Specific InDel molecular marker primer for identifying gender of new trachinotus ovatus variety and application of specific InDel molecular marker primer
By designing the gender-specific InDel molecular marker primers InDel-W and InDel-ZW of the new ovate pompeople variety, the problem of gender identification of ovate pompeople in the prior art is solved, and rapid and accurate gender identification and strain distinction are achieved, which promotes gender determination research and whole female breeding, and improves breeding efficiency and corporate interests.
Patent Information
- Application Number
- CN202510448048.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art is difficult to accurately identify the genetic gender of new ovate pompa, especially male and female individuals cannot pass morphological identification in the embryo and juvenile stages, which limits the study of gonad differentiation and the implementation of whole female breeding, and the existing SNP gender marker specificity is insufficient.
The sex-specific InDel molecular marker primers InDel-W and InDel-ZW of the new ovate pompeople were designed to identify the gender of the new ovate pompeopleple variety by PCR amplification and gel electrophoresis. The primer InDel-W was used for female individual identification and InDel-ZW was used for male and female individual identification.
The rapid and accurate gender identification of new ovate pomrose varieties has been achieved, the benefits of breeding for all females have been improved, the new varieties and wild varieties have been distinguished, the variety identification and gender determination research have been supported, and the breeding efficiency and corporate interests have been improved.
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Figure CN120442770A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of molecular biology, and in particular relates to a sex-specific InDel molecular marker primer for identifying a new species of oval pomfret and its application. Background Art
[0002] The ovate pomfret (Trachinotus ovatus) belongs to the order Perciformes, family Trachinotidae, genus Trachinotus, commonly known as golden pomfret, yellow wax pomfret, and pomfret. It is a warm-water, pelagic, migratory, carnivorous fish found primarily in tropical and subtropical waters. With few bones and tender, delicious meat, it is a high-quality marine edible fish. It grows rapidly and is readily marketable within the same year. It is an important marine aquaculture species in Fujian, Guangdong, Guangxi, and Hainan provinces. Its aquaculture output and economic scale have increased rapidly and steadily in recent years. According to the 2024 China Fisheries Statistical Yearbook, the ovate pomfret is now China's largest marine aquaculture species.
[0003] Therefore, it is urgent to develop a new fast-growing breed of oval pomfret. Hainan Blue Grain Technology Co., Ltd. has developed a new breed of oval pomfret with a shorter growth cycle and better quality using the sixth generation of oval pomfret bred as a parent after group selection. However, there is currently no molecular marker that can accurately identify the genetic sex of this new breed.
[0004] The oval pomfret belongs to the ZZ / ZW female heterogametic sex determination system fish, with sexual growth dimorphism. The growth traits of female individuals are significantly better than those of male individuals. If all-female farming is achieved in production, the breeding efficiency will be significantly improved. However, the sex of male and female individuals of the oval pomfret cannot be identified by morphology during the embryonic, juvenile, and even adult stages. This brings great difficulties to the research on gonad differentiation and the breeding of single-sex seedlings of the oval pomfret, and also greatly restricts the development of the industry of this species. In addition, the development of molecular markers for the oval pomfret is currently lacking. Although there are sex markers designed based on the single nucleotide polymorphism (SNP) site of the candidate sex-determining gene hsd17b1, the primers designed based on this SNP site have shortcomings such as lack of specificity and easy binding to the male homologous segment of the oval pomfret, which need further improvement. At the same time, the sex molecular marker designed based on the insertion-deletion sequence InDel site in the oval pomfret has not yet been identified. Therefore, the present invention intends to develop sex-specific InDel markers for the new breed of oval pomfret cultivated by Hainan Blue Grain Technology Co., Ltd. Summary of the Invention
[0005] The purpose of the present invention is to provide a sex-specific InDel molecular marker primer for identifying a new species of oval pomfret.
[0006] The present invention also aims to provide a method for identifying the sex of the new species of oval pomfret.
[0007] The last object of the present invention is to provide the application of the above primers or methods in sex identification of the new species of oval pomfret and in distinguishing the new species of oval pomfret from wild oval pomfret.
[0008] The first object of the present invention can be achieved by the following technical solution: a sex-specific InDel molecular marker primer for identifying a new species of oval pomfret, wherein the primer is a primer InDel-W or a primer InDel-ZW, wherein the primer InDel-W includes an upstream primer InDel-WF and a downstream primer InDel-WR, and the primer InDel-ZW includes an upstream primer InDel-ZW-F and a downstream primer InDel-ZW-R, and the nucleotide sequences of the primers InDel-WF, InDel-WR, InDel-ZW-F and InDel-ZW-R are shown in SEQ ID NOs: 3 to 6, respectively.
