A SNP molecular marker associated with resistance to bacterial septicemia in Sussini catfish and its application

Abstract

The present invention discloses a SNP molecular marker associated with the bacterial septicemia resistance trait of Su's round-bellied catfish and its application. The SNP molecular marker of the present invention includes two SNP sites; wherein, SNP site 1 is located at the nucleotide site 20215478 of the Su's round-bellied catfish reference genome CM018548.1, and SNP site 2 is located at the nucleotide site 20215688 of the Su's round-bellied catfish reference genome CM018548.1, and the polymorphism of the two SNP sites is both A / G. Based on the SNP molecular marker of the present invention, the present invention also provides a method for screening Su's round-bellied catfish that is resistant to bacterial septicemia, that is, selecting Su's round-bellied catfish whose SNP site 1 allele is G and / or whose SNP site 2 genotype is AG. The molecular marker of the present invention can be used in molecular marker-assisted breeding of Su's round-bellied catfish resistant varieties to accelerate the selection of Su's round-bellied catfish varieties that are resistant to bacterial septicemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and more specifically relates to a SNP molecular marker associated with the bacterial sepsis resistance trait of Su's round-bellied catfish and its application. Background Art

[0002] Pangasianodon hypophthalmus is an important economic fish. In recent years, with the continuous growth of consumer demand, the aquaculture scale of Pangasianodon hypophthalmus has also been expanding.

[0003] Diseases are a major bottleneck limiting the long-term, stable development of the Su's round-bellied catfish aquaculture industry. Among them, bacterial septicemia, caused by infection with Aeromonas hydrophila, is one of the main diseases threatening the Su's round-bellied catfish aquaculture industry. Catfish suffering from bacterial septicemia experience symptoms such as congestion, ulcers, intestinal bleeding, pallor of the liver, ascites, and ulcers throughout the body. Once the disease develops, it is difficult to control and treat with medication, ultimately leading to mass mortality and significant economic losses for fish farmers. Breeding Su's round-bellied catfish varieties resistant to bacterial septicemia is an important approach to addressing this disease.

[0004] Discovering molecular markers associated with the Sussippino catfish's resistance to bacterial septicemia is fundamental to modern biological breeding, improving selection accuracy and shortening the breeding cycle. Single nucleotide polymorphisms (SNPs) are molecular markers commonly found in human and animal genomes. They primarily refer to DNA sequence polymorphisms caused by single nucleotide variations at the genomic level, such as deletions, transversions, insertions, and transitions, and are the most common type of heritable variation. However, molecular markers such as SNPs associated with Sussippino catfish's resistance to bacterial septicemia are randomly distributed in the genome, not evenly distributed across every gene. The primary challenge to overcome is how to obtain SNP molecular markers associated with Sussippino catfish's resistance to bacterial septicemia from the vast genome. Currently, there are few reports on molecular markers associated with Sussippino catfish's resistance to bacterial septicemia. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a SNP molecular marker associated with the bacterial sepsis resistance trait of Catfish Su's round belly and an application thereof.

[0006] The first object of the present invention is to provide a SNP molecular marker associated with the bacterial sepsis resistance trait of Catfish Su's.

[0007] The second object of the present invention is to provide a reagent for detecting the allele or genotype of the SNP molecular marker.

[0008] The third object of the present invention is to provide the use of the reagent in identifying the bacterial septicemia resistance trait of Catfish Su's.

[0009] The fourth object of the present invention is to provide the use of the reagent in preparing a product for identifying the bacterial septicemia resistance of catfish.

[0010] The fifth object of the present invention is to provide the use of the reagent in screening catfish resistant to bacterial sepsis.

[0011] The sixth object of the present invention is to provide the use of the reagent in preparing a product for screening catfish resistant to bacterial sepsis.

[0012] The seventh object of the present invention is to provide the use of the reagent in molecular marker-assisted breeding of Su's round-bellied catfish varieties resistant to bacterial sepsis.

