Molecular markers and products for screening disease-resistant p. spinosus
By identifying SNP molecular markers and primers in the SOCS5b gene of *Catella suis*, the problem of screening *Catella suis* resistant to Aeromonas hydrophila septicemia in existing technologies has been solved, enabling rapid and accurate breeding of disease-resistant varieties.
Patent Information
- Application Number
- CN202410959744.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-17
- Publication Date
- 2025-11-28
- Estimated Expiration
- 2044-07-17
AI Technical Summary
There is a lack of effective molecular markers in the current technology for screening catfish thuringiensis resistant to bacterial septicemia caused by Aeromonas hydrophila. Traditional breeding methods are time-consuming and inaccurate, and cannot meet the need for rapid breeding of disease-resistant varieties.
SNP molecular markers associated with resistance to bacterial septicemia were discovered in the SOCS5b gene of *Catella suis*. Corresponding primers were designed to identify and screen disease-resistant *Catella suis* by detecting the genotype of the SNP loci, providing corresponding detection products and breeding methods.
This method enables rapid and accurate screening and breeding of *Catfish suis* resistant to bacterial septicemia, avoiding the time and subjective errors of traditional breeding and promoting the development of disease-resistant varieties.
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Figure CN118834960B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of screening of fish molecular markers and breeding of disease-resistant varieties. More specifically, it relates to a molecular marker for screening disease-resistant Pangasianodon hypophthalmus and a product. BACKGROUND
[0002] Bacterial septicemia caused by Aeromonas hydrophila seriously endangers the development of Pangasianodon hypophthalmus aquaculture and brings serious economic losses to breeders. Although antibiotics can have some preventive and therapeutic effects on bacterial diseases, the large-scale use of antibiotics can lead to the emergence of antibiotic-resistant bacteria, antibiotic residues, and environmental pollution. To solve the above problems caused by antibiotics, on the one hand, antibiotic substitutes need to be developed, and on the other hand, varieties resistant to bacterial septicemia need to be selected.
[0003] Traditional breeding methods mainly rely on visual observation of phenotypic variation of breeding materials, and screening of breeding materials with desired traits for production is based on experience and subjective judgment. Such methods require a long time for selection and breeding of varieties. Molecular marker-assisted breeding is a method that uses the characteristics of molecular markers being closely linked to genes determining target traits to select target traits by detecting molecular markers, which has the advantages of being fast, accurate, and not affected by environmental conditions. However, there are few reported molecular markers related to bacterial septicemia resistance in Pangasianodon hypophthalmus, which cannot meet the needs of biological breeding. To promote the breeding of bacterial septicemia-resistant Pangasianodon hypophthalmus varieties, it is still necessary to continuously explore related molecular markers. SUMMARY
[0004] The present application provides a molecular marker for screening disease-resistant Pangasianodon hypophthalmus and a product to address the lack of molecular markers related to bacterial septicemia resistance in Pangasianodon hypophthalmus.
[0005] The first object of the present application is to provide a molecular marker for screening disease-resistant Pangasianodon hypophthalmus.
[0006] The second object of the present application is to provide a primer for detecting the genotype of the SNP site of the molecular marker.
[0007] The third object of the present application is to provide the use of the primer in the preparation of a product for identifying the bacterial septicemia resistance trait of Pangasianodon hypophthalmus.
[0008] The fourth object of the present application is to provide a method for identifying the bacterial septicemia resistance trait of Pangasianodon hypophthalmus.
[0009] A fifth object of the present application is to provide use of the primer in the preparation of a product for screening P. spinibarba against bacterial septicemia.
[0010] A sixth object of the present application is to provide a method for screening P. spinibarba against bacterial septicemia.
[0011] A seventh object of the present application is to provide a product for screening P. spinibarba against disease.
[0012] An eighth object of the present application is to provide use of the product in marker-assisted breeding of P. spinibarba against bacterial septicemia.
[0013] The above objects of the present application are achieved by the following technical solutions.
[0014] The present application finds a SNP molecular marker related to the trait of P. spinibarba against bacterial septicemia in the SOCS5b gene of P. spinibarba, which can be used for identification of the trait of P. spinibarba against bacterial septicemia and screening of P. spinibarba with stronger disease resistance. Based on the molecular marker, the present application further provides a primer for detecting the genotype of the SNP site, which can be used to prepare a product for screening P. spinibarba against bacterial septicemia, which is conducive to the breeding of P. spinibarba against bacterial septicemia. Therefore, the present application claims the molecular marker and related detection products and applications.
[0015] The present application provides a molecular marker for screening P. spinibarba against disease, which is a SNP molecular marker, and the SNP site is located at the 21496497th nucleotide site of the reference genome CM018543.1 of P. spinibarba, and the polymorphism is C / T.
[0016] Specifically, the SNP molecular marker is located in the SOCS5b gene of P. spinibarba, and the SNP site of the SNP molecular marker is located at the 139th position of the sequence with reference to the SOCS5b gene of P. spinibarba shown in SEQ ID NO. 3.
[0017] Specifically, the disease resistance is against bacterial septicemia.
[0018] Specifically, the bacterial septicemia is caused by infection of Aeromonas hydrophila.
[0019] The present application further provides a primer for detecting the genotype of the SNP site of the SNP molecular marker.
[0020] As an optional implementation, the nucleotide sequence of the primer is shown in SEQ ID NO. 1-2.
[0021] As an optional implementation, the primer is an SNaPshot typing primer, and the nucleotide sequence of the SNaPshot typing primer is shown as SEQ ID NO. 4-5.
[0022] The primer can be used to detect the SNP site genotype, thereby identifying the bacterial septicemia resistance trait of the P. sutchi. Therefore, the application of the primer in the preparation of a product for identifying the bacterial septicemia resistance trait of the P. sutchi is protected.
[0023] The application further provides a method for identifying the bacterial septicemia resistance trait of the P. sutchi, which comprises the following steps: detecting the SNP site genotype of the SNP molecular marker; and if the SNP site genotype of the P. sutchi is CC, the P. sutchi has the bacterial septicemia resistance trait; and if the SNP site genotype of the P. sutchi is TC or TT, the P. sutchi does not have the bacterial septicemia resistance trait.
[0024] The application further protects the application of the primer in the preparation of a product for screening the P. sutchi with the bacterial septicemia resistance trait.
[0025] The application further provides a method for screening the P. sutchi with the bacterial septicemia resistance trait, which comprises the following steps: detecting the SNP site genotype of the SNP molecular marker, and selecting the P. sutchi with the SNP site genotype of CC.
[0026] The application further provides a product for screening the P. sutchi with the bacterial septicemia resistance trait, and the product contains the primer.
[0027] Specifically, the bacterial septicemia resistance trait is the bacterial septicemia resistance trait.
[0028] The application further protects the application of the product in the molecular marker assisted breeding of the P. sutchi with the bacterial septicemia resistance trait.
[0029] The application has the following beneficial effects:
[0030] The application provides a molecular marker that can be used for screening the P. sutchi with the bacterial septicemia resistance trait, and the molecular marker is a SNP molecular marker found in the SOCS5b gene of the P. sutchi, the SNP site of which is located at the 21496497th nucleotide site of the P. sutchi reference genome CM018543.1, and the polymorphism is C / T. The molecular marker is related to the bacterial septicemia resistance trait of the P. sutchi, and the application further provides a corresponding detection product based on the molecular marker, which can be used for screening the P. sutchi with the bacterial septicemia resistance trait, so as to promote the breeding of the P. sutchi with the bacterial septicemia resistance trait. BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 The sequencing peak map of the SNP site of the SNP molecular marker; the position indicated by the arrow in the figure is the SNP site, the single peak indicates the homozygous genotype, and the double peaks indicate the heterozygous genotype; the genotype corresponding to a in the figure is CC type; the genotype corresponding to b is TC type; and the genotype corresponding to c is TT type. DETAILED DESCRIPTION
[0032] The present application will be further described below in conjunction with the drawings of the specification and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.
[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
[0034] Example 1 PCR amplification of SOCS5b gene and screening and typing of SNP site
[0035] 1. Collection and processing of samples
[0036] The animals used in the experiments of the present application are Pimephales promelas weighing about 100 g per tail. After the collected Pimephales promelas is fished from the breeding barrel to a small barrel for stable cultivation, 0.2 mL of Aeromonas hydrophila bacterial solution with a concentration of 1×10 7 cfu / mL is injected into each tail to artificially induce infection; Pimephales promelas individuals that die within 24 h after infection are collected as a susceptible group, a total of 20 individuals; Pimephales promelas individuals that still have no infection symptoms after 7 d of infection are collected as a resistant group, a total of 20 individuals; the fin rays of the collected Pimephales promelas in the susceptible group and the resistant group are cut and stored in anhydrous ethanol.
