries. There are rich varieties of goldfish breeding. The existing goldfish have four major strains of "grass species, literary species, dragon species, and egg species", with more than 300 varieties. Raising palace goldfish at home not only beautifies the home environment, but also cultivates sentiment, and has the beautiful meaning of "a house full of gold and jade". Therefore, the palace goldfish has important ornamental value and economic value.

[0003] Correct identification of sex is crucial for the breeding management and reproduction plan of palace goldfish. Only by knowing the sex of palace goldfish can we better grasp the proportion of the population and avoid the problem of too many males or too many females. At present, the sex identification of palace goldfish mainly relies on appearance characteristics and gender judgment. It is often wrong to make judgments by observing and judging through body characteristics and fin morphology. Especially in infancy, the morphological differences are very small and difficult to distinguish. After sexual maturity, observation through sex characteristics is highly accurate, but the cycle is long. Therefore, it is particularly important to carry out screening of palace goldfish genetic sex-specific markers and quickly realize sex identification for the industrial breeding management of palace goldfish. Summary of the invention

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

[0005] The present invention provides multiple molecular markers for identifying or assisting in identifying the genetic sex of palace goldfish, wherein the multiple molecular markers include the following A1) and A2),

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

[0007] A2) Using the genomic DNA of the royal goldfish as a template, a single-stranded DNA molecule shown in SEQ ID NO.3 and a universal primer consisting of a single-stranded DNA molecule shown in SEQ ID NO.4 were used to amplify a DNA fragment with a length of 204 bp.

[0008] The present invention provides a primer composition for identifying or assisting in identifying the genetic sex of palace goldfish, wherein the primer composition comprises the following B1) and B2),

[0009] B1) a male chromosome-specific primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2;

[0010] B2) A universal primer pair consisting of the single-stranded DNA molecule shown in SEQ ID NO.3 and the single-stranded DNA molecule shown in SEQ ID NO.4.

[0011] The use of the above-mentioned molecular markers or the above-mentioned primer pair combination in the preparation of products for identifying or assisting in identifying the genetic sex of palace goldfish should also be within the protection scope of the present invention.

[0012] The present invention provides a kit, which contains the primer composition.

[0013] Furthermore, the kit also includes a PCR reaction system, which includes TaqDNA polymerase, dNTPs, magnesium chloride, potassium chloride, betaine and glycerol, wherein the concentration of the TaqDNA polymerase is 0.3U / μL, the concentration of dNTPs is 200μM, the concentration of magnesium chloride is 3.5mM, the concentration of potassium chloride is 50mM, the concentration of betaine is 0.3M, and the concentration of glycerol is 5%.

[0014] The use of the above-mentioned molecular marker, the above-mentioned primer pair combination or the above-mentioned kit in identifying or assisting in identifying the genetic sex of palace goldfish should also be within the protection scope of the present invention.

[0015] The present invention provides a method for identifying or assisting in identifying the genetic sex of palace goldfish, using the above primer combination or the above kit.

[0016] Furthermore, the method is:

[0017] Using the genomic DNA of the sample to be tested as a template, amplification is performed using a male chromosome-specific primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2, and a universal primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.3 and a single-stranded DNA molecule shown in SEQ ID NO.4;

[0018] When only a 204 bp-long royal goldfish universal chromosome-specific molecular marker is amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic female royal goldfish, and the sex chromosome genotype is XX;

[0019] When a 204 bp palace goldfish universal chromosome-specific molecular marker and a 486 bp palace goldfish genetic male chromosome-specific molecular marker are simultaneously amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic male palace goldfish, and the sex chromosome genotype is XY;

[0020] When the universal chromosome-specific molecular marker of the royal goldfish with a length of 204 bp cannot be amplified from the genomic DNA of the sample to be tested, the sample to be tested is not the royal goldfish.

[0021] Furthermore, the method is:

[0022] Perform multiplex PCR amplification on the sample to be tested using the above kit;

[0023] When only a 204 bp-long royal goldfish universal chromosome-specific molecular marker is amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic female royal goldfish, and the sex chromosome genotype is XX;

[0024] When a 204 bp palace goldfish universal chromosome-specific molecular marker and a 486 bp palace goldfish genetic male chromosome-specific molecular marker are simultaneously amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic male palace goldfish, and the sex chromosome genotype is XY;

[0025] When the universal chromosome-specific molecular marker of the royal goldfish with a length of 204 bp cannot be amplified from the genomic DNA of the sample to be tested, the sample to be tested is not the royal goldfish.