[0013] The eighth object of the present invention is to provide the use of the reagent in preparing a product for molecular marker-assisted breeding of a Su's round-bellied catfish variety resistant to bacterial sepsis.

[0014] The ninth object of the present invention is to provide a product for identifying the bacterial septicemia resistance trait of Su's round belly catfish or for screening Su's round belly catfish resistant to bacterial septicemia.

[0015] The tenth object of the present invention is to provide a method for screening catfish resistant to bacterial sepsis.

[0016] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0017] The present invention discovers a single-nucleotide polymorphism (SNP) molecular marker in the TLR9 gene of Sussini catfish that is associated with its resistance to bacterial septicemia. The present invention also provides information on the relationship between the alleles or genotypes of the SNP molecular marker and the resistance to bacterial septicemia in Sussini catfish. This SNP molecular marker can be used to select varieties of Sussini catfish that are resistant to bacterial septicemia. Therefore, the present invention seeks protection for this SNP molecular marker and its use.

[0018] The present invention provides a SNP molecular marker associated with the bacterial septicemia resistance trait of Su's round-bellied catfish (caused by Aeromonas hydrophila infection), comprising SNP site 1 and / or SNP site 2; the SNP site 1 is located at the nucleotide site 20215478 of the Su's round-bellied catfish reference genome CM018548.1, and the polymorphism is A / G (SNP1A>G); the SNP site 2 is located at the nucleotide site 20215688 of the Su's round-bellied catfish reference genome CM018548.1, and the polymorphism is A / G (SNP2A>G).

[0019] The present invention also provides a reagent for detecting the allele or genotype of the SNP molecular marker, wherein the reagent contains a primer for amplifying the SNP molecular marker or a primer for detecting the genotype of the SNP molecular marker.

[0020] Specifically, the primers are designed with the sequence containing the SNP molecular marker of the present invention as the target, and the primers can amplify SNP site 1 and SNP site 2 simultaneously, or can amplify SNP site 1 and SNP site 2 separately.

[0021] As an optional embodiment, the present invention provides primers for amplifying SNP site 1 and SNP site 2, respectively, that is, the primers provided by the present invention for amplifying the SNP molecular marker described in claim 1 include two pairs of primers; wherein, the primers used to amplify SNP site 1 are TLR9-120F and TLR9-120R, and their sequences are shown in SEQ ID NO.2 and SEQID NO.3 respectively; the primers used to amplify SNP site 2 are TLR9-F and TLR9-R, and their sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0022] As an optional embodiment, the primers provided by the present invention for detecting the genotype of the SNP molecular marker are SNaPshot typing primers, including an amplification primer SNP1-P1F and an extension primer SNP1-E, whose sequences are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0023] Based on the SNP molecular marker of the present invention, the allele or genotype of the SNP molecular marker can be obtained by using a primer (or reagent) for amplifying the SNP molecular marker or a primer (or reagent) for detecting the genotype of the SNP molecular marker, and then the bacterial sepsis resistance trait of Su's round-bellied catfish can be identified or Su's round-bellied catfish resistant to bacterial sepsis can be obtained by screening.

[0024] Therefore, the present invention claims protection for the use of the reagent in identifying the bacterial sepsis resistance trait of Catfish Su's round belly.

[0025] The present invention also claims to protect the use of the reagent in preparing a product for identifying the bacterial septicemia resistance trait of catfish.

[0026] The present invention also claims to protect the use of the reagent in screening Su's round-bellied catfish resistant to bacterial sepsis.

[0027] The present invention also claims to protect the use of the reagent in preparing a product for screening catfish resistant to bacterial sepsis.

[0028] The present invention also claims to protect the use of the reagent in molecular marker-assisted breeding of Su's round-bellied catfish varieties resistant to bacterial sepsis.

[0029] The present invention also claims protection for the use of the reagent in preparing a product for molecular marker-assisted breeding of Su's round-bellied catfish species resistant to bacterial sepsis.

[0030] The present invention also provides a product for identifying the bacterial septicemia resistance trait of catfish Suckeri or for screening catfish Suckeri that are resistant to bacterial septicemia, which contains the reagent of the present invention.

[0031] Specifically, among the SNP molecular markers described in the present invention, the probability that the Su's round-bellied catfish population with the SNP site 1 allele G is resistant to bacterial septicemia is significantly higher than that of the Su's round-bellied catfish population with the allele A (P<0.05); the probability that the Su's round-bellied catfish with the SNP site 2 genotype AG is resistant to bacterial septicemia is significantly higher than that of the Su's round-bellied catfish with the genotype GG or AA (P<0.05). Therefore, when identifying the bacterial septicemia resistance trait of Su's round-bellied catfish, if the SNP site 1 allele of the Su's round-bellied catfish tested is G and / or the SNP site 2 genotype is AG, it is more likely to have the bacterial septicemia resistance trait. When screening Su's round-bellied catfish that are resistant to bacterial septicemia, Su's round-bellied catfish with the SNP site 1 allele containing G and / or the SNP site 2 genotype being AG should also be selected.

[0032] The present invention also provides a method for screening Su's round-bellied catfish resistant to bacterial sepsis, the method comprising: detecting the allele or genotype of the SNP molecular marker in the tested Su's round-bellied catfish, and selecting Su's round-bellied catfish with SNP site 1 allele G and / or SNP site 2 genotype AG.

[0033] Alternatively, the primers shown in SEQ ID NOs. 2 and 3 and the primers shown in SEQ ID NOs. 5 and 6 of the present invention may be used to amplify the SNP molecular marker.

[0034] Optionally, the genotype of the SNP molecular marker can be obtained by using the SNaPshot typing primers shown in SEQ ID NOs. 7 and 8 of the present invention in combination with a SNaPshot sequencing analyzer.

[0035] The present invention has the following beneficial effects:

[0036] The present invention discovered a SNP molecular marker in the TLR9 gene of Su's catfish that is associated with its resistance to bacterial septicemia. The SNP molecular marker includes two SNP sites; among them, SNP site 1 is located at nucleotide position 20215478 of the Su's catfish reference genome CM018548.1, and SNP site 2 is located at nucleotide position 20215688 of the Su's catfish reference genome CM018548.1. The polymorphisms of the two SNP sites are both A / G. Based on the SNP molecular markers of the present invention, the present invention also provides a method for screening Su's catfish that is resistant to bacterial septicemia, that is, selecting Su's catfish whose SNP site 1 allele is G and / or whose SNP site 2 genotype is AG. The molecular markers of the present invention can be used in molecular marker-assisted breeding of Su's catfish resistant varieties to accelerate the selection of Su's catfish varieties that are resistant to bacterial septicemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the sequencing information of SNP1 A>G; the genotype corresponding to a in the figure is AG type; the genotype corresponding to b is GG type; and the genotype corresponding to c is AA type.

[0038] Figure 2 This is the sequencing information of SNP2 A>G; the genotype corresponding to a in the figure is AG type; the genotype corresponding to b is AA type; and the genotype corresponding to c is GG type. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0041] Example 1 Cloning of the TLR9 gene sequence of Catfish Su's round belly and screening and typing of SNP site 1

[0042] 1. Collection of samples of susceptible and resistant catfish

[0043] Several healthy Su's round-bellied catfish weighing about 100 g were collected from an aquaculture farm. After artificial infection with Aeromonas hydrophila was injected, their survival status was observed. Twenty Su's round-bellied catfish from each of the susceptible and resistant groups were collected for SNP site screening.

[0044] The specific process is as follows: about 100g of Su's round-bellied catfish collected from aquaculture farms were salvaged from the culture barrels and placed in small barrels. After stabilization, they were injected with Aeromonas hydrophila to induce infection artificially. The injection concentration of Aeromonas hydrophila was 1×107 cfu / mL, the injection volume was 0.2mL / fish, and the culture water temperature was maintained at 27-29℃; the Su's round-bellied catfish that died within 24 hours of infection were collected as the susceptible group, and the Su's round-bellied catfish that still had no infection symptoms 7 days after infection were collected as the disease-resistant group; among them, 20 Su's round-bellied catfish were collected from the susceptible group, and 20 Su's round-bellied catfish were collected from the disease-resistant group.

[0045] Fin ray samples from susceptible and resistant groups of Su's round-bellied catfish were cut and preserved in anhydrous ethanol.

[0046] 2. PCR amplification of TLR9 gene sequence

[0047] (1) Design of PCR amplification primers

[0048] PCR primers for amplifying the TLR9 gene sequence were designed based on the TLR9 gene sequence of Catfish Su's published in the GenBank database (NCBI Gene ID: 113533129) (the amplified TLR9 gene sequence is shown in SEQ ID NO. 1). The designed PCR primers are shown in Table 1.

[0049] Table 1 PCR primers for amplifying the sequence shown in SEQ ID NO.1

[0050]

[0051] (2) PCR amplification of TLR9 gene sequence

[0052] The genomic DNA of the susceptible group and the disease-resistant group were extracted respectively. The concentration of the extracted genomic DNA was detected by ultraviolet spectrophotometer and then diluted to 100 ng / μL.

[0053] The diluted genomic DNA was used as a template to amplify the TLR9 gene sequence using the PCR amplification primers shown in Table 1. The PCR reaction system used for amplification is shown in Table 2.

[0054] Table 2 PCR reaction system

[0055]

[0056] PCR reaction conditions included pre-denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, followed by extension at 72°C for 5 min. PCR products were checked for purity and integrity using 1% agarose gel electrophoresis. Products with a single, bright band were selected and Sanger sequenced using an ABI 3730xl DNA Analyzer.

[0057] 3. Screening and typing of SNP site 1

[0058] The sequencing results of the PCR amplification products of the susceptible group and the disease-resistant group of Su's round-bellied catfish were compared and analyzed with the reference sequence (NCBIGene ID: 113533129), and the SNP sites were determined to have a ratio of different bases greater than 1 / 3 at the same site. After comparative analysis, the present invention obtained a SNP site (position 623 of the sequence shown in SEQ ID NO.1) in the sequence shown in SEQ ID NO.1, named SNP site 1 (SNP1A>G). Its nucleotide position in the Su's round-bellied catfish reference genome CM018548.1 is 20215478, located in the exon region of the TLR9 gene, and is not involved in encoding amino acids.

[0059] The relevant information is summarized in Table 3.

[0060] Table 3 Information on SNP site 1 in the exon region of the TLR9 gene in Catfish Su's round belly

[0061]

[0062] Note: “-” means no (the SNP site is not located in the CDS region and does not encode amino acids).

[0063] The present invention also determines the genotype of SNP site 1 by sequencing peak analysis. The sequencing information of SNP1 A>G is as follows: Figure 1 As shown. Figure 1 It can be seen that SNP1 T>G has three genotypes, namely AG / GG / AA.

[0064] The sequencing results of the PCR amplification products of the susceptible group and the disease-resistant group were imported into the GeneMarker analysis software. According to the signal value of each site and the peak situation, the primer ratio of each site was adjusted to ensure that the signal value of each site was basically consistent with the peak situation. Subsequently, statistics were performed according to the sample and site situation to obtain the typing results of each sample site and derive the genotype statistical table. According to the results shown in the genotype statistical table, the generalized linear model (GLM) program and t-test of SPSS analysis software were used to perform association analysis on the genotype of the SNP site 1 and the anti-bacterial septicemia trait of the Su's round belly catfish. The characteristics of the significant differences were tested by analysis of variance, and the Duncan method was used for multiple comparisons.

