Molecular marker primer for identifying genetic sex of centrarchus macrocephalus, identification method and application

Abstract

This invention relates to molecular marker primers, identification methods, and applications for identifying the genetic sex of pikeperch, belonging to the field of aquatic animal molecular genetics and breeding technology. This invention solves the problems of early sex identification in pikeperch, including inability to identify sex early on, high invasiveness, poor timeliness, low efficiency, and limited accuracy. It describes the application of three pairs of specific primers for identifying the genetic sex of pikeperch. The method involves either individual or combined identification, with separate amplification for combined identification, and differentiation between genetically female and male individuals based on the amplification results. The molecular markers of this invention offer multiple verification mechanisms, strong early identification capabilities, low detection costs, simple operation, non-invasiveness, high stability, wide applicability, and high throughput, making them suitable for large-scale commercial applications.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and breeding technology for aquatic animals, specifically involving molecular markers, identification methods, and applications for identifying the genetic sex of pikeperch. Background Technology

[0002] The pikeperch (Sander lucioperca) belongs to the order Perciformes, family Percidae, and genus Percioperca. Originally distributed in rivers and lakes of the Aral Sea, Black Sea, Caspian Sea, and Baltic Sea basins in Eurasia, it is an important freshwater aquaculture species in Europe and Central Asia. A large carnivorous fish, the pikeperch is stout, with thick, tender flesh, no intramuscular bones, rapid growth, and strong adaptability, exhibiting a high degree of dispersal ability. In my country, pikeperch populations originated from the natural dispersal of the border river Irtysh River and the border lake Khanka, subsequently forming stable populations in lakes such as Ulungur and Khanka. Breeding and aquaculture of pikeperch in my country began in the 1990s, with broodstock sourced from the Ulungur Lake population. Some provinces and cities cultivate pikeperch as a premium fish species.

[0003] Female pikeperch grow significantly faster than males, reach sexual maturity later, have a longer breeding cycle, and are larger in market size. Achieving all-feminized breeding can effectively increase breeding yield by 20-30%, which is a key technology for improving the quality and efficiency of the pikeperch industry. However, female and male pikeperch cannot be distinguished by appearance, and there are no obvious secondary sexual characteristics during the breeding season, which brings inconvenience to genetic breeding. At present, the sex identification of pikeperch mainly relies on dissection to observe the gonad morphology. This method has the following prominent defects: (1) Highly invasive: dissection leads to the death of the fish and cannot be used for the preservation of parent stock; (2) Poor timeliness: the gonads of juvenile fish (<60 days old) are not differentiated and cannot be identified; (3) Low efficiency: not suitable for large-scale population screening; (4) Limited accuracy: highly dependent on experience and has a high misjudgment rate.

[0004] Molecular marker technology offers a new approach for sex identification in fish. While single nucleotide polymorphism (SNP) markers have been used for sex identification, detection relies on techniques such as sequencing, fluorescent probes, or high-resolution melting curves (HRM), which are demanding in terms of equipment and cost. Insertion / deletion (InDel) markers offer advantages such as abundance, wide distribution, and intuitive detection (based on fragment length differences), but research on sex-related InDel markers in pikeperch is still lacking. The current lack of efficient, economical, and accurate molecular sex identification methods suitable for pikeperch hinders the industrial application of pikeperch parthenogenesis breeding technology. Summary of the Invention

[0005] To address the problems of early sex identification in pikeperch, such as inability to identify sex, high invasiveness, poor timeliness, low efficiency, and limited accuracy, this invention provides molecular marker primers, identification methods, and applications for identifying the genetic sex of pikeperch.

[0006] This invention provides specific primer pairs for identifying the genetic sex of pikeperch using molecular markers, wherein the molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel73, the forward primer SL-Sex-InDel73 F is: 5'-CAAACACCCTCAAACAGCCC-3', and the reverse primer SL-Sex-InDel73 R is: 5'-GTTTATGCGTACCTGCTGCC-3'.

[0007] The molecular marker for identifying the genetic sex of pikeperch in this invention is SL-Sex-InDel73, whose nucleotide sequence is SL-Sex-InDel73A or SL-Sex-InDel73B, wherein SL-Sex-InDel73A is shown in SEQ ID NO.1 and SL-Sex-InDel73B is shown in SEQ ID NO.2; this molecular marker is located on chromosome 3 of the pikeperch reference genome, and SEQ ID NO.2 has a 73 bp fragment deleted from positions 59 to 131 bp compared to SEQ ID NO.1.

