Crassostrea gigas disease resistance related gene marker and application thereof

Abstract

The invention belongs to the technical field of aquatic organisms, and particularly relates to a crassostrea gigas disease resistance related gene marker and application thereof. The crassostrea gigas disease resistance related gene marker is located in an RNA helicase promoter region, and the sequence of the crassostrea gigas disease resistance related gene marker is shown as a base sequence SEQ ID No.1. According to the invention, polymorphic sites in a promotor region of a disease-resistant related RNA helicase gene of crassostrea gigas are screened, a disease-resistant related gene marker is explored, and a disease-resistant gene marker-assisted breeding method is established. The method has the characteristics of strong pertinence, high breeding efficiency, simplicity, convenience and rapidness in operation and the like, and is suitable for screening related disease-resistant markers of shellfish and breeding good disease-resistant varieties.

Description

Technical Field

[0001] The present invention belongs to the field of aquaculture biotechnology, and specifically relates to a gene marker related to disease resistance of the Pacific oyster (Crassostrea gigas) and its application. Background Art

[0002] Shellfish, especially oysters, play an important role in China's mariculture. In 2022, the aquaculture output of oysters in China was 6.1995 million tons, accounting for 39.50% of the mariculture output of shellfish (China Fisheries Statistical Yearbook 2023). The Pacific oyster (Crassostrea gigas) is a widely cultured variety worldwide. However, in recent years, with the continuous expansion of aquaculture density and scale, diseases of cultured Pacific oysters have occurred frequently due to reasons such as germplasm degradation, environmental stress, and disease infection. In particular, large-scale deaths often occur in summer, and the disease problem in the Pacific oyster aquaculture industry has become increasingly prominent, causing significant economic losses and seriously threatening the healthy development of the shellfish aquaculture industry. Cultivating stress-resistant and disease-resistant excellent varieties is an urgent need for the green and healthy development of China's Pacific oyster aquaculture industry.

[0003] Currently, conventional selective breeding and crossbreeding are still important means for cultivating new varieties. However, due to their dependence on phenotypic selection, they have disadvantages such as long cycle and instability, and cannot meet the current urgent demand for improved varieties. Therefore, it is necessary to accelerate the cultivation of stress-resistant and disease-resistant excellent varieties through molecular means. Marker-assisted selection (MAS) refers to a method of assisting in screening target traits through genetic markers linked to target trait genes. The prerequisite for MAS breeding is to identify genetic markers co-segregating or tightly linked to the target gene. Currently, the most commonly used genetic markers are DNA molecular markers that directly reflect genetic variation at the DNA level. Among them, SNPs have advantages such as wide distribution, large quantity, and easy large-scale batch detection, and have quickly become a research hotspot in MAS.

[0004] RNA helicase is a class of proteins that can bind to or reconstruct RNA using ATP and is an essential part of all organisms. It participates in many important biological processes such as cell transcription, ribosome assembly, mRNA translation, and double-stranded RNA unwinding. In recent years, more and more studies have shown that RNA helicase can enhance the disease resistance of organisms by regulating gene expression. The present invention has developed SNP markers related to the disease resistance of the Pacific oyster for RNA helicase genes, laying a foundation for MAS of disease resistance in the Pacific oyster. Summary of the Invention

[0005] The purpose of the present invention is to provide a gene marker related to the disease resistance of the Pacific oyster and its application.

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

[0007] A Crassostrea gigas disease resistance-related gene marker, the Crassostrea gigas disease resistance-related gene marker is located in the promoter region of RNA helicase, and its sequence is shown as the nucleotide sequence of SEQ ID No. 1.

[0008] Furthermore, the Crassostrea gigas disease resistance-related gene marker locus is one or several of -224T, -371A, -383C, -448A, -487T, -716A, -737T, and -825C upstream of the start codon of the RNA helicase promoter.

[0009] Even further, the Crassostrea gigas disease resistance-related gene marker locus is -224T, -371A, -383C, -448A, -487T, -716A, -737T, and -825C upstream of the start codon of the RNA helicase promoter.

[0010] A method for obtaining the above-mentioned Crassostrea gigas disease resistance-related gene marker, by analyzing the polymorphism of the promoter region of the Crassostrea gigas RNA helicase gene; through the screening of gene phenotypes in the disease-resistant population and the normal population, and then obtaining the disease resistance-related gene marker.

[0011] The genomic DNA is extracted from the adductor muscle of Crassostrea gigas as the gene amplification template for the RNA helicase promoter region, and using the primers CgHelicF0, CgHelicR0 and CgHelicF1, CgHelicR1, the nucleotide sequence shown as SEQ ID No. 1 is amplified by nested PCR technology;

[0012] The primer sequence CgHelicF0 is 5′-taacgctgagggtaatagaacgg-3′, CgHelicR0 is 5′-atggcttacctgaacgctgacc-3′, CgHelicF1 is 5′-tctgtctcaagttattgcttccatc-3′, and CgHelicR1 is 5′-catatccctatcgttattcttttgc-3′.

[0013] An application of the above-mentioned Crassostrea gigas disease resistance-related gene marker, the application of the Crassostrea gigas disease resistance-related gene marker in screening disease-resistant individuals.

[0014] An application of the above-mentioned Crassostrea gigas disease resistance-related gene marker, the application of the Crassostrea gigas disease resistance-related gene marker in assisted breeding.

[0015] A method for breeding disease-resistant varieties, using the disease-resistant related RNA helicase gene marker as described above, breeding the Pacific oyster carrying this disease-resistant related gene marker, and cultivating the offspring; at the same time, performing pathogen stress treatment, and screening out the oysters that carry both the disease-resistant related RNA helicase gene marker and disease resistance ability for multiple generations of breeding and cultivation, which are the disease-resistant new varieties.

[0016] The characteristics of the present invention compared with the existing technologies are as follows:

[0017] By means of molecular biology techniques, the present invention uses the Pacific oyster as the material to discover the disease-resistant related RNA helicase gene marker of cultured shellfish in China, and preliminarily establishes the molecular marker-assisted breeding technology for the disease-resistant related RNA helicase gene of the Pacific oyster. This technology has the characteristics of simple and rapid operation, high breeding efficiency, short cycle, etc., opens up a new molecular breeding technology approach for the cultivation of disease-resistant shellