Application of pacific oyster disease-resistant gene SNP (Single Nucleotide Polymorphism) marker in identifying individual antibacterial pacific oyster

Abstract

The invention belongs to the technical field of aquatic organisms, and relates to application of a pacific oyster anti-disease gene SNP marker in identification of an anti-bacterial-disease pacific oyster individual, the SNP marker is located at the 1275th base, the 1281th base, the 1285th base, the 1386th base, the 1623th base and the 1701th base from an initiation codon of a hypoxia inducible factor CgHIF-1alpha gene as shown in SEQ ID No.1, and the mutation types are respectively + 1275G / A, + 1281A / G, + 1285C / T, + 1356A / T, + 1386C / T, + 1623C / T and + 1701C / G. The polymorphic sites of the hypoxia-inducible factor CgHIF-1alpha gene in the crassostrea gigas are screened, the SNP marker related to the crassostrea gigas bacterial disease resistance character is developed, and the SNP marker can be used for identification of crassostrea gigas bacterial disease resistance individuals and parent screening in bacterial disease resistance strain breeding.

Description

Technical Field

[0001] This invention belongs to the field of aquatic organism technology, specifically relating to the application of SNP markers for disease resistance genes in Pacific oysters in identifying Pacific oysters resistant to bacterial diseases. Background Technology

[0002] The Pacific oyster (Crassostrea gigas) is an important economic shellfish, playing a vital role in global aquaculture. In recent years, outbreaks of diseases caused by environmental stress and pathogen infection have occurred frequently, resulting in significant economic losses to the Pacific oyster farming industry and severely hindering its green and high-quality development. Therefore, creating superior breeds with strong resistance and high quality has become an urgent need for the industry's development.

[0003] Marker-assisted selection (MAS) utilizes molecular markers closely linked to target traits to identify genotypes in individuals at an early stage, precisely selecting individuals carrying genes for desirable traits. It offers advantages such as speed, accuracy, and independence from environmental conditions. Among these, single nucleotide polymorphisms (SNPs) stand out due to their ease of operation, low cost, and large-scale production, gradually becoming a hot topic in MAS research. Developing SNP molecular markers associated with disease resistance traits is a crucial technological requirement for advancing disease-resistant breeding in the Pacific oyster.

[0004] Screening for disease-resistance-related SNP markers, starting with immune-related genes, is a crucial method for creating resistant germplasm. Hypoxia-inducible factor HIF-1α is a core regulatory factor in cellular responses to hypoxia, participating in physiological and pathological processes such as inflammation, metabolism, and immune responses. HIF-1α SNP sites can influence gene expression, protein stability, and function, thereby regulating disease susceptibility, drug response, and personalized treatment strategies. Therefore, developing SNP markers for hypoxia-inducible factor HIF-1α can screen for sites associated with disease resistance traits in the Pacific oyster, providing an important molecular basis for the creation of disease-resistant germplasm and disease-resistant breeding. Summary of the Invention

[0005] The purpose of this invention is to provide the application of disease resistance gene markers in Pacific oysters in identifying Pacific oysters resistant to bacterial diseases.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Application of SNP markers for disease resistance genes in Pacific oysters in identifying Pacific oysters resistant to bacterial diseases, wherein the SNP markers for disease resistance genes in Pacific oysters are at least one of the bases at positions 1275, 1281, 1285, 1356, 1386, 1623, and 1701 in the coding region of the Pacific oyster hypoxia-inducible factor HIF-1α (hypoxia-inducible factor CgHIF-1α) gene, with mutation types of +1275G / A, +1281A / G, +1285C / T, +1356A / T, +1386C / T, +1623C / T, and +1701C / G, respectively.

[0008] The sequence of the hypoxia-inducible factor HIF-1α of the Pacific oyster is shown in SEQ ID No. 1.

[0009] Preferably, the genotypes at the 1275th marker position are GG or GA, the genotypes at the 1281st marker position are AA, AG or GG, the genotypes at the 1285th marker position are CC or CT, the genotypes at the 1356th marker position are AA, AT or TT, the genotypes at the 1386th marker position are CC or CT, the genotypes at the 1623rd marker position are CC, CT or TT, and the genotypes at the 1701st marker position are CC, CG or GG.