[0037] 2. Obtaining of SOCS5b gene
[0038] The SOCS5b gene is obtained by PCR amplification and sequencing in the present application.
[0039] (1) Design of PCR amplification primer
[0040] The PCR amplification primer for amplifying the SOCS5b gene is designed according to the DNA (NCBI GeneID: 113542077) of the SOCS5b gene of Pimephales promelas published in the GenBank database, and the nucleotide sequence of the designed PCR amplification primer is shown in Table 1.
[0041] Table 1 PCR amplification primer of SOCS5b gene of Pimephales promelas
[0042]
[0043] (2) PCR amplification and sequencing of SOCS5b gene
[0044] Genomic DNA of the collected susceptible group and resistant group P. sutchi samples was extracted respectively, and the obtained DNA was diluted to 100 ng / μL after detecting the concentration by ultraviolet spectrophotometer.
[0045] The diluted genomic DNA was used as a template to amplify the SOCS5b gene by using the PCR amplification primers shown in Table 1, and the PCR reaction system used for amplification is shown in Table 2.
[0046] Table 2 PCR reaction system
[0047]
[0048]
[0049] PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles; 72℃ extension for 5 min.
[0050] The PCR amplification product was checked for purity and integrity by 1% agarose gel electrophoresis, and the product with a single and bright band was selected to be sequenced by Guangzhou Tianyi Huirong Gene Technology Co., Ltd. According to the sequencing results, the nucleotide sequence of the SOCS5b gene of P. sutchi is shown as SEQ ID NO. 3.
[0051] 3. Screening and typing of SNP sites
[0052] The sequencing results of the PCR amplification products of the susceptible group and resistant group P. sutchi samples were compared and analyzed with the reference sequence (NCBI Gene ID: 113542077), and the same site with a proportion of different bases greater than 1 / 3 was determined as a SNP site. Through comparative analysis, the present application obtained one SNP site in the SOCS5b gene of P. sutchi, which was named SNP site 1 (SNP1C>T), and its nucleotide site was at 21496497 in the reference genome CM018543.1 of P. sutchi. If the SOCS5b gene shown in SEQ ID NO. 3 is used as a reference, the SNP site is located at its 139th position, and the polymorphism is C / T. The SNP site is located in the exon region of the SOCS5b gene, participates in encoding amino acids, but does not change the type of amino acids, which is a SNP synonymous mutation. The relevant information is shown in Table 3.
[0053] Table 3 Related information of SNP site of SOCS5b gene of P. sutchi
[0054]
[0055] The genotype of the SNP site is determined by the sequencing peak analysis simultaneously, and the sequencing peak chart of SNP1 C>T is shown in the following figure. Figure 1 As shown in the figure, SNP1 C>T has three genotypes, which are CC / TC / TT. Figure 1
[0056] According to the results shown in the genotype statistical table, the genotype of the SNP site and the bacterial septicemia resistance of P. spinosus are verified by using the General Linear Model (GLM) program and t test of SPSS analysis software, and the variance analysis test shows significant difference, and the Duncan method is used for multiple comparisons.
[0057] The allele and genotype of the SNP site in the susceptible group and the resistant group of P. spinosus are shown in Table 4. As shown in Table 4, the frequency of the SNP site allele T / C is significantly different (p<0.05) in the susceptible group and the resistant group of P. spinosus, and the disease resistance of P. spinosus with genotype CC is significantly higher than that of P. spinosus with genotype TC or TT (p<0.05), and the SNP molecular marker can be used to distinguish the susceptible and resistant groups.
[0058] Table 4 Allele and genotype of SNP site in susceptible group and resistant group
[0059]
[0060] Example 2 Genotype typing of SNP site and correlation verification with bacterial septicemia resistance
[0061] 1. Genotype typing of SNP site
[0062] The same method as in Example 1 is used, and 211 susceptible P. spinosus samples and 215 resistant P. spinosus samples are collected. The genotype of the SNP site in the collected 426 P. spinosus samples is detected by SNaPshot typing method.
[0063] According to the sequence information of the SNP site in Example 1, the corresponding SNaPshot typing primer is designed, as shown in Table 5.
