Application of SNP (Single Nucleotide Polymorphism) marker of crassostrea gigas tumor necrosis factor TNF-3 related to bacterial disease resistance

Abstract

The invention discloses application of a pacific oyster tumor necrosis factor TNF-3 (Tumor Necrosis Factor 3) and an anti-bacterial disease related SNP (Single Nucleotide Polymorphism) marker in breeding of an anti-bacterial disease pacific oyster variety, the SNP marker is located in the 17103 base, the 17111 base, the 17132 base, the 17145 base, the 17169 base, the 17175 base and the 17337 base from an initiation codon of a pacific oyster tumor necrosis factor TNF-3 gene as shown in SEQ ID No.1, and the mutation types are respectively + 17103G / A, + 17111 A / G, + 17132 T / A, + 17145 C / T, + 17169 A / G, + The SNP marker related to the disease resistance of the crassostrea gigas is developed aiming at the tumor necrosis factor MgTNF-3 gene in the crassostrea gigas, the marker-assisted breeding technology of the tumor necrosis factor gene related to the bacterial disease resistance of the crassostrea gigas is preliminarily established, and the marker-assisted breeding method has the characteristics of simplicity, convenience and rapidness in operation, high breeding efficiency, short period and the like; the method opens up a new molecular breeding technical approach for cultivation of bacterial disease resistant shellfish varieties, and has important theoretical significance and application value for breeding of bacterial disease resistant shellfish varieties.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetic breeding technology, specifically involving the application of SNP markers related to tumor necrosis factor TNF-3 and antibacterial disease resistance in oysters. Background Technology

[0002] The Pacific oyster (Magallana gigas), also known as the giant oyster, is an important marine aquaculture species worldwide. In recent years, due to factors such as germplasm degradation, deteriorating aquaculture environments, and pathogen outbreaks, oyster diseases have become increasingly serious. In 2019, a large-scale oyster mortality event due to red shell disease occurred at a large-scale aquaculture farm in Dalian, Liaoning Province, causing significant economic losses and severely impacting the green and healthy development of the Pacific oyster aquaculture industry. Through pathogen isolation, identification, and reinfection experiments, a pathogenic bacterium strain—Haloxymonas—was obtained. The resistant oyster population of this invention was developed based on challenge with this pathogenic Haloxymonas strain.

[0003] Because Pacific oysters lack adaptive immunity and are typically farmed in open waters, effective defense against pathogens in these areas cannot be achieved through medication. Therefore, breeding disease-resistant superior varieties is imperative. Marker-assisted selection (MAS) is a breeding method based on molecular markers for genotypic selection, particularly suitable for selecting complex quantitative traits such as disease resistance and quality traits. A prerequisite for MAS breeding is obtaining genetic markers that co-segregate or are closely linked to the target gene. Single nucleotide polymorphisms (SNPs) have rapidly become a hot topic in MAS research due to their wide distribution, large number, and ease of large-scale detection. Currently, the number of SNP molecular markers associated with Pacific oyster resistance to halophilic bacteria is very limited, hindering the development of disease-resistant superior varieties. Therefore, it is urgent to develop SNP molecular markers related to halophilic bacteria resistance.

[0004] Tumor necrosis factor (TNF) possesses a typical TNF homologous domain, comprising both TNF-α and TNF-β forms. TNF-β is an important mediator in inflammatory and immune responses. It belongs to the TNF superfamily of ligands and transmits signals through two receptors, TNFR1 and TNFR2. TNF-β is produced by activated T cells and B cells. Multiple studies have found that TNF-β plays a crucial role in various immune defense processes in invertebrates, especially shellfish, including cell activation, survival, proliferation, necrosis, apoptosis, and inflammatory responses. Therefore, in disease-resistant breeding research and practice in aquatic animals, tumor necrosis factor shows promise as a candidate gene for improving susceptibility to pathogens. Summary of the Invention

[0005] The purpose of this invention is to provide the application of tumor necrosis factor TNF-3 in oysters as a SNP marker associated with antibacterial disease.

[0006] To achieve the above objectives, the technical solution adopted in this invention is: the application of SNP markers related to tumor necrosis factor TNF-3 and antibacterial disease resistance in Pacific oysters in the breeding of Pacific oyster varieties resistant to bacterial diseases.

[0007] The SNP marker is located in the TNF domain region of the TNF-3 gene in the oyster tumor necrosis factor, and its sequence is shown in SEQ ID No. 1.