[0008] This invention provides specific primer pairs for identifying the genetic sex of pikeperch using molecular markers, wherein the molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel34, the forward primer SL-Sex-InDel34 F: 5'-AGGTAGTCAAGAGCCCACAC-3', and the reverse primer SL-Sex-InDel34 R: 5'-GAATTAACCACTCGGGGCTGT-3'.

[0009] The molecular marker for identifying the genetic sex of pikeperch in this invention is SL-Sex-InDel134, whose nucleotide sequence is SL-Sex-InDel134A or SL-Sex-InDel134B. SL-Sex-InDel134A is shown in SEQ ID NO.3, and SL-Sex-InDel134B is shown in SEQ ID NO.4. This molecular marker is located on chromosome 3 of the pikeperch reference genome. SEQ ID NO.4 has inserted fragments of 4 bp and 1 bp at positions 27 bp and 48 bp, respectively, compared to SEQ ID NO.3. A fragment of 39 bp is deleted at positions 105-143 bp. The sequence length of SEQ ID NO.4 is 34 bp shorter than that of SEQ ID NO.3.

[0010] This invention relates to specific primer pairs for identifying the genetic sex of pikeperch using molecular markers. The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel66. The forward primer of the primer pair is SL-Sex-InDel66 F: 5'-AGAGACCTCAGTTCCTGACGA-3', and the reverse primer of the primer pair is SL-Sex-InDel66 R: 5'-GTAACTGCAAGAAAGGGGCAT-3'.

[0011] The molecular marker for identifying the genetic sex of pikeperch in this invention is SL-Sex-InDel166, whose nucleotide sequence is SL-Sex-InDel66A or SL-Sex-InDel66B, wherein SL-Sex-InDel66A is shown in SEQ ID NO.5 and SL-Sex-InDel66B is shown in SEQ ID NO.6; this molecular marker is located on chromosome 3 of the pikeperch reference genome, and SEQ ID NO.6 has a 66 bp fragment inserted at position 297 bp relative to SEQ ID NO.5.

[0012] The method for identifying the genetic sex of pikeperch according to the present invention is carried out according to the following steps:

[0013] 1. Collect samples to be tested and extract genomic DNA;

[0014] 2. Amplify SL-Sex-InDel73, SL-Sex-InDel34 and / or SL-Sex-InDel66 using the specific primers described above to obtain PCR amplification products;

[0015] 3. Agarose gel electrophoresis is used to analyze genotype data in the amplified pattern. Based on the specific insertion or deletion of allele fragments in the genetically male individuals, the genetically female and male individuals can be distinguished and identified.

[0016] Furthermore, in the SL-Sex-InDel73 primer amplification pattern, individuals amplifying a 335 bp band were female, while those amplifying 335 bp and 262 bp bands were male. Male individuals exhibited a specific 73 bp deletion compared to female individuals. In the SL-Sex-InDel34 amplification pattern, individuals amplifying a 285 bp band were female, while those amplifying 285 bp and 251 bp bands were male. Male individuals exhibited a specific 34 bp deletion compared to female individuals. In the SL-Sex-InDel66 amplification pattern, individuals amplifying a 450 bp band were female, while those amplifying 450 bp and 516 bp bands were male. Male individuals exhibited a specific 66 bp insertion compared to female individuals.

[0017] The application of the molecular marker-specific primers described in this invention in identifying the genetic sex of pikeperch.

[0018] Furthermore, the specific primer pairs SL-Sex-InDel73, SL-Sex-InDel34, and SL-Sex-InDel66 were used for genetic sex identification in pikeperch, employing either individual or combined identification methods. In combined identification, each primer pair was amplified separately, and the genetic distinction between male and female individuals was made based on the amplification results.

[0019] The kit for detecting the genetic sex of pikeperch of the present invention contains specific primer pairs for the molecular markers SL-Sex-InDel73, SL-Sex-InDel34 or SL-Sex-InDel66 used to identify the genetic sex of pikeperch, PCR reaction reagents, a positive control and a negative control. The PCR reaction reagents are Taq DNA polymerase, reaction buffer and ddH2O. The positive control is a male pikeperch DNA template and the negative control is a female pikeperch DNA template.

[0020] The present invention has the following beneficial effects:

[0021] 1. Multiple validation mechanism: Cross-validation with 3 independent InDel markers, achieving 100% accuracy, significantly outperforming single-marker systems;

[0022] 2. Strong early identification capability: Applicable to juvenile fish aged 25-30 days, more than 60 days earlier than traditional methods;

[0023] 3. Low detection cost: No sequencing or fluorescent probes are required, and the cost per sample is less than 2 yuan;

[0024] 4. Simple operation: Only a PCR instrument and electrophoresis equipment are needed, and it can be carried out in ordinary laboratories;

[0025] 5. Non-invasive: A small amount of fin tissue (5-10 mg) does not affect the survival and growth of the fish;

[0026] 6. High stability: The InDel mutation is genetically stable and there is no risk of reversion mutation;

[0027] 7. Wide applicability: Suitable for different geographical populations, farmed populations, and wild populations;

[0028] 8. High throughput: A single PCR reaction takes only 2-3 hours, making it suitable for large-scale commercial applications.