[0065] Table 4 shows the alleles and genotypes of SNP locus 1 in susceptible and resistant populations of Sussippino catfish. As shown in Table 4, the frequency of the G allele at SNP locus 1 in the resistant group (0.825) was significantly higher than that in the susceptible group (0.6), indicating that populations of Sussippino catfish carrying the G allele at SNP locus 1 had significantly increased resistance to bacterial septicemia (P < 0.05). Specifically, a higher frequency of the G allele at SNP locus 1 in a population of Sussippino catfish significantly increased the probability that the population was resistant to bacterial septicemia; conversely, a higher frequency of the A allele in a population of Sussippino catfish significantly decreased the probability that the population was resistant to bacterial septicemia (P < 0.05).

[0066] Table 4 Alleles and genotypes of SNP locus 1 in susceptible and resistant populations of Catfish Su's

[0067]

[0068] Example 2 Cloning of the TLR9 gene sequence of Catfish Su's round belly and screening and typing of SNP site 2

[0069] The catfish sample and genomic DNA template used in this example are the same as those in Example 1.

[0070] 1. PCR amplification of TLR9 gene sequence

[0071] (1) Design of PCR amplification primers

[0072] PCR primers for amplifying the TLR9 gene sequence were designed based on the TLR9 gene sequence of Catfish Su's published in the GenBank database (NCBI Gene ID: 113533129) (the amplified TLR9 gene sequence is shown in SEQ ID NO. 4). The designed PCR primers are specifically shown in Table 5.

[0073] Table 5 PCR primers for amplifying the sequence shown in SEQ ID NO.2

[0074]

[0075] (2) PCR amplification of TLR9 gene sequence

[0076] The TLR9 gene sequence was amplified using the same genomic DNA template as in Example 1 and the PCR amplification primers shown in Table 5. The PCR reaction system used for amplification was the same as that in Table 2.

[0077] PCR reaction conditions included pre-denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by extension at 72°C for 5 min. PCR products were checked for purity and integrity using 1% agarose gel electrophoresis. Products with a single, bright band were selected and Sanger sequenced using an ABI 3730xl DNA Analyzer.

[0078] 2. Screening and typing of SNP site 2

[0079] The sequencing results of the PCR amplification products of the susceptible and resistant groups of Sussini catfish were compared with the reference sequence (NCBI Gene ID: 113533129), and the SNP sites were identified if the ratio of different bases at the same site was greater than 1 / 3. After comparative analysis, the present invention obtained a SNP site (position 243 of the sequence described in SEQ ID NO.4) in the sequence shown in SEQ ID NO.4, named SNP site 2 (SNP2A>G). Its nucleotide position in the Sussini catfish reference genome CM018548.1 is 20215688, located in the exon region of the TLR9 gene, and does not participate in encoding amino acids.

[0080] The relevant information is summarized in Table 6.

[0081] Table 6 Information on SNP site 2 in the exon region of the TLR9 gene in Catfish Su's roundbellied catfish

[0082]

[0083] Note: “-” means no (the SNP site is not located in the CDS region and does not encode amino acids).

[0084] The present invention also determines the genotype of SNP site 2 by sequencing peak analysis. The sequencing information of SNP2 A>G is as follows: Figure 2 As shown. Figure 2 It can be seen that SNP2 A>G has three genotypes, namely AG / AA / GG.

[0085] Using the same method as in Example 1, the present invention conducted an association analysis between the genotype of SNP locus 2 and the bacterial septicemia resistance trait of Sussippinosus catfish based on sequencing results of PCR amplification products from susceptible and resistant groups of Sussippinosus catfish. The alleles and genotypes of SNP locus 2 in susceptible and resistant groups of Sussippinosus catfish are shown in Table 7. As shown in Table 7, Sussippinosus catfish with the AG genotype at SNP locus 2 exhibited significantly higher disease resistance than those with the GG or AA genotypes (P < 0.05).