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

[0027] The beneficial effects of the present invention are as follows: 1) The present invention is the first to conduct genetic sex research on 19 representative varieties of domestic palace goldfish, screen genetic sex-specific molecular markers, establish a multiple PCR identification method for the genetic sex of palace goldfish, and apply this method to the genetic sex identification in the breeding process of palace goldfish. 2) The multiple PCR identification method established by the present invention can independently prepare a PCR reaction system, and the whole process is accurate, efficient, stable, convenient, and low-cost, and has important application value in the sex control of palace goldfish and the large-scale breeding of palace goldfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Example 1 Agarose gel electrophoresis quality inspection image of representative samples of palace goldfish;

[0029] Figure 2 Example 1: GWAS analysis results of genetic sex of palace goldfish;

[0030] Figure 3 Example 1: Results of genetic sex-specific multiple molecular markers of palace goldfish;

[0031] Figure 4 . Result diagram of Example 2 Palace goldfish genetic sex-specific multiple molecular marker kit;

[0032] Figure 5 Example 3: Results of the application of multiple molecular markers specific to genetic sex of palace goldfish. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0036] The experimental methods for which specific conditions are not specified in the following examples are generally performed according to conventional conditions, such as the conditions described in Molecular Cloning Laboratory Manual (3rd edition, written by J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002), or according to the conditions recommended by the reagent manufacturers.

[0037] Example 1: Obtaining Genetic Sex-Specific Multiple Molecular Markers for Palace Goldfish

[0038] 1. Genome sequencing of representative varieties of imperial goldfish

[0039] 19 representative varieties of palace goldfish were selected, namely, black butterfly, red dragon eye, five-color 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 big eyes, pearl, blue bubble, goose head red, king tiger head, egg type fluff ball (the above test varieties are all from the Tongzhou base of Beijing Fisheries Technology Extension Station), 2-3 purebred samples were selected for each variety, a total of 48, including 1-2 male and female samples. DNA was extracted from the above samples, and the test kit used in the experiment was purchased from Beijing Tongnong Testing Technology Co., Ltd., item number DE302-MB-100. The experimental process is as follows:

[0040] ①Cut about 50 mg of the tail of the royal goldfish and put it in a 2.0 mL centrifuge tube. Add 500 μL of tissue lysis buffer and 20 μL (20 mg / mL) of proteinase K. Vortex to mix 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 to mix, and let stand for 10 minutes; then place the centrifuge tube on a magnetic rack for 1 minute to remove the waste liquid.

[0042] ③ Add 500 μL of washing solution I, fully shake and suspend the magnetic beads, and wash for 3 minutes; then place the centrifuge tube on the magnetic rack for 1 minute and remove the waste liquid.

[0043] ④ Add 500 μL of washing solution II, shake the magnetic beads thoroughly, and wash for 3 minutes; then place the centrifuge tube on the magnetic rack for 1 minute to remove the waste liquid; and let it dry for 5 minutes.

[0044] ⑤ Add 100 μL of double distilled water, fully shake and suspend the magnetic beads, and elute at 56°C for 10 minutes; then place the centrifuge tube on the magnetic rack for 1 minute, and save the DNA solution for later use.

[0045] ⑥ Perform Nanodrop quality check on the DNA solution to check the concentration and purity of the DNA, and perform 1.5% agarose gel electrophoresis to check the integrity of the DNA.

[0046] After quality inspection, the average concentration of sample DNA was higher than 100ng / μL, OD260 / OD280=1.80~1.90, OD2360 / OD280=1.70~1.90, and the electrophoresis bands were clear without tailing. Figure 1 The extracted DNA was entrusted to Beijing Tongnong Testing Technology Co., Ltd. for library construction and whole genome sequencing. The sequencing depth of each sample was 5×, and the sequencing volume was 8G / sample.