[0029] This invention screens for insertion / deletion allele sequences on the Y chromosome relative to the X chromosome from comparative genomic sequences of female and male pikeperch individuals. Specifically, it identifies three InDel markers in the pikeperch genome, which, compared to the X chromosome, amplify specific insertion / deletion fragments on the Y chromosome. This allows for rapid and accurate differentiation between female and male pikeperch individuals, with a comprehensive identification accuracy rate as high as 99.5%. This invention requires only a small amount of fin tissue and can achieve rapid and accurate identification of the genetic sex of pikeperch through conventional PCR amplification and agarose gel electrophoresis, resulting in low detection costs. This invention solves the technical problems of early sex identification in pikeperch and the high invasiveness of traditional methods, and has significant application value in single-sex breeding, sex ratio regulation, breeding of superior varieties, and research on sex determination mechanisms.

[0030] The molecular markers, primers, and reagent kits for identifying the genetic sex of pikeperch described in this invention establish a rapid and reliable technique for sex identification in pikeperch. This technique can be applied to the identification of genetic sex in pikeperch asexual breeding and is of great significance to the development of the pikeperch aquaculture industry. Attached Figure Description

[0031] Figure 1 Sequence alignment diagram of SL-Sex-InDel73 molecular markers;

[0032] Figure 2 Sequence alignment diagram of SL-Sex-InDel34 molecular markers;

[0033] Figure 3 Sequence alignment diagram of SL-Sex-InDel66 molecular markers;

[0034] Figure 4 Electrophoresis results for SL-Sex-InDel73;

[0035] Figure 5 Electrophoresis results for SL-Sex-InDel34;

[0036] Figure 6 Electrophoresis results for SL-Sex-InDel66;

[0037] Figure 7 This is a representative electrophoresis result image. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0040] Example 1: Development and Detection Method of Specific InDel Molecular Markers for Identifying the Genetic Sex of Pikeperch

[0041] Twelve male and female pikeperch broodstock samples were collected from the Hulan test site of the Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences. Blood was extracted, and genomic DNA was extracted using the phenol-chloroform extraction method. The resulting DNA solution was subjected to electrophoresis on a 1% agarose gel to check DNA integrity. DNA concentration was measured using a Nanodrop 2000 micro-volume spectrophotometer (Thermo Scientific), and DNA quality was assessed by the A260 nm / A280 nm ratio. Library construction and genome resequencing were performed by the BGI Qingdao Institute. A brief experimental procedure is as follows: DNA samples that passed quality control were randomly fragmented, target fragments were screened and purified, sequencing adapters were ligated, and DNA nanoballs (DNBs) were prepared using rolling circle amplification (RCA) technology. The prepared DNBs were loaded onto an arrayed sequencing chip, and high-throughput sequencing was performed using an MGISEQ series sequencer. The raw sequencing data was filtered for adapters and low-quality data using SOAPnuke (v2.1.5) to obtain clean data. The filtered clean reads were then aligned to the reference genome (GCA_008315115.2) using the "MEM" algorithm in BWA alignment software. Sequence alignment between males and females yielded 300 significantly different sequences of 1000 bp each. Further analysis identified three sequences located on chromosome 3 with significant length differences between the X and Y chromosomes, which can be used to identify male and female pikeperch individuals. Figure 1 The image shows the sequence alignment of the SL-Sex-InDel73 molecular marker (the aligned sequences are SEQ ID NO.1 and SEQ ID NO.2). Figure 2 The image shows the sequence alignment of the SL-Sex-InDel34 molecular marker (the aligned sequences are SEQ ID NO.3 and SEQ ID NO.4). Figure 3 The sequence alignment diagram of the SL-Sex-InDel66 molecular marker (the aligned sequences are the reverse complementary sequences of SEQ ID NO.5 and SEQ ID NO.6). Figure 1 , Figure 2 , Figure 3Insertion / deletion differences between male and female individuals were shown. Primers were designed for the three sequences using the Primer 3 online tool and used as InDel markers (Table 1).

[0042] Table 1 InDel-labeled primer information

[0043]

[0044] Example 2: Identification of genetic sex in pikeperch using three InDel molecular markers alone.