[0086] Table 7 Alleles and genotypes of SNP locus 2 in susceptible and resistant populations of Catfish Su's

[0087]

[0088] Example 3 Genotyping of SNP sites 1 and 2 and verification of their association with resistance to bacterial sepsis

[0089] 1. Genotyping of SNP loci

[0090] Using the same method as in Example 1, the present invention collected 241 susceptible and 223 resistant samples of Sussini catfish. Genomic DNA was extracted from the Sussini catfish samples, and genotyping was performed at SNP site 1 and SNP site 2 using the SNaPshot genotyping method.

[0091] Based on the sequence information of the SNP sites described in Examples 1 and 2, the present invention designed corresponding SNaPshot typing primers, including amplification primer SNP1-P1F (shown in SEQ ID NO.7) and extension primer SNP1-E (shown in SEQ ID NO.8). More information is shown in Table 8.

[0092] Table 8 SNaPshot typing primers

[0093]

[0094] Note: The lowercase bases in the extension primer SNP1-E are the repeats of the extended sequence "gact", and the uppercase bases are the flanking specific sequences of the SNP site to be detected.

[0095] The genomic DNA of the 464 samples of Su's round-bellied catfish was extracted respectively, and PCR amplification was performed using the genomic DNA as a template using the primers shown in Table 8. The PCR reaction system is shown in Table 9.

[0096] Table 9 PCR reaction system of SNaPshot

[0097]

[0098]

[0099] PCR reaction conditions: 95°C initial denaturation for 5 min; 35 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; final extension at 72°C for 3 min. After the PCR reaction, the PCR product was treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs. Following the SAP treatment, a single-base extension reaction was performed using the reaction system shown in Table 10.

[0100] Table 10 Reaction system for single base extension reaction

[0101]

[0102] The single-base extension reaction conditions were as follows: initial denaturation at 95°C for 30 seconds, followed by 35 cycles of denaturation at 95°C for 5 seconds, annealing at 52°C for 5 seconds, and extension at 60°C for 5 seconds; and a final extension at 72°C for 3 minutes. The reaction products were sequenced and analyzed using a 96-channel automated ABI 3730xL genetic analyzer.

[0103] Export raw data in .fsa format from the 3730xL instrument, categorize and archive them by test reaction, and then import them into GeneMarker analysis software. Based on the signal values and peak patterns at each site, adjust the primer ratios at each site to ensure consistent signal values and peak patterns at each site. Perform statistics by sample and site to obtain typing results for each sample site, and export a genotype statistics table.

[0104] 2. Association Verification Analysis

[0105] According to the results shown in the genotype statistical table, the association verification analysis between the genotype of the SNP site and the bacterial sepsis resistance trait of Su's round belly catfish was performed using the same method as in Example 1. The variance analysis test showed significant differences, and the Duncan method was used for multiple comparisons.

[0106] The alleles and genotypes of SNP site 1 in susceptible and resistant populations of S. suis are shown in Table 11. As shown in Table 11, the frequency of allele G at SNP site 1 in the resistant group (0.686) was significantly higher than that in the susceptible group (0.551), confirming that the gene frequency of allele G at SNP site 1 was high in the candidate breeding population, and that this population had significantly improved resistance to bacterial septicemia (P < 0.05).

[0107] Table 11 Alleles and genotypes of SNP1 in susceptible and resistant populations of Aeromonas hydrophila

[0108]

[0109] The allele and genotype conditions of SNP locus 2 in the susceptible and resistant populations of Pangasianodon hypophthalmus are shown in Table 12. As can be seen from Table 12, the disease resistance of Pangasianodon hypophthalmus with the genotype AG at SNP locus 2 is significantly higher than that of Pangasianodon hypophthalmus with the genotypes GG or AA (P < 0.05), indicating that the SNP molecular markers described in the present invention can indeed significantly distinguish between susceptible and resistant populations.

