Method for extracting DNA (deoxyribonucleic acid) from muscular tissue of Alosa sapidissima, five gender-related molecular markers and application

Abstract

The invention discloses a method for extracting DNA from muscular tissues of Alosa sapidissima, five gender-related molecular markers and application, the muscular tissues of Alosa sapidissima are fixed and frozen by adopting a specific pretreatment method, then DNA extraction is performed on the muscular tissues, and a high-quality DNA template is obtained; then, five molecular marker molecules capable of distinguishing the sex of Alosa sapidissima are obtained through further screening of the DNA template extracted through the method, and specific primers capable of amplifying the five pairs of molecular markers are further obtained based on the five molecular markers, and the five pairs of specific primers obtained through the method can accurately distinguish the hereditary sex; the invention further provides a molecular marker method for identifying the sex of Alosa sapidissima, the method for extracting DNA from muscle tissue is adopted, the sex specific molecular marker can be more accurately screened out, and it is guaranteed that the sex identification accuracy of the primer pair is 100%.

Description

Technical Field

[0001] The present invention belongs to the field of fish genetics, and specifically relates to a method for extracting DNA from the muscle tissue of American shad, five sex-related molecular markers and their applications. The present invention is a divisional application of a patent application with patent number 202210678354.6 and the invention name is a method for extracting DNA from the muscle tissue of American shad, five sex-related molecular markers and their applications. Background Art

[0002] The most valuable shad species in the world include American shad, Chinese shad and Indian shad. The breeding and reproduction of shad has always been a problem that has troubled fish biologists and breeding technicians at home and abroad, and there are few successful cases of breeding and reproduction at home and abroad. Because shad has completely different physiological characteristics from ordinary farmed fish, it is extremely sensitive to changes in the external environment, has a delicate body, is squeamish and irritable, has a strong stress response, and is very easy to be injured and killed. At the end of the last century, Chinese scholars conducted preliminary research on the breeding and artificial breeding of Chinese shad, but the results were not satisfactory. The resources of Chinese shad are becoming increasingly depleted and it is difficult to see any traces. In the case of the endangered Chinese shad, our team has been conducting research and development in the introduction, domestication, artificial breeding and seedling cultivation of American shad and germplasm improvement since 2003, when there was a gap in shad biology, behavioral characteristics and breeding technology. Over the past 20 years, our team has achieved a number of results in the biology, reproduction and seedling raising, and germplasm improvement of American shad with the spirit of "ten years of hard work" and "crossing the river by feeling the stones", leading the development of the shad farming industry. On the basis of successful farming, we have carried out attempts in shad breeding and industrialized it, completely getting rid of the situation in which the shad farming industry has long relied on imported fertilized eggs.

[0003] Based on the selection and breeding of American shad strains, whole genome sequencing and simplified genome sequencing technology, our team isolated 5 male molecular markers of American shad, identified xx pseudomale fish (neomales), and successfully applied them to the production of all-female shad. All-female farming takes advantage of the fast growth of female fish and produces expensive large-sized shad. More importantly, it can effectively avoid the large-scale death and weight loss caused by male fish chasing in spring, thereby improving the breeding efficiency by more than 28%, becoming an important measure to promote the development of shad farming. This technology provides key technical support from the source of the shad farming industry chain and the renewal of the farming model, sustainably improving the efficiency of the shad farming industry, and is of great significance to the development of the farming industry of other shad species in the world.

[0004] The specific molecular marker technology of other fish bodies mostly uses the tail fin and scales to extract DNA, but in fact, the tail fin and scales of fish are hard, and incomplete grinding can easily lead to low DNA extraction concentration, causing inaccurate primer extension sequence. In addition, the fiber structure, bone layer, sampling weight, sample freshness and storage method in the tail fin or scales all affect the quality of extracted DNA.

[0005] Therefore, there is an urgent need for a method to extract high-quality DNA from American shad and to obtain more molecular markers for sex identification. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for extracting DNA from muscle tissue of American shad, which pre-treats the muscle tissue by immobilization and freezing, thereby obtaining a complete and high-quality DNA template. The quality of the template DNA in the present invention directly affects the identification result. Using high-quality DNA can avoid false negatives in PCR reactions and improve the sensitivity and specificity of the experiment. Finally, by using primers of different concentrations and DNA verification, the accuracy of the experiment is increased.