[0045] Twenty-four parent fish of known sex were collected from a group of pikeperch at the Hulan test site of the Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences. Fin samples were taken, and genomic DNA was extracted and amplified using three InDel markers. Figure 4 Electrophoresis results for SL-Sex-InDel73 Figure 5 Electrophoresis results for SL-Sex-InDel34 Figure 6 The electrophoresis results for SL-Sex-InDel66 Figure 4 , Figure 5 and Figure 6 M: DNA Marker; female: female individual (single band); male: male individual (double band).

[0046] Figure 4 In the SL-Sex-InDel73 primer amplification pattern, individuals that amplified a 335 bp band were female, while individuals that amplified both 335 bp and 262 bp bands were male. Male individuals had a specific deletion of a 73 bp band compared to female individuals. Figure 5 In the SL-Sex-InDel34 amplification pattern, individuals amplifying a 285 bp band are female, while individuals amplifying both 285 bp and 251 bp bands are male. Male individuals have a specific deletion of a 34 bp band compared to female individuals. Figure 6 In the SL-Sex-InDel66 amplification pattern, individuals amplifying a 450 bp band are female, while individuals amplifying both 450 bp and 516 bp bands are male. Male individuals have a specific insertion of a 66 bp band compared to female individuals.

[0047] from Figure 4 , Figure 5 and Figure 6 It can be seen that the markers SL-Sex-InDel73, SL-Sex-InDel34, and SL-Sex-InDel66, whether used independently or in combination, can accurately identify the sex of the sample, with an accuracy rate of 100%.

[0048] Example 3: Preparation and application of a reagent kit containing the InDel molecular marker described in this invention.

[0049] 1. Kit preparation and usage methods

[0050] 1.1 Kit Components (100 tests):

[0051] 2×PCR Master Mix (containing Taq enzyme, dNTPs, Mg²⁺, and buffer): 1.0 mL

[0052] ddH2O: 1.0 mL

[0053] SL-Sex-InDel73 forward and reverse primers (10 μmol / L): 100 μL each

[0054] SL-Sex-InDel34 forward and reverse primers (10 μmol / L): 100 μL each

[0055] SL-Sex-InDel66 forward and reverse primers (10 μmol / L): 100 μL each

[0056] Positive control (male fish DNA, 50 ng / μL): 50 μL

[0057] Negative control (female fish DNA, 50 ng / μL): 50 μL

[0058] 1.2 Usage Instructions:

[0059] To prepare a 20 μL PCR reaction system, add the following to the PCR tube:

[0060] 2×PCR Master Mix 10.0 μL

[0061] ddH2O 6.4 μL

[0062] Forward primer (10 μM) 0.8 μL

[0063] Reverse primer (10 μM) 0.8 μL

[0064] 2.0 μL DNA template

[0065] PCR amplification: 95℃ for 8 min; 33×(94℃ for 30 s, 56℃ for 30 s, 72℃ for 45 s); 72℃ for 10 min

[0066] Take 5 μL of the product for 2.0–3.0% agarose gel electrophoresis.

[0067] Result Interpretation: Gender was determined according to the standards of Example 2.

[0068] 1.3 Storage conditions: Store at −20℃ for 12 months, avoiding repeated freeze-thaw cycles.

[0069] 2. Large-scale validation experiments were conducted using a kit containing the InDel molecular marker described in this invention.

[0070] 2.1 Experimental population: 300 pikeperch were randomly selected from the farmed population (100 from Heilongjiang, 100 from Shandong, and 100 from Liaoning), covering different ages (2-3 years old).

[0071] 2.2 Blind testing: The experimenters are unaware of the sex of the organism after dissection and conduct molecular identification independently.

[0072] 2.3 Detection Procedure: After DNA was extracted from each fish, PCR was performed using the three pairs of primers in the kit, followed by detection by 2.0% agarose gel electrophoresis.

[0073] 2.44 Result Interpretation Criteria:

[0074] Female: All three markers are single bands (335 / 285 / 450 bp).

[0075] Male: ≥2 markers showing double bands (335 / 262 bp, 285 / 251 bp, 516 / 450 bp)

[0076] 2.5 Anatomical Verification: All samples were dissected and examined after anesthesia to confirm the actual sex. Figure 7 The image shows a representative electrophoresis result (electrophoresis of a portion of the samples detected by SL-Sex-InDel73 labeling). In the image, M represents the DNA Marker; female represents female individuals (single band); and male represents male individuals (double band).