[0008] The SNP markers shown are located at one or more of the following bases from the start codon of the TNF-3 gene in the oyster: position 17103, position 17111, position 17132, position 17145, position 17169, position 17175, and position 17337. The mutation types are +17103G / A, +17111A / G, +17132T / A, +17145C / T, +17169A / G, +17175A / G, and +17337C / T, respectively.

[0009] The genotypes at the 17103 marker position are AA, AG, or GG; the genotypes at the 17111 marker position are AA, AG, or GG; the genotypes at the 17132 marker position are TA or TT; the genotypes at the 17145 marker position are TC or TT; the genotypes at the 17169 marker position are AA, AG, or GG; the genotypes at the 17175 marker position are AA, AG, or GG; and the genotypes at the 17337 marker position are CC, CT, or TT.

[0010] The dominant genotype at position 17103 is +17103GG, the dominant genotype at position 17111 is +17111AA, the dominant genotype at position 17132 is +17132AT, the dominant genotype at position 17145 is +17145CT, the dominant genotype at position 17169 is +17169AA, the dominant genotype at position 17175 is +17175GG, and the dominant genotype at position 17337 is +17337TT.

[0011] Another object of the present invention relates to primer pairs for detecting SNP markers associated with tumor necrosis factor TNF-3 and antibacterial disease in oysters.

[0012] The primer pair is:

[0013] MgTNF-3-F: 5'-ATCAGCAACATGAAATACCAG-3',

[0014] MgTNF-3-R: 5'-AAAAGTGACGGTTTGATGAAT-3'.

[0015] Another object of the present invention relates to a kit for detecting SNP markers associated with tumor necrosis factor TNF-3 and antibacterial disease in oysters.

[0016] Specifically, the kit contains the primer pairs described above.

[0017] Another object of the present invention relates to the application of the primer pairs or the kits described herein.

[0018] Specifically, this relates to the application of the primer pairs or the kit in identifying resistant individuals of Pacific oysters to bacterial diseases. Another object of the invention relates to a method for identifying resistant individuals of Pacific oysters to bacterial diseases by detecting tumor necrosis factor-TNF-3 and SNP markers associated with antibacterial disease in the oyster, determining the genotype of the oyster, and then identifying resistant individuals based on the genotype.

[0019] Furthermore, genomic DNA was extracted from the oysters to be tested and used as a template for PCR amplification to obtain fragments containing the target SNP sites. Then, direct sequencing was used to perform genotyping of the SNP sites to identify the genotypes of the oysters to be tested. Individuals with genotypes of +17103GG, +17111AA, +17132AT, +17145CT, +17169AA, +17175GG, and +17337TT at seven loci (17103, 17111, 17132, 17145, 17169, 17175GG, and 17337TT) were identified as resistant to bacterial diseases.

[0020] In PCR amplification, the specific primer pairs are:

[0021] MgTNF-3-F: 5'-ATCAGCAACATGAAATACCAG-3',

[0022] MgTNF-3-R: 5'-AAAAGTGACGGTTTGATGAAT-3'.

[0023] PCR amplification conditions: denaturation at 94℃ for 5 min; followed by 35 cycles of constant amplification, i.e., denaturation at 94℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s; and finally incubation at 72℃ for 10 min.

[0024] Another object of the present invention relates to a method for breeding disease-resistant varieties of Pacific oysters.

[0025] Specifically, a method for breeding disease-resistant varieties of Pacific oysters involves detecting SNP markers related to tumor necrosis factor-3 (TNF-3) and resistance to bacterial diseases in Pacific oysters, screening out Pacific oysters carrying the SNP markers for propagation, and cultivating offspring; simultaneously, bacterial stress treatment is performed, and oysters that carry both the antibacterial disease-related TNF-3 marker and antibacterial disease ability are selected and propagated and cultivated for multiple generations to obtain new disease-resistant varieties.

[0026] The antibacterial disease described is against halomonas infection. The bacteria are halomonas.

[0027] Advantages of this invention:

[0028] This invention develops SNP markers related to disease resistance in the Pacific oyster by targeting the tumor necrosis factor MgTNF-3 gene, laying the foundation for the creation of disease-resistant Pacific oyster germplasm.

[0029] This invention utilizes molecular biology techniques to discover tumor necrosis factor gene markers related to disease resistance in farmed shellfish in my country, using the Pacific oyster as the material. It has preliminarily established a gene marker-assisted breeding technology for tumor necrosis factor gene markers related to bacterial disease resistance in Pacific oysters. This technology is characterized by its simple and rapid operation, high breeding efficiency, and short cycle, opening up a new molecular breeding technology approach for the cultivation of bacterial disease-resistant shellfish varieties. It has important theoretical significance and application value for the breeding of bacterial disease-resistant varieties of farmed shellfish. Attached Figure Description

[0030] Figure 1 The TNF domain sequence and +17103G / A, +17111G / A, +17132T / A, +17145C / T, +17169G / A, +17175G / A, and +17337C / T sites in the coding region of tumor necrosis factor TNF-3, which is related to antibacterial disease in oysters, are shown in the embodiments of the present invention.

[0031] Figure 2 Sequencing maps of different genotypes at the TNF domain of the tumor necrosis factor TNF-3 coding region, which is related to antibacterial disease in oysters, provided in embodiments of the present invention, for the TNF domain of the TNF domain. These genotypes are at the +17103G / A, +17111G / A, +17132T / A, +17145C / T, +17169G / A, +17175G / A, and +17337C / T sites.

[0032] Figure 3 The distribution frequency of different genotypes of the TNF domain in the coding region of tumor necrosis factor TNF-3 in the antibacterial disease population and the normal population of oysters provided in the embodiments of the present invention, and the corresponding chi-square test plots. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0034] Example 1

[0035] The gene marker of tumor necrosis factor-3 (TNF-3), which is associated with antibacterial disease resistance in Pacific oysters, was obtained through the following steps:

[0036] a) Cloning of the TNF domain sequence in the coding region of the tumor necrosis factor TNF-3 gene, which is associated with antibacterial disease resistance in the Pacific oyster.

[0037] Following the methods described in Molecular Cloning, genomic DNA was extracted from the adductor muscle of the Pacific oyster as a template for amplification. Primers MgTNF-3-F and MgTNF-3-R were designed based on the known TNF domain sequence of the coding region of the tumor necrosis factor-3 gene, which is associated with disease resistance. PCR amplification was performed according to the following procedure: denaturation at 94℃ for 5 min; followed by 35 cycles of constant amplification (denaturation at 94℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s); and finally, incubation at 72℃ for 10 min. The PCR amplification products were sequenced to obtain their nucleotide sequences (see [link to relevant documentation]). Figure 1 The cloned tumor necrosis factor TNF-3 coding region TNF domain sequence is 440bp in length.

[0038] MgTNF-3-F: 5'-ATCAGCAACATGAAATACCAG-3',

[0039] MgTNF-3-R: 5'-AAAAGTGACGGTTTGATGAAT-3'.

[0040] b) Molecular marker screening for tumor necrosis factor-TNF-3 in Pacific oyster

[0041] Genomic DNA was extracted from 35 disease-resistant individuals and 35 normal individuals and used as templates for gene amplification for gene marker screening. Using primers MgTNF-3-F and MgTNF-3-R, the TNF domain gene encoding the tumor necrosis factor-3 coding region was amplified by PCR in individuals from the disease-resistant and normal groups, respectively. The TNF domain sequence product obtained from the PCR amplification was purified and sequenced.

[0042] Statistical analysis of the frequency of different genotypes in the resistant bacterial disease group and the general population (see Table 1) and subsequent Chi-square test analysis using SPSS 11.5 software revealed that individuals with the genotypes +17103GG, +17111AA, +17132AT, +17145CT, +17169AA, +17175GG, and +17337TT were significantly more frequent in the resistant bacterial disease group than in the general population.

[0043] Therefore, +17103G, +17111A, +17132T, +17145T, +17169A, +17175G, and +17337T were identified as SNP markers associated with resistance to bacterial diseases. +17103GG, +17111AA, +17132AT, +17145CT, +17169AA, +17175GG, and +17337TT were identified as genotypes associated with resistance to bacterial diseases.

[0044] Table 1. Chi-square test of the distribution frequency of different tumor necrosis factor-3 genotypes in the general and antibacterial populations of Pacific oyster.

[0045]

[0046] Example 2: Marker-Assisted Breeding Method for Antibacterial Disease Resistance in Crassula ovata

[0047] 25 mg of mantle membrane of oysters was cut using a non-destructive sampling method, and DNA was extracted and used as a template. PCR amplification was performed using specific primers MgTNF-3-F and MgTNF-3-R according to the following procedure: denaturation at 94℃ for 5 min; followed by 35 cycles of constant amplification: denaturation at 94℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s; and finally incubation at 72℃ for 10 min.

[0048] MgTNF-3-F: 5'-ATCAGCAACATGAAATACCAG-3',

[0049] MgTNF-3-R: 5'-AAAAGTGACGGTTTGATGAAT-3'.