[0047] 2. GWAS analysis to develop genetic sex-linked molecular markers

[0048] After obtaining the sequencing data of the above samples, the rawdata was filtered by Fastp software to obtain cleandata, with a ratio of 98.71%; the cleandata was aligned to the reference genome of the royal goldfish GCF_003368295.1 by BWA software, and the royal goldfish genome-wide INDEL and SNP mutation sites were obtained by Samtools and GATK software. The distribution of sex-linked SNPs was statistically analyzed by GWAS, and the genetic sex-linked interval of the royal goldfish was located at the chromosome position of CHR22: 20-30M (see Figure 2 ), then sort and index the aligned BAM files and then open them with the visualization software IGV for viewing. Genetic sex-linked primers were designed where there were obvious structural variations in male and female individuals, and universal primers were designed where there were no obvious mutations in male and female individuals. PCR amplification was performed on male and female fish respectively to screen for sex-specific molecular markers.

[0049] 22 samples of the above 48 goldfish were selected for primer testing, and 1-2 samples of each species were selected, with samples of koi, carp, and sturgeon (the above test species were all from the Tongzhou base of Beijing Aquatic Technology Extension Station) as controls. The above-mentioned universal primers and genetic sex-linked primers were used for PCR detection, and the PCR products were mixed together and then tested by 1.5% agarose gel electrophoresis. The PCR system was: 2μL DNA (30ng / μL), 0.4μL primer mixture (10mM), 5μL2×PCRMix, 2.6μL double distilled water. The PCR reaction program was: S1, 95℃ reaction for 10min, 1cycle; S2, 95℃ reaction for 30s, 57℃ reaction for 30s, 72℃ reaction for 30s, 35cycles; S3, 72℃ reaction for 5min. The PCR reagent used was the kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., item 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 palace goldfish samples, but could not amplify in koi, colored carp, and sturgeon ( Figure 3 ); The primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2 have obvious gender specificity and can only amplify a 486 bp specific band in XY individuals, but cannot amplify in XX individuals, koi, colored carp, and sturgeon ( Figure 3 ). The test results are consistent with the gender results of the palace goldfish samples, indicating that the primers of the present invention have a good application prospect in the genetic gender identification of palace goldfish.

[0051]

[0052]

[0053] Example 2: Obtaining a Genetic Sex-Specific Multiple Molecular Marker Kit for Palace Goldfish

[0054] Since the use of single-plex PCR reactions and commercial PCR reagents is costly and has low detection efficiency, in order to improve detection efficiency and reduce detection costs, the above-mentioned PCR reaction reagents and multiplex PCR reaction systems are self-developed. The self-developed 2×PCRMix is ​​prepared as follows: the concentration of TaqDNA polymerase is 0.6U / μL, the concentration of dNTPs is 400μM, the concentration of magnesium chloride is 8mM, the concentration of potassium chloride is 100mM, the concentration of betaine is 0.6M, and the concentration of glycerol is 10%. In order to adjust the optimal multiplex PCR effect, the amount of genetic sex-specific molecular marker mixture and universal chromosome-specific molecular marker mixture of the PCR system was adjusted and tested, and the optimal PCR reaction system was finally determined to be: 2μL DNA (30ng / μL), 0.3μL genetic sex-specific molecular marker mixture (10mM), 0.25μL universal chromosome-specific molecular marker mixture (10mM), 5μL 2×PCRMix, 2.45μL double distilled water. TaqDNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number ET106. Other general chemical reagents that meet the experimental conditions are available.

[0055] The 25 samples in Table 1 were tested according to the self-developed PCR reaction system and multiple PCR reaction. The results are as follows: Figure 4 The results of sex detection are consistent with those of commercial reagents, and the specificity and uniformity of the two pairs of primers are better, which shows that the multiple molecular marker kit of the present invention is highly universal in the genetic sex identification of different varieties of palace goldfish and has a good application prospect.

[0056] Example 3: Application of the genetic sex-specific multiple molecular marker method for palace goldfish

[0057] The genetic sex-specific multiple molecular marker kit for palace goldfish developed based on Examples 1 and 2 was used to identify the genetic sex of the palace goldfish to be tested. A total of 48 palace goldfish from Beijing Xianglong Technology Co., Ltd., Hengfeng Ornamental Fish Farm in Dianshan Lake Town, Kunshan City, and Pan's Ornamental Fish Farm in Jingxi Guanzhong, Minhou County were collected as samples to be tested, and the sex was identified using the method of the present invention. The results are shown in Table 1. Figure 5And Table 2. A total of 46 samples were detected to be able to amplify the 204bp band, and 2 samples were unable to amplify the 204bp band; among the above 46 samples, 9 samples were detected to be able to amplify the 486bp band, and 37 samples were unable to amplify the 486bp band, as shown in the following table. According to the comparison of the expanded band and the actual gender in the table, it can be seen that the amplification result is completely consistent with the actual gender. The identification conclusion of the method of the present invention is completely consistent with the actual situation.