[0077] 2.6 The verification results are shown in Table 2 (Table 2 is a statistical summary of the sex identification results of 300 pikeperch):

[0078]

[0079] Combination Figure 7 As shown in Table 2, the accuracy of the combination of the three molecular markers described in this invention reaches 99.3%, which is significantly higher than that of single marker detection. Both false positives were male chimeras.

Claims

1. A specific primer pair for identifying the genetic sex of pikeperch, characterized in that, The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel73. The forward primer SL-Sex-InDel73 F is 5'-CAAACACCCTCAAACAGCCC-3', and the reverse primer SL-Sex-InDel73 R is 5'-GTTTATGCGTACCTGCTGCC-3'.

2. Molecular markers for identifying the genetic sex of pikeperch, characterized in that, The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel73, with nucleotide sequences of SL-Sex-InDel73A or SL-Sex-InDel73B. SL-Sex-InDel73A is shown in SEQ ID NO.1, and SL-Sex-InDel73B is shown in SEQ ID NO.

2. This molecular marker is located on chromosome 3 of the pikeperch reference genome. SEQ ID NO.2 has a 73 bp segment deleted from positions 59 to 131 bp compared to SEQ ID NO.

1.

3. A specific primer pair for identifying the genetic sex of pikeperch, characterized in that, The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel34, with the primer pair consisting of the forward primer SL-Sex-InDel34 F: 5'-AGGTAGTCAAGAGCCCACAC-3' and the reverse primer SL-Sex-InDel34 R: 5'-GAATTAACCACTCGGGGCTGT-3'.

4. Molecular markers for identifying the genetic sex of pikeperch, characterized in that, The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel134, whose nucleotide sequence is SL-Sex-InDel134A or SL-Sex-InDel134B. SL-Sex-InDel134A is shown in SEQ ID NO.3, and SL-Sex-InDel134B is shown in SEQ ID NO.

4. This molecular marker is located on chromosome 3 of the pikeperch reference genome. SEQ ID NO.4 has inserted fragments of 4 bp and 1 bp at positions 27 bp and 48 bp, respectively, and has deleted a fragment of 39 bp at positions 105-143 bp. The sequence length of SEQ ID NO.4 is 34 bp shorter than that of SEQ ID NO.

3.

5. A specific primer pair for identifying the genetic sex of pikeperch, characterized in that, The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel66, with the forward primer SL-Sex-InDel66 F: 5'-AGAGACCTCAGTTCCTGACGA-3' and the reverse primer SL-Sex-InDel66 R: 5'-GTAACTGCAAGAAAGGGGCAT-3'.

6. Molecular markers for identifying the genetic sex of pikeperch, characterized in that, The molecular marker for identifying the genetic sex of pikeperch is SL-Sex-InDel166, with nucleotide sequences of SL-Sex-InDel66A or SL-Sex-InDel66B, where SL-Sex-InDel66A is shown in SEQ ID NO.5 and SL-Sex-InDel66B is shown in SEQ ID NO.

6. This molecular marker is located on chromosome 3 of the pikeperch reference genome, and SEQ ID NO.6 has a 66 bp fragment inserted at position 297 bp relative to SEQ ID NO.

5.

7. A method for identifying the genetic sex of pikeperch using the molecular markers described in claims 2, 4 and / or 6, characterized in that... The method for determining the genetic sex of pikeperch is as follows:

1. Collect samples to be tested and extract genomic DNA; 2. Amplify SL-Sex-InDel73, SL-Sex-InDel34 and / or SL-Sex-InDel66 respectively using the specific primers described in claims 1, 3 and / or 5 to obtain PCR amplification products; 3. Agarose gel electrophoresis is used to analyze genotype data in the amplified pattern. Based on the specific insertion or deletion of allele fragments in the genetically male individuals, the genetically female and male individuals can be distinguished and identified.

8. The application of the specific primers of the molecular markers described in claim 1, 3 or 5 in identifying the genetic sex of pikeperch.

9. The application according to claim 8, characterized in that, The specific primer pairs SL-Sex-InDel73, SL-Sex-InDel34, and SL-Sex-InDel66 were used for genetic sex identification in pikeperch, employing either individual or combined identification methods. In combined identification, each primer pair was amplified separately, and the genetic distinction between male and female individuals was made based on the amplification results.

10. A kit for detecting the genetic sex of pikeperch, characterized in that, The kit contains specific primer pairs for identifying the genetic sex of pikeperch using molecular markers as described in claims 1, 3, or 5, PCR reaction reagents, a positive control, and a negative control. The PCR reaction reagents are Taq DNA polymerase, reaction buffer, and ddH2O. The positive control is a male pikeperch DNA template, and the negative control is a female pikeperch DNA template.

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