[0009] Based on the new variety of oval pomfret with a shorter growth cycle and better quality bred by Hainan Blue Grain Technology Co., Ltd., the present invention designed InDel molecular markers and primers that can accurately identify the genetic sex of the new variety. This not only provides great assistance for the cultivation of an all-female breeding population of the new variety of oval pomfret, but also can distinguish the new strain population from other source populations, providing support for identifying and distinguishing the source of the oval pomfret population.
[0010] The above-mentioned second object of the present invention can be achieved by the following technical scheme: a method for identifying the sex of a new variety of oval pomfret, comprising the following steps: extracting genomic DNA of the new variety of oval pomfret, using the primer InDel-W or the primer InDel-ZW, performing PCR amplification with the genomic DNA as a template, detecting the amplified product by gel electrophoresis, and identifying the male and female individuals of the new variety of oval pomfret according to the number of gel electrophoresis bands.
[0011] Preferably, the PCR reaction system used in PCR amplification includes: 1.0 μL DNA template, 10 μL 2×Es TaqMasterMix, 0.5 μL each of the forward and reverse primers of primer InDel-W or primer InDel-ZW, and 8 μL ddH2O, a total of 20 μL.
[0012] Preferably, the PCR reaction program used in PCR amplification is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, 32 cycles, extension at 72°C for 5 minutes, and holding at 12°C for 2 minutes.
[0013] Preferably, the gel electrophoresis results of amplification using primer InDel-W are no bands in male individuals and a single band of 389 bp in female individuals; the gel electrophoresis results of amplification using primer InDel-ZW are a single band of 243 bp in male individuals and double bands of 243 bp and 738 bp in female individuals.
[0014] The last object of the present invention can be achieved by the following technical solution: application of the primers or methods in sex identification of new varieties of oval pomfret.
[0015] The present invention also provides application of the primer or method in distinguishing the new species of oval pomfret from wild oval pomfret.
[0016] The successful development of sex-specific InDel molecular marker primers for the new strain of oval pomfret in the present invention has, on the one hand, broadened the technical path for genetic sex identification of oval pomfret and, on the other hand, enabled the first technological breakthrough in the identification of oval pomfret strains. The application of this technology enables sex identification of new strains of oval pomfret throughout their developmental period, thereby promoting research on sex determination and the development of sex-controlled breeding, thereby improving the breeding efficiency of this strain and reducing breeding costs. The application of this technology also facilitates the rapid identification of different oval pomfret breeding strains, providing unique molecular markers for the cultivation of new varieties, protecting the interests of new variety breeding companies, and providing a typical example for genetic breeding work in other breeding categories.
[0017] Compared with the prior art, the present invention has the following advantages:
[0018] (1) The present invention provides for the first time a specific InDel molecular marker and primer for sex identification of a new species of oval pomfret, breaking through the limitations of the original SNP sex marker, and the identification method is simple and easy with good specificity;
[0019] (2) The method of the present invention can quickly and accurately identify the genetic sex of a new species of oval pomfret in batches at one time. Compared with the previous SNP sex markers, the InDel sex markers in the present invention are more accurate in genetic sex identification. The two pairs of primers confirm each other, with a high accuracy rate of up to 100%;
[0020] (3) The InDel molecular markers provided by the method of the present invention have a dual function. While identifying sex, they can also be used to distinguish new varieties of oval pomfret from wild oval pomfret and to identify different oval pomfret culture strains.
[0021] (4) The present invention overcomes the problem of the inability to identify the source of seedlings. The developed new candidate strain of oval pomfret can be used to quickly identify and distinguish seedlings from other sources, effectively protecting the economic interests of the enterprise.
[0022] (5) The primers and methods of the present invention have important scientific research value and practical application value for sex determination and sex control of oval pomfret. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The positions of the tag sequence in step (3) of Example 1 and the sequences and positions of three pairs of primers designed based on the scaffold sequence at the position of the tag;
[0024] Figure 2 The following are gel electrophoresis images of PCR amplification of individuals from the new family of silver pomfret using the Tag primers in step (3) of Example 1 and the PCR products, as well as gel electrophoresis images of PCR amplification of individuals from the new family of silver pomfret using the three pairs of InDel primers in step (5) of Example 1, with the gel electrophoresis images of the PCR products of the Tag primers, InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R from top to bottom.