[0010] More preferably, the dominant genotype at position 1275 is +1275GA, the dominant genotype at position 1281 is +1281GG, the dominant genotype at position 1285 is +1285CT, the dominant genotype at position 1356 is +1356TT, the dominant genotype at position 1386 is +1386CT, the dominant genotype at position 1623 is +1623TT, and the dominant genotype at position 1701 is +1701GG.

[0011] The bacteria in question are pathogenic halomonas.

[0012] A second objective of this invention is to provide primers for detecting the aforementioned SNP markers of the disease resistance gene in the Pacific oyster.

[0013] The primer sequences are:

[0014] CgHIF-1α-F: 5'-TCAATGAAGATGGACGAGACCG-3',

[0015] CgHIF-1α-R: 5'-TGAATCGTTTCTTGCTGGGAGG-3'.

[0016] A third objective of this invention is to provide the application of the above-mentioned primers in identifying individuals of the Pacific oyster resistant to bacterial diseases.

[0017] A fourth objective of this invention is to provide a kit containing the primers described above for identifying individuals of resistant oysters.

[0018] A fifth objective of this invention is to provide the application of the above-described kit in the identification of individuals with resistance to bacterial diseases in oysters.

[0019] The sixth objective of this invention is to provide a method for identifying individuals of the Pacific oyster resistant to bacterial diseases.

[0020] Specifically, the method for identifying bacterial-resistant individuals of the Pacific oyster involves using the genomic DNA of the oyster to be tested as a template, and performing PCR amplification using primers CgHIF-1α-F and CgHIF-1α-R to obtain gene fragments containing the aforementioned SNP marker sites. Then, direct sequencing is used to perform genotyping at these sites, thereby identifying the genotype of the oyster. Individuals with genotypes of +1275GA, +1281GG, 1285CT, +1356TT, +1386CT, +1623TT, and +1701GG at positions 1275, 1281, 1285, 1356, 1386, 1623, and 1701 are considered bacterial-resistant individuals.

[0021] CgHIF-1α-F: 5'-TCAATGAAGATGGACGAGACCG-3',

[0022] CgHIF-1α-R: 5'-TGAATCGTTTCTTGCTGGGAGG-3'.

[0023] PCR amplification conditions: denaturation at 94℃ for 5 min; followed by 35 cycles of constant amplification (denaturation at 94℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 50 s); and finally incubation at 72℃ for 10 min.

[0024] The seventh objective of this invention is to provide a method for breeding disease-resistant varieties of Pacific oyster.

[0025] The method for breeding disease-resistant varieties of Pacific oysters involves detecting SNP markers of disease-resistant genes in Pacific oysters, screening out Pacific oyster individuals carrying the SNP markers for reproduction, and cultivating offspring; at the same time, bacterial stress treatment is carried out, and oysters with bacterial disease resistance are selected for multiple generations of reproduction and cultivation to obtain new disease-resistant varieties.

[0026] The beneficial effects of this invention are:

[0027] This invention utilizes molecular biology techniques to discover a marker for the key gene HIF-1α, a hypoxia-inducible factor associated with disease resistance in the Pacific oyster. By precisely screening oyster individuals with potential disease resistance through SNP loci, a gene marker-assisted breeding technology has been preliminarily established. This technology is characterized by its simplicity, speed, and short cycle, which improves yield and quality, strongly promotes the sustainable development of the industry, and provides a new direction for disease resistance breeding of shellfish and other aquaculture organisms through its innovative breeding approach. Attached Figure Description

[0028] Figure 1 The coding region sequence of the hypoxia-inducible factor HIF-1α in oyster provided in the embodiments of the present invention and the distribution map of polymorphic sites of +1275G / A, +1281A / G, +1285C / T, +1356A / T, +1386C / T, +1623C / T and +1701C / G.