[0110] Table 12 Allele and genotype conditions of SNP2 in the susceptible and resistant populations of Aeromonas hydrophila

[0111]

[0112] Example 4 Genetic analysis of SNP loci

[0113] The results of the genetic analysis of SNP locus 1 of the TLR9 gene in Pangasianodon hypophthalmus are shown in Table 13. As can be seen from Table 13, SNP1 A>G is moderately polymorphic in both the susceptible and disease-resistant populations (0.25 < PIC < 0.5), indicating that SNP1 A>G can provide relatively reasonable genetic information as a genetic marker.

[0114] Table 13 Genetic information of SNP locus 1 of the TLR9 gene in Pangasianodon hypophthalmus

[0115]

[0116] Note: Na is the number of alleles; He is the observed heterozygosity; Ne is the expected heterozygosity; PIC is the polymorphism information content.

[0117] The results of the genetic analysis of SNP locus 2 of the TLR9 gene in Pangasianodon hypophthalmus are shown in Table 14. As can be seen from Table 14, SNP2 A>G is moderately polymorphic in both the susceptible and disease-resistant populations (0.25 < PIC < 0.5), indicating that SNP2 A>G can provide relatively reasonable genetic information as a genetic marker.

[0118] Table 14 Genetic information of SNP locus 2 of the TLR9 gene in Pangasianodon hypophthalmus

[0119]

[0120] Note: Na is the number of alleles; He is the observed heterozygosity; Ne is the expected heterozygosity; PIC is the polymorphism information content.

[0121] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting alleles or genotypes of SNP molecular markers in identifying the bacterial sepsis resistance trait of Su's round-bellied catfish, characterized in that: The SNP molecular marker includes SNP site 1 and SNP site 2; the SNP site 1 is located at the nucleotide site 20215478 of the Su's round-bellied catfish reference genome CM018548.1, and the polymorphism is A / G; the SNP site 2 is located at the nucleotide site 20215688 of the Su's round-bellied catfish reference genome CM018548.1, and the polymorphism is A / G; the probability that the Su's round-bellied catfish population with the SNP site 1 allele G is resistant to bacterial septicemia is significantly higher than the Su's round-bellied catfish population with the SNP site 1 allele A; the probability that the Su's round-bellied catfish with the SNP site 2 genotype AG is resistant to bacterial septicemia is significantly higher than the Su's round-bellied catfish with the genotype GG or AA.

2. The use of the reagent described in claim 1 in preparing a product for identifying the anti-bacterial septicemia properties of catfish, characterized in that: The probability that the Su's round-bellied catfish population with SNP site 1 allele G is resistant to bacterial septicemia is significantly higher than that with allele A; the probability that the Su's round-bellied catfish population with SNP site 2 genotype AG is resistant to bacterial septicemia is significantly higher than that with genotype GG or AA.

3. The use of the reagent described in claim 1 in screening for resistance to bacterial septicemia of Catfish Sue, characterized in that: The Su's round-bellied catfish with SNP site 1 allele containing G and SNP site 2 genotype of AG were selected.

4. The use of the reagent described in claim 1 in preparing a product for screening catfish of the Su's family for resistance to bacterial septicemia, characterized in that: The Su's round-bellied catfish with SNP site 1 allele containing G and SNP site 2 genotype of AG were selected.

5. Use of the reagent described in claim 1 in molecular marker-assisted breeding of Su's round-bellied catfish varieties resistant to bacterial septicemia.

6. Use of the reagent according to claim 1 in preparing a product for molecular marker-assisted breeding of a species of catfish resistant to bacterial septicemia.

7. A method for screening catfish resistant to bacterial sepsis, characterized in that: The allele or genotype of the SNP molecular marker described in claim 1 is detected in the tested Su's round-bellied catfish, and the Su's round-bellied catfish with SNP site 1 allele G and SNP site 2 genotype AG is selected.

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