[0025] Figure 3 This is an excerpt of the results of PCR amplification of the new family of oval pomfret using three pairs of InDel-labeled primers in step (6) of Example 1 and the gel electrophoresis of the PCR products, from top to bottom: the gel electrophoresis images of InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R;
[0026] Figure 4 PCR amplification of wild oval pomfret from Guangxi using three pairs of InDel-labeled primers in Example 2 and gel electrophoresis images of the PCR products, from top to bottom: InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R;
[0027] Figure 5 PCR amplification of wild oval pomfret in Zhanjiang area using three pairs of InDel-labeled primers in Example 3 and gel electrophoresis images of PCR products, from top to bottom are the gel electrophoresis images of InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R. DETAILED DESCRIPTION
[0028] The technical solution of the present invention will be described in detail below through specific examples, which is intended to help those skilled in the art to more accurately understand and smoothly implement the technical solution of the present invention. It should be understood that the following examples and drawings are only exemplary and are by no means limiting of the scope of the present invention. The reagents or materials used in the examples, unless otherwise specified, are all derived from commercial sources. In addition, unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.
[0029] Example 1
[0030] The present embodiment provides a method for screening specific InDel molecular marker primers for identifying the sex of a new species of oval pomfret and for genetic sex identification, comprising the following five steps: (1) extracting genomic DNA from individuals of the new species of oval pomfret; (2) screening sex-specific InDel molecular marker sequences based on 2b-RAD sequencing; (3) designing a set of sex molecular marker primers based on the InDel molecular marker sequences; (4) performing PCR amplification using the three pairs of synthesized InDel molecular marker primers; and (5) performing agarose gel electrophoresis on the PCR products and analyzing the results to infer the genetic sex of the individuals, as follows:
[0031] (1) Extraction of genomic DNA
[0032] The full-sib family of the new breed of oval pomfret developed by Hainan Blue Grain Technology Co., Ltd. using the sixth generation of oval pomfret as the parent after population selection was collected, including 2 parents and 300 offspring individuals. The physiological sex was identified after dissection, and then about 1 square centimeter of fin rays were clipped and preserved in 95% alcohol. The tissue DNA was extracted using a commercially available DNA extraction kit or conventional phenol imitation method.
[0033] (2) Screening of sex-specific InDel molecular marker sequences
[0034] A total of 302 genetic samples from the parents and offspring of the oval pomfret family were sent to Sangon Biotech (Shanghai) Co., Ltd. The oval pomfret genome sequence (NCIB accession number: SAMEA104304869) was used as a reference sequence for marker development of the parents and offspring. The 2b-RAD five-tag tandem technology was used for library construction and sequencing, and the output tag sequence was aligned to the reference genome to screen for sex-specific tag sequences. A total of three F group tag tags were screened.
[0035] The scaffold sequences at the positions of the three F group Tag tags, i.e., indel molecular markers (i.e., Z sequence, W sequence, and W sequence), are shown in SEQ ID NO: 7 to SEQ ID NO: 9, respectively.
[0036] (3) Design of InDel sex marker primers
[0037] According to the Scaffold sequences of the three F group Tag tags screened in step (2), random primers were designed on both sides of the tags to develop sex-specific InDel molecular marker primers. The designed primers were used to conduct PCR experiments and gel electrophoresis experiments in the oval pomfret family population to verify that a pair of Tag tag primers with obvious male and female differences were finally screened (the specific results are shown in Figure 2). Figure 2 The Tag-Z / R of the Tag primer was then used to perform gel electrophoresis and cloning experiments to obtain the male and female differential sequences of the oval pomfret family (see Figure 1 ), primers were designed for the male-female differential sequence, and a total of three primer pairs were designed: InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R (for specific sequences and design positions, see Figure 1 ).
[0038] Specifically, the names, base sequences and product band sizes of the three pairs of primers are shown in Table 1:
[0039] Table 1 Three pairs of primers InDel-ZF / R, InDel-WF / R, InDel-ZW-F / R and their amplification results
[0040]
[0041] (4) PCR amplification
[0042] The forward and reverse primers of the three primer pairs InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R were used as amplification primers, and the extracted oval pomfret DNA was used as a template, and PCR amplification was performed using 2×Es Taq MasterMix (Dye).
[0043] PCR reaction system (20 μL):
[0044]
[0045] The PCR reaction procedure is as follows:
[0046]
[0047] (5) Agarose gel electrophoresis analysis
[0048] The amplified product in step (2) was subjected to gel electrophoresis. The gel electrophoresis procedure was as follows: voltage U: 180V, current I: 180mA, power P: 50W, time: 15min. In the InDel-ZF / R diagram, both male and female individuals showed a single band of 486bp (as shown in SEQ ID NO: 10); in the InDel-WF / R diagram, both male individuals had no bands, while both female individuals showed a single band of 389bp (as shown in SEQ ID NO: 11); in the InDel-ZW-F / R diagram, both male individuals showed a single band of 243bp (as shown in SEQ ID NO: 12), while both female individuals showed a double band of 243bp (as shown in SEQ ID NO: 12) and 738bp (as shown in SEQ ID NO: 13) (see the specific results). Figure 2 ).