[0029] Figure 2 The sequencing maps of the HIF-1α coding region of the Pacific oyster provided in this embodiment of the invention are +1275G / A, +1281A / G, +1285C / T, +1356A / T, +1386C / T, +1623C / T and +1701C / G.

[0030] Figure 3 The distribution frequencies of different genotypes of the hypoxia-inducible factor HIF-1α coding region SNP sites 1275G / A, 1281A / G, 1285C / T, 1356A / T, 1386C / T, 1623C / T and 1701C / G in the disease-resistant population and normal population of Pacific oysters provided in the embodiments of the present invention. Detailed Implementation

[0031] 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.

[0032] Example 1: Obtaining the gene marker of HIF-1α, a disease resistance-related hypoxia-inducible factor, in oysters.

[0033] a) Cloning of the coding region sequence of the hypoxia-inducible factor HIF-1α gene in the Pacific oyster

[0034] Following the methods described in Molecular Cloning, genomic DNA was extracted from the adductor muscle of 30 Pacific oysters from a general population and six families as amplification templates. Primers CgHIF-1α-F and CgHIF-1α-R were designed based on the known coding region sequence of the hypoxia-inducible factor HIF-1α gene in Pacific oysters, and 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 52℃ for 30 s, extension at 72℃ for 50 s); and finally, incubation at 72℃ for 10 min. The PCR amplification products were sequenced to obtain their nucleotide sequences. The cloned hypoxia-inducible factor HIF-1α coding region sequence is 627 bp in length, and a total of 14 SNP sites were found, namely +1275G, +1281A, +1285C, +1347C, +1356A, +1386C, +1413G, +1575C, +1587C, +1590C, +1623C, +1626C, +1698G, and +1701C.

[0035] CgHIF-1α-F: 5'-TCAATGAAGATGGACGAGACCG-3',

[0036] CgHIF-1α-R: 5'-TGAATCGTTTCTTGCTGGGAGG-3'.

[0037] b) Molecular marker screening of HIF-1α, a hypoxia-inducible factor in Pacific oyster

[0038] Thirty disease-resistant individuals and 30 normal individuals were randomly selected, and genomic DNA was extracted as templates for gene amplification and screening of gene markers. The hypoxia-inducible factor HIF-1α coding region gene from the disease-resistant and normal groups was amplified by PCR using primers CgHIF-1α-F and CgHIF-1α-R, respectively. The PCR-amplified HIF-1α coding region sequence product was purified and sequenced.

[0039] Statistical analysis of the frequency of different genotypes in the disease-resistant and normal populations (see Table 1) and subsequent Chi-square test analysis using SPSS 11.5 software revealed that individuals with the genotypes +1275GA, +1281GG, +1285CT, +1356TT, +1386CT, +1623TT, and +1701GG were significantly more frequent in the disease-resistant population than in the normal population. Among these, individuals with the genotypes +1275GA and +1285CT were only found in the disease-resistant population.

[0040] Therefore, +1275G, +1281G, +1285T, +1356T, +1386T, +1623T, and +1701G were identified as disease resistance-related SNP markers. +1275GA, +1281GG, +1285CT, +1356TT, +1386CT, +1623TT, and +1701GG were identified as disease resistance genotypes.

[0041] Table 1. Chi-square test of the distribution frequency of different genotypes of hypoxia-inducible factor HIF-1α in Pacific oysters in the normal and antibacterial populations.

[0042]

[0043]

[0044] Example 2: Disease Resistance-Related Molecular Marker-Assisted Breeding Method for Crassula ovata

[0045] 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 CgHIF-1α-F and CgHIF-1α-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 52℃ for 30 s, extension at 72℃ for 50 s); and finally, incubation at 72℃ for 10 min.

[0046] CgHIF-1α-F: 5'-TCAATGAAGATGGACGAGACCG-3',

[0047] CgHIF-1α-R: 5'-TGAATCGTTTCTTGCTGGGAGG-3'.