[0064] Table 5 SNaPshot typing primer
[0065]
[0066] Genomic DNA of the 426 P. szechuanense samples was extracted respectively, and the obtained genomic DNA was used as a template to perform PCR amplification with the SNaPshot typing primers shown in Table 5, and the PCR amplification reaction system is shown in Table 6.
[0067] Table 6 PCR amplification reaction system of SNaPshot
[0068]
[0069] PCR amplification reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 20 s, 60°C annealing for 30 s, 72°C extension for 30 s, a total of 35 cycles; 72°C final extension for 3 min. After the PCR amplification reaction was completed, the PCR amplification product was treated with SAP (shrimp alkaline phosphatase), and the free dNTPs in the system were removed. After the shrimp alkaline phosphatase treatment was completed, a single base extension reaction was performed, and the reaction system is shown in Table 7.
[0070] Table 7 Reaction system of single base extension reaction
[0071]
[0072] Single base extension reaction conditions: 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 52°C annealing for 5 s, 60°C extension for 5 s, a total of 35 cycles; 72°C final extension for 3 min.
[0073] The reaction product was sequenced and analyzed by using a 96-channel full-automatic ABI 3730xL genetic analyzer. The.fsa format raw data was exported from the 3730xL instrument, classified and archived according to the detection reaction, and then imported into the GeneMarker analysis software. According to the signal value and peak situation of each site, the primer ratio of each site was adjusted to ensure that the signal value and peak situation of each site were basically consistent. According to the sample and site situation, the typing results of each site of the sample were obtained, and a genotype statistical table was exported.
[0074] 2. Association verification
[0075] According to the results shown in the genotype statistical table, the genotype of the SNP site and the anti-bacterial septicemia trait of P. szechuanense were verified by using the general linear model (GLM) program and t test of the SPSS analysis software. The characteristics of significant difference were tested by variance analysis, and multiple comparisons were performed by using the Duncan method.
[0076] The allelic and genotypic conditions of the SNP site in the susceptible population and the resistant population of P. steindachneri are shown in Table 8. As shown in Table 8, the disease resistance of P. steindachneri with the SNP site genotype CC is significantly higher than that of P. steindachneri with the SNP site genotype TC or TT (p<0.05), indicating that the SNP molecular marker of the present application can indeed distinguish the susceptible and resistant populations.
[0077] Table 8 Allelic and genotypic conditions of the SNP site in the susceptible and resistant populations
[0078]
[0079] Example 3 Genetic analysis of the SNP site
[0080] The genetic analysis results of the SNP site of the SOCS5b gene of P. steindachneri according to the present application are shown in Table 9. As shown in Table 9, the SNP site SNP1 C>T is a moderate polymorphism (0.15<PIC<0.5) in the susceptible population and the disease-resistant population, indicating that SNP1 C>T can provide reasonable genetic information as a genetic marker.
[0081] Table 9 Genetic information of the SNP site of the SOCS5b gene of P. steindachneri
[0082]
[0083] Note: Na is the number of alleles; He is the observed heterozygosity; Ne is the expected heterozygosity; and PIC is the polymorphic information content.
[0084] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.
Claims
1. A method of identifying a bacterial sepsis resistant trait in Pseudopimplus sp. characterized in that, Detecting the SNP site genotype of the SNP molecular marker; the SNP site of the SNP molecular marker is located at the nucleotide site 21496497 of Pseudobagrus vandeveerdenu reference genome CM018543.1, and the polymorphism is C / T; the disease resistance of Pseudobagrus vandeveerdenu with the SNP site genotype of CC to bacterial septicemia is significantly higher than that of Pseudobagrus vandeveerdenu with the SNP site genotype of TC or TT; the bacterial septicemia is caused by Aeromonas hydrophila infection.
2. A method of selecting more resistant S. soldatii against bacterial septicemia, characterized in that, Detecting the SNP site genotype of the SNP molecular marker, selecting Pseudobagrus vandeveerdenu with the SNP site genotype of CC, and the disease resistance of Pseudobagrus vandeveerdenu with the SNP site genotype of CC to bacterial septicemia is significantly higher than that of Pseudobagrus vandeveerdenu with the SNP site genotype of TC or TT; the SNP site of the SNP molecular marker is located at the nucleotide site 21496497 of Pseudobagrus vandeveerdenu reference genome CM018543.1, and the polymorphism is C / T; the bacterial septicemia is caused by Aeromonas hydrophila infection.