[0050] Subsequently, the genotypes of different individuals were detected using direct sequencing. Individuals with genotypes of +17103GG, +17111AA, +17132AT, +17145CT, +17169AA, +17175GG, and +17337TT at base positions 17103, 17111, 17132, 17145CT, +17169AA, +17175GG, and +17337TT at base positions 17103, 17111, 17132, 17145, 17169, 17175GG, and 17337TT were selected as bacterial disease resistant individuals. They were then bred using conventional methods, while simultaneously subjected to bacterial stress treatment, and offspring were cultivated to develop new bacterial disease resistant varieties.

Claims

1. Application of TNF-3 and SNP markers related to bacterial disease resistance in breeding oyster varieties resistant to bacterial disease, characterized in that: The SNP markers are located at the 17103rd, 17111th, 17132nd, 17145th, 17169th, 17175th and 17337th bases from the start codon of the long oyster tumor necrosis factor TNF-3 gene shown in SEQ ID No.1, and the mutation types are +17103G / A, +17111A / G, +17132T / A, +17145C / T, +17169A / G, +17175A / G, and +17337C / T, respectively.

2. The use of the SNP marker associated with the tumor necrosis factor TNF-3 of the long oyster and anti-bacterial disease according to claim 1 in breeding long oyster varieties resistant to bacterial diseases, characterized in that: The base genotype at the 17103rd marker is AA, AG or GG, the base genotype at the 17111th marker is AA, AG or GG, the base genotype at the 17132nd marker is TA or TT, the base genotype at the 17145th marker is TC or TT, the base genotype at the 17169th marker is AA, AG or GG, the base genotype at the 17175th marker is AA, AG or GG, and the base genotype at the 17337th marker is CC, CT or TT.

3. The use of the SNP marker associated with tumor necrosis factor TNF-3 and bacterial disease resistance in the long oyster according to claim 1 in breeding long oyster varieties resistant to bacterial diseases, characterized in that: The dominant genotype at position 17103 is +17103GG genotype, the dominant genotype at position 17111 is +17111AA, the dominant genotype at position 17132 is +17132AT genotype, the dominant genotype at position 17145 is +17145CT genotype, the dominant genotype at position 17169 is +17169AA genotype, the dominant genotype at position 17175 is +17175GG genotype, and the dominant genotype at position 17337 is +17337TT genotype.

4. A primer pair for detecting the SNP marker associated with tumor necrosis factor TNF-3 and anti-bacterial disease in the long oyster according to any one of claims 1 to 3, characterized in that: The primer pairs are: MgTNF-3-F: 5'-ATCAGCAACATGAAATACCAG-3', MgTNF-3-R: 5'-AAAAGTGACGGTTTGATGAAT-3'.

5. Use of the primer pair according to claim 4 in identifying individuals of Crassostrea gigas that are resistant to bacterial diseases.

6. A kit for detecting SNP markers associated with tumor necrosis factor TNF-3 and resistance to bacterial diseases in Crassostrea gigas, comprising the primer pair according to claim 4.

7. Use of the kit according to claim 6 in identifying individuals of Crassostrea gigas that are resistant to bacterial diseases.

8. A method for identifying individuals of Crassostrea gigas that are resistant to bacterial disease, characterized in that: The genotype of the oyster to be tested is determined by detecting the tumor necrosis factor TNF-3 and the SNP marker related to anti-bacterial disease as described in claim 1, and then the anti-bacterial disease individual is determined according to the genotype.

9. The method for identifying individuals of Crassostrea gigas that are resistant to bacterial diseases according to claim 8, characterized in that: Extract the genomic DNA of the long oyster to be tested as a template, perform PCR amplification, obtain a fragment containing the target SNP site, and then use direct sequencing to perform typing detection on the SNP site to identify the genotype of the long oyster to be tested. The individuals whose genotypes at seven sites, namely, 17103, 17111, 17132, 17145, 17169, 17175 and 17337, are +17103GG, +17111AA, +17132AT, +17145CT, +17169AA, +17175GG and +17337TT, are one or more of the individuals resistant to bacterial disease; In PCR amplification, the specific primer pairs are: MgTNF-3-F: 5'-ATCAGCAACATGAAATACCAG-3', MgTNF-3-R: 5'-AAAAGTGACGGTTTGATGAAT-3'.

10. A method for breeding disease-resistant varieties of long oysters, characterized by: Detect the long oyster tumor necrosis factor TNF-3 and SNP markers related to anti-bacterial disease described in claim 1 of the long oyster, screen out the long oysters carrying the SNP markers for reproduction and cultivation of offspring; at the same time, carry out bacterial stress treatment, and screen out oysters with both anti-bacterial disease-related tumor necrosis factor TNF-3 markers and anti-bacterial disease ability. After multiple generations of reproduction and cultivation, a new disease-resistant variety is obtained.

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