[0007] The technical problem that the present invention needs to solve is to provide a method for obtaining molecular markers related to the sex of American shad.

[0008] The technical problem that the present invention needs to solve is to provide a method for obtaining gene fragments of molecular markers related to sex of American shad.

[0009] The technical problem that the present invention also aims to solve is to provide a molecular marker related to the sex of American shad obtained according to a method for obtaining a molecular marker and a gene fragment of a molecular marker related to the sex of American shad obtained according to a method for obtaining a gene fragment.

[0010] The technical problem that the present invention needs to solve is to provide a specific primer pair and a kit for detecting the molecular markers related to the sex of the American shad or the gene fragment.

[0011] The technical problem that the present invention needs to solve is to provide a DNA marking method for identifying the sex of American shad.

[0012] Technical solution: In order to solve the above technical problems, the present invention provides a method for extracting DNA from muscle tissue of American shad, which comprises pretreating the muscle tissue, wherein the pretreatment comprises fixing the muscle tissue of American shad with 50% alcohol or 20% formaldehyde and then freezing it at -20°C or -80°C.

[0013] The present invention also includes a method for obtaining sex-related molecular markers of American shad, wherein the method uses the extraction method to extract muscle tissue DNA of male American shad and female American shad respectively, and then obtains sex-related molecular markers of American shad by sequencing using a simplified genome method.

[0014] The present invention also includes a method for obtaining a gene fragment of a molecular marker associated with the sex of American shad, the method comprising comparing the molecular marker obtained by the molecular marker obtaining method with the assembled genome sequence of male shad in the NCBI database to obtain the base sequence of the gene fragment of the molecular marker associated with the sex of American shad.

[0015] The nucleotide sequence of the molecular marker related to sex of American shad obtained according to the method for obtaining molecular markers is shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5.

[0016] The base sequence of the gene fragment of the molecular marker related to sex of American shad obtained according to the method for obtaining the gene fragment is shown in SEQ ID NO: 6 or 7 or 8 or 9 or 10.

[0017] A specific primer pair for detecting the molecular markers related to the sex of the American shad or the gene fragment, wherein the sequences of the specific primer pair are shown as SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0018] The present invention also includes the application of the specific primer pair in preparing a kit for identifying the genetic sex of American shad.

[0019] The present invention also includes a kit for identifying the genetic sex of American shad, and the kit includes the specific primer pair.

[0020] The present invention also includes a DNA marker method for identifying the sex of American shad, the method comprising the following steps:

[0021] (1) Extract DNA from muscle tissue of American shad of unknown sex;

[0022] (2) performing PCR amplification using the specific primer pair or the kit;

[0023] (3) When the PCR amplification product shows a band of 350 bp, 463 bp, 430 bp, 170 bp or 185 bp, it can be determined to be a male American shad; when the PCR amplification product does not show a band of 350 bp, 463 bp, 430 bp, 170 bp or 185 bp, it can be determined to be a female American shad.

[0024] The PCR amplification system comprises: 2×Taq Plus Master Mix IDye Plus, the specific primer pair described in claim 6, DNA, and water; the PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and final extension at 72°C for 5 minutes.

[0025] The invention firstly uses a plurality of American shads of known male and female as samples, extracts total DNA from muscle tissue, constructs sequencing libraries according to a simplified genome method, and performs sequencing on a machine after the sequencing library is qualified; filters the raw data (Raw data) in FASTQ format generated by the sequencing machine to obtain clean data (clean data), clusters using the ustacks software (version 1.34) in the Stacks software package, and constructs a reference sequence. SOAP software (version 2.21) is used to align the sequencing data to the reference sequence, and the maximum likelihood method (ML) is used to perform site typing. Finally, five short sequences specific to male American shads are obtained, which exist in male shads but not in female shads, and the sequences are molecular marker sequences of the sex of American shads, as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0026] As one of the specific embodiments of the present invention, the present invention also proposes to use the muscle tissue of American shad to perform quality analysis of extracted DNA, compare the quality of DNA extracted by different fixing solutions (50% alcohol or 20% formaldehyde fixation) and storage temperatures (-20°C freezing, -80°C freezing), improve the key steps of the kit according to specific examples, and finally select agarose gel electrophoresis and ultraviolet spectrophotometer to perform quality analysis on the extracted DNA, and perform subsequent verification experiments with the highest accuracy, so that the sex-specific molecular markers can be obtained more accurately.