[0058]

[0059]

[0060]

[0061] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Multiple molecular markers for identifying or assisting in identifying the genetic sex of palace goldfish, characterized in that: The multiple molecular markers include the following A1) and A2), A1) using the genomic DNA of the royal goldfish as a template, and using the male chromosome-specific primers consisting of the single-stranded DNA molecule shown in SEQ ID NO.1 and the single-stranded DNA molecule shown in SEQ ID NO.2, to amplify a DNA fragment of 486 bp in length; A2) Using the genomic DNA of the royal goldfish as a template, a single-stranded DNA molecule shown in SEQ ID NO.3 and a single-stranded DNA molecule shown in SEQ ID NO.4 were used as universal primers to amplify a DNA fragment of 204 bp in length.

2. A primer composition for identifying or assisting in identifying the genetic sex of a palace goldfish, characterized in that: The primer composition comprises the following B1) and B2), B1) a male chromosome-specific primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2; B2) A universal primer pair consisting of the single-stranded DNA molecule shown in SEQ ID NO.3 and the single-stranded DNA molecule shown in SEQ ID NO.

4.

3. Use of the molecular marker according to claim 1 or the primer pair composition according to claim 2 in preparing a product for identifying or assisting in identifying the genetic sex of palace goldfish.

4. A kit, characterized in that: The kit comprises the primer combination according to claim 2.

5. The kit according to claim 4, characterized in that The kit also includes a PCR reaction system, which includes TaqDNA polymerase, dNTPs, magnesium chloride, potassium chloride, betaine and glycerol, wherein the concentration of the TaqDNA polymerase is 0.3U / μL, the concentration of dNTPs is 200μM, the concentration of magnesium chloride is 3.5mM, the concentration of potassium chloride is 50mM, the concentration of betaine is 0.3M, and the concentration of glycerol is 5%.

6. Use of the molecular marker according to claim 1, the primer pair composition according to claim 2 or the kit according to claim 4 in identifying or assisting in identifying the genetic sex of palace goldfish.

7. A method for identifying or assisting in identifying the genetic sex of a palace goldfish, characterized in that: Use the primer composition according to claim 2 or the kit according to any one of claims 4 or 5.

8. The method according to claim 7, characterized in that The method is: Using the genomic DNA of the sample to be tested as a template, amplification is performed using a male chromosome-specific primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2, and a universal primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.3 and a single-stranded DNA molecule shown in SEQ ID NO.4; When only a 204 bp-long royal goldfish universal chromosome-specific molecular marker is amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic female royal goldfish, and the sex chromosome genotype is XX; When a 204 bp palace goldfish universal chromosome-specific molecular marker and a 486 bp palace goldfish genetic male chromosome-specific molecular marker are simultaneously amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic male palace goldfish, and the sex chromosome genotype is XY; When the universal chromosome-specific molecular marker of the royal goldfish with a length of 204 bp cannot be amplified from the genomic DNA of the sample to be tested, the sample to be tested is not the royal goldfish.

9. The method according to claim 7, characterized in that: The method is: Performing multiple PCR amplification on the sample to be tested using the kit according to claim 4 or 5; When only a 204 bp-long royal goldfish universal chromosome-specific molecular marker is amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic female royal goldfish, and the sex chromosome genotype is XX; When a 204 bp palace goldfish universal chromosome-specific molecular marker and a 486 bp palace goldfish genetic male chromosome-specific molecular marker are simultaneously amplified from the genomic DNA of the sample to be tested, the sample to be tested is judged to be a genetic male palace goldfish, and the sex chromosome genotype is XY; When the universal chromosome-specific molecular marker of the royal goldfish with a length of 204 bp cannot be amplified from the genomic DNA of the sample to be tested, the sample to be tested is not the royal goldfish.

10. Use of the molecular marker according to claim 1, the primer pair composition according to claim 2 or the kit according to claim 3 in breeding of palace goldfish.

Images