[0048] Subsequently, the genotypes of different individuals were detected using direct sequencing. Individuals with genotypes of +1275GA, +1281GG, +1285CT, +1356TT, +1386CT, +1623TT, and +1701GG at base positions 1275, 1281, 1285, 1356, 1386, 1623TT, and +1701GG at base positions 1275, 1281, 1285, 1356TT, 1386CT, +1623TT, and +1701GG were selected as disease-resistant individuals. They were then bred using conventional methods to produce offspring, thus developing new disease-resistant varieties.

Claims

1. The application of SNP markers for disease resistance genes in Pacific oysters in identifying Pacific oysters resistant to bacterial diseases, characterized in that, The disease resistance gene marker of the Pacific oyster is at least one of the bases at positions 1275, 1281, 1285, 1356, 1386, 1623, and 1701 of the hypoxia-inducible factor CgHIF-1α gene shown in SEQ ID No. 1, with mutation types of +1275G / A, +1281A / G, +1285C / T, +1356A / T, +1386C / T, +1623C / T, and +1701C / G, respectively.

2. The application of the SNP marker for disease resistance genes in Pacific oysters according to claim 1 in identifying Pacific oysters resistant to bacterial diseases, characterized in that, The genotype at the 1275th marker is GG or GA, the genotype at the 1281st marker is AA, AG or GG, the genotype at the 1285th marker is CC or CT, the genotype at the 1356th marker is AA, AT or TT, the genotype at the 1386th marker is CC or CT, the genotype at the 1623rd marker is CC, CT or TT, and the genotype at the 1701st marker is CC, CG or GG.

3. The application of the SNP marker for disease resistance genes in Pacific oysters according to claim 2 in identifying Pacific oysters resistant to bacterial diseases, characterized in that, The dominant genotype at position 1275 is +1275GA, the dominant genotype at position 1281 is +1281GG, the dominant genotype at position 1285 is +1285CT, the dominant genotype at position 1356 is +1356TT, the dominant genotype at position 1386 is +1386CT, the dominant genotype at position 1623 is +1623TT, and the dominant genotype at position 1701 is +1701GG.

4. The application of the SNP marker for disease resistance genes in Pacific oysters according to claim 1, 2, or 3 in identifying Pacific oysters resistant to bacterial diseases, characterized in that, The bacteria in question are pathogenic halomonas.

5. Primers for detecting the SNP marker of the disease resistance gene in the Pacific oyster according to any one of claims 1-4, characterized in that, The primer sequence is as follows: CgHIF-1α-F: 5'-TCAATGAAGATGGACGAGACCG-3', CgHIF-1α-R: 5'-TGAATCGTTTCTTGCTGGGAGG-3'.

6. The use of the primers according to claim 5 in identifying individuals of the Pacific oyster resistant to bacterial diseases.

7. A kit comprising the primers of claim 5 for identifying individuals of resistant oysters.

8. The use of the kit according to claim 7 in identifying individuals of resistant Oyster disease.

9. A method for identifying individuals of the Pacific oyster resistant to bacterial diseases, characterized in that: Using the genomic DNA of the oyster *Crassostrea gigas* as a template, PCR amplification was performed using primers CgHIF-1α-F and CgHIF-1α-R to obtain fragments containing the SNP marker sites of the gene described in claim 1. Then, direct sequencing was used to perform genotyping at these sites, thereby identifying the genotype of the oyster. Individuals with genotypes of +1275GA, +1281GG, 1285CT, +1356TT, +1386CT, +1623TT, and +1701GG at positions 1275, 1281, 1285, 1356, 1386, 1623, and 1701 were identified as resistant to bacterial diseases. CgHIF-1α-F: 5'-TCAATGAAGATGGACGAGACCG-3', CgHIF-1α-R: 5'-TGAATCGTTTCTTGCTGGGAGG-3'.

10. A method for breeding disease-resistant varieties of Crassula ovata, characterized in that, The method involves detecting the SNP marker of the disease-resistant gene in the Pacific oyster as described in claim 1, screening out Pacific oyster individuals carrying the SNP marker for reproduction, and cultivating offspring; simultaneously, bacterial stress treatment is performed, and oysters with bacterial disease resistance are selected for multi-generation reproduction and cultivation to obtain new disease-resistant varieties.

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