[0027] As one of the embodiments of the present invention, the obtained molecular markers of male sex of American shad were compared with the assembled genome sequence of male shad obtained in NCBI (JAHTKL010000000) by basic local alignment search tool (BLAST), and male shad-specific long sequences of 350 bp, 463 bp, 430 bp, 170 bp and 185 bp were obtained. The nucleic acid sequence of this long sequence is SEQ ID NO: 6, 7, 8, 9, 10.

[0028] As one of the specific embodiments of the present invention, PCR primers are designed based on the above-mentioned male shad specific long sequence SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 to obtain five pairs of specific marker primers that can 100% distinguish the sex of American shad, and their names are Tag-2, Tag-3, Tag-9, Tag-10, and Tag-11, and their sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the present invention fixes and freezes the muscle tissue of the American shad by adopting a specific pretreatment method, and then extracts DNA from the muscle tissue to obtain a high-quality DNA template, and then further screens the DNA template extracted by the method to obtain five molecular marker molecules that can distinguish the sex of the American shad, and based on the above five molecular markers, further obtains specific primers that can amplify five pairs of molecular markers. The five pairs of specific primers obtained by the present invention can accurately distinguish genetic sex. The present invention also provides a molecular marker method for identifying the sex of the American shad. The present invention adopts a method for extracting DNA from muscle tissue, which can more accurately screen out sex-specific molecular markers, ensure that the accuracy of the primer pair sex identification in the present invention is 100%, and ensure the accuracy of subsequent enterprises using specific primers for non-invasive sex identification of fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 1. DNA extraction under different fixative solutions and storage temperatures.

[0031] Figure 2 Performance verification of primer pair Tag-2 for shad with known sex;

[0032] Figure 3Performance verification of primer pair Tag-3 for shad with known sex;

[0033] Figure 4 Performance verification of primer pair Tag-9 for shad with known sex;

[0034] Figure 5 Performance verification of primer pair Tag-10 for shad with known sex;

[0035] Figure 6 Performance verification of primer pair Tag-11 for shad with known sex;

[0036] Figure 7 Verification of the genetic sex of Tag-2 in the offspring culture population of American shad;

[0037] Figure 8 Verification of the genetic sex of Tag-3 in the offspring culture population of American shad;

[0038] Fig. 9 Verification of the genetic sex of Tag-9 in the offspring culture population of American shad;

[0039] Fig.10 Verification of the genetic sex of Tag-10 in the offspring culture population of American shad;

[0040] Fig.11 Genetic sex verification of Tag-11 in the offspring culture population of American shad. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described in detail below through specific embodiments. However, it is necessary to point out that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the protection scope of the present invention.

[0042] Example 1 DNA extraction from different tissues of American shad under different conditions

[0043] 32 female and 32 male American shad of known sex were used as samples. Muscle, caudal fin and scale tissues were taken respectively. Tissue / blood genomic DNA (Easy Biological, Cat. No. DR0301050) was used to extract total DNA. For specific operations, refer to the instructions of the kit:

[0044] The present invention improves the key steps in the kit as follows:

[0045] (1) First, the sampling site was in an outdoor greenhouse, so it was not possible to directly extract DNA. Thirty-two female American shad and thirty-two male American shad were collected. The experimental groups included four groups, each with five males and five females. 20 mg of muscle tissue was taken from each shad. Two of the experimental groups were fixed with 2 mL of 50% alcohol for 3 hours, and the other two groups were fixed with 20% formaldehyde for 3 hours. The samples were then stored in 5 mL centrifuge tubes and then stored under different storage conditions (-20°C or -80°C) for 2 days before being analyzed. The muscle tissue was used for subsequent DNA extraction. The control group included four groups, each with 3 males and 3 females. 30 mg of muscle tissue was taken from control group 1 and control group 2 without fixation. The muscle tissue was stored under different storage conditions (-20°C or -80°C frozen) for 2 days before subsequent DNA extraction. 20 mg of caudal fin and scale tissue was taken from control group 3 and control group 4, respectively, and fixed in 2 mL of 50% alcohol for 3 hours. The caudal fin and scale tissue were stored at -80°C for 2 days before subsequent DNA extraction.

[0046] Then, in the animal tissue digestion step in the selected kit, each tissue sample treated above is taken out and placed in a 2mL centrifuge tube, 200μL buffer solution G1 and 20μL proteinase K are added and mixed, and then the tissue is fully stirred with a homogenizer until it is completely broken, and then digested at 56°C for 20 minutes, and finally 200μL buffer solution G2 is added and mixed by inversion. After this improvement, DNA can be repeatedly released from the tissue, and the DNA extraction concentration is increased.

[0047] (2) During the steps of column loading, washing, evaporation and elution by centrifugation, the centrifuge was modified to be performed at 4°C in a refrigerated centrifuge, the speed of the centrifuge was changed to 8000 rpm, and all operations were placed on ice. This modification ensured that high-concentration intact DNA fragments were extracted.

[0048] The experimental groups are shown in Table 1:

[0049] Table 1

[0050]

[0051]

[0052] From the above comparison, it can be seen that in the DNA extraction of the present invention, the concentration of the fish tail fin and scale tissue extraction is very low, and the DNA chain extracted by other schemes is incomplete and easily damaged, and the subsequent PCR results are not good. Figure 1 Therefore, the best solution is to extract DNA from muscle tissue in the 50% alcohol-fixed -80°C cryopreservation group. The DNA extracted by this solution is more complete and not easy to break. The DNA extracted by this method was subsequently diluted for further experiments.

[0053] Example 2

[0054] Refer to the patent publication number CN114574600A for enzyme digestion, ligation, enrichment, purification, PCR introduction of index sequence, library mixing and sequencing.

[0055] Finally, five male shad-specific short sequences were obtained, and their nucleic acid sequences were shown in SEQ ID NO.1, 2, 3, 4, and 5, respectively.

[0056] SEQ ID NO.1:

[0057] SEQ ID NO.2:

[0058] SEQ ID NO.3:

[0059] SEQ ID NO.4:

[0060] SEQ ID NO.5:

[0061] Example 3 Extension of the male-specific sequence of American male shad

[0062] 1. The American shad male-specific short sequence identified in Example 1 was compared with the assembled genome sequence of male shad (JAHTKL010000000) obtained in NCBI by basic local alignment search tool (BLAST) to obtain five long sequences of 350 bp, 463 bp, 430 bp, 170 bp and 185 bp in length, whose nucleic acid sequences are shown in SEQ ID NOs. 6, 7, 8, 9 and 10.

[0063] SEQ ID NO.6:

[0064]

[0065] SEQ ID NO.7:

[0066]

[0067] SEQ ID NO.8:

[0068]

[0069] SEQ ID NO.9:

[0070]

[0071] SEQ ID NO.10:

[0072]

[0073]

[0074] Example 4 Primer design based on male shad-specific long sequence and its performance verification

[0075] PCR primers were designed based on the long sequences shown in SEQ ID NO. 6, 7, 8, 9, and 10, and five pairs of specific marker primers capable of distinguishing the sex of shad were obtained, namely Tag-2, Tag-3, Tag-9, Tag-10, and Tag-11. The sequences are shown in Table 2.

[0076] The male shad specific marker primer information table is as follows:

[0077] Table 2 Primer sequences

[0078]

[0079] 100 shad (50 males and 50 females) of known sex were selected from the group of shad cultured in this laboratory and divided into five groups. DNA was extracted from the group fixed with 50% alcohol and frozen at -80℃, and then PCR was performed for verification. The experimental conditions were as follows:

[0080]

[0081] The PCR reaction conditions are as follows:

[0082]

[0083] The best effect was achieved after 35 cycles of amplification.