[0049] Therefore, the primers InDel-ZF / R cannot distinguish between males and females, while the primers InDel-WF / R and InDel-ZW-F / R can distinguish between males and females in the new species of oval pomfret. Therefore, InDel-WF / R and InDel-ZW-F / R were selected as specific InDel molecular marker primers for identifying the sex of the new species of oval pomfret.
[0050] Example 2
[0051] Specific InDel molecular marker primers for identifying the sex of a new species of oval pomfret were expanded and validated in a population of candidate lines of a fast-growing new species of oval pomfret.
[0052] Fifty male and female DNA samples of 50 new varieties of oval pomfret bred by Hainan Blue Grain Technology Co., Ltd. were randomly selected and PCR amplified and gel electrophoresed using the three primer pairs InDel-ZF / R, InDel-WF / R, and InDel-ZW-F / R to expand the verification. The experimental results were consistent with those of Figure 2 The gel electrophoresis band patterns of ZF / R, WF / R, and ZW-F / R were the same (see Figure 3 , 24 samples were selected, and the remaining results were the same for these 24 samples). Therefore, using the primers and method of Example 1 of the present invention, the accuracy rate of identifying the sex of this family's oval pomfret was 100%.
[0053] Example 3
[0054] Expanded validation of specific InDel molecular marker primers for sex identification of new species of oval pomfret in wild populations of oval pomfret
[0055] Forty wild ovate pomfret were purchased from Guangxi and Zhanjiang, respectively. After dissection and observation of their gonadal tissue to determine their physiological sex, approximately 1 square centimeter of fin rays were excised and preserved in 95% ethanol. Tissue DNA was extracted using a commercially available DNA extraction kit or a conventional phenol-imitation method.
[0056] The three pairs of primers were used to perform PCR amplification and gel electrophoresis experiments on the DNA extracted from wild pomfret (part of the experimental results are shown in Figure 4 、 Figure 5 ), and found that the three primer pairs produced different results in wild and resident populations of oval pomfret. Therefore, using the primers and method of Example 1 of the present invention, it is possible to distinguish between a candidate fast-growing new pomfret strain and a wild population of pomfret.
[0057] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention. Any improvements and modifications based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection shall be subject to the claims.
Claims
1. A sex-specific InDel molecular marker primer for identifying a new species of oval pomfret, characterized in that: The primer is a primer InDel-W or a primer InDel-ZW, wherein the primer InDel-W includes an upstream primer InDel-WF and a downstream primer InDel-WR, and the primer InDel-ZW includes an upstream primer InDel-ZW-F and a downstream primer InDel-ZW-R. The nucleotide sequences of the primers InDel-WF, InDel-WR, InDel-ZW-F and InDel-ZW-R are shown in SEQ ID NOs: 3 to 6, respectively.
2. A method for identifying the sex of a new species of oval pomfret, characterized in that: The following steps are involved: The genomic DNA of the new species of oval pomfret is extracted, and PCR amplification is performed using the primer InDel-W or the primer InDel-ZW described in claim 1 with the genomic DNA as a template. The amplified product is detected by gel electrophoresis, and the male and female individuals of the new species of oval pomfret are identified based on the number of gel electrophoresis bands.
3. The method according to claim 2, characterized in that The PCR reaction system used in PCR amplification included: 1.0 μL of DNA template, 10 μL of 2×Es Taq MasterMix, 0.5 μL each of the forward and reverse primers of primer InDel-W or primer InDel-ZW, and 8 μL of ddH2O, for a total of 20 μL.
4. The method according to claim 2, characterized in that The PCR reaction program used in PCR amplification was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 30 sec, 32 cycles, extension at 72°C for 5 min, and holding at 12°C for 2 min.
5. The method according to claim 2, characterized in that The gel electrophoresis results of the amplification using primer InDel-W showed no band in male individuals and a single band of 389 bp in female individuals; the gel electrophoresis results of the amplification using primer InDel-ZW showed a single band of 243 bp in male individuals and double bands of 243 bp and 738 bp in female individuals.
6. Use of the primers according to claim 1 in sex identification of new varieties of ovate pomfret.
7. Use of the method according to any one of claims 2 to 5 in sex identification of new varieties of oval pomfret.
8. Use of the primers according to claim 1 in distinguishing new varieties of ovate pomfret from wild ovate pomfret.
9. Use of the method according to any one of claims 2 to 5 in distinguishing new varieties of oval pomfret from wild oval pomfret.