[0084] The results are as follows Figure 2-6 As shown, Figure 1-5 100 fish were verified for Tag-2, Tag-3, Tag-9, Tag-10 and Tag-11 respectively, including 50 females and 50 males.

[0085] In this embodiment, primer sensitivity detection was also performed, which specifically included the following contents:

[0086] Sample preparation: The shad muscle DNA basic sample extracted by the above optimal extraction scheme was diluted with deionized water in multiple ratios. The sample concentrations after dilution were 200ng / μL, 20ng / μL, 2ng / μL, 0.2ng / μL, and 0.02ng / μL, and 1μL of each sample was used for detection. The above kit 2×Taq Plus Master Mix II (Dye Plus) (PCR-fluorescent probe method) was used to test the sensitivity of five primers and analyze the detection rate of each concentration sample. The results are shown in Table 3.

[0087] Table 3 Detection of five pairs of primer sequences on male fish DNA samples of different concentrations

[0088]

[0089] From the above results, it can be seen that the present invention can detect samples with a minimum DNA concentration of 0.2 ng / μL, while the primer sensitivity in patent publication number CN114574600A has a minimum DNA concentration of only 2 ng / μL, and its sensitivity is far higher than the level of existing technologies.

[0090] Example 4 Identification of genetic sex of the second generation of American shad based on male shad-specific marker primers

[0091] 100 female and 100 male shad were randomly collected from four shad breeding ponds for gender verification using male shad specific marker primers. First, DNA was extracted using a 50% alcohol fixation -80°C cryopreservation group, followed by PCR verification. The primer sequences are shown in Table 2. The experimental conditions are as follows:

[0092]

[0093] The PCR reaction conditions are as follows:

[0094]

[0095]

[0096] The best effect was achieved after 35 cycles of amplification.

[0097] The results are as follows Figure 7-11The results showed that the amplified specific marker bands (350bp, 463bp, 430bp, 170bp and 185bp bands) were all shown in the electrophoresis patterns of the PCR amplification products of all male individuals; the amplified specific marker bands (i.e., 350bp, 463bp, 430bp, 170bp and 185bp bands) were not found in the electrophoresis patterns of the PCR amplification products of all female individuals. The sex identified by the sex-specific molecular marker method was consistent with the sex identified by the anatomical gonad, indicating that the accuracy of the sex identification method was 100%.

Claims

1. A gene fragment of a molecular marker related to sex of American shad, characterized in that: The base sequence of the gene fragment is shown in SEQ ID NO: 7 or 8 or 9 or 10.

2. A specific primer pair for detecting the gene fragment according to claim 1, characterized in that: The sequences of the specific primer pairs are shown in SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

3. Use of the specific primer pair according to claim 2 in preparing a kit for identifying the genetic sex of American shad.

4. A kit for identifying the genetic sex of American shad, characterized in that: The kit comprises the specific primer pair according to claim 2.

5. A DNA marker method for identifying the sex of American shad, characterized in that: The method comprises the following steps: (1) Extract DNA from muscle tissue of American shad of unknown sex; (2) using the specific primer pair described in claim 2 or the kit described in claim 4 to perform PCR amplification; (3) When the PCR amplification product shows a 463 bp, 430 bp, 170 bp, or 185 bp band, it can be identified as a male American shad; When no 463bp, 430bp, 170bp or 185bp bands are found in the PCR amplification product, it can be determined to be a female American shad.

6. The DNA labeling method for identifying the sex of American shad according to claim 5, characterized in that: The PCR amplification system comprises: 2 × Taq Plus Master Mix I Dye Plus, the specific primer pair described in claim 2, DNA, and water; the PCR reaction conditions are as follows: 95° C. pre-denaturation for 3 min, 95° C. denaturation for 15 s, 60° C. annealing for 30 s, 72° C. extension for 1 min, and 72° C. final extension for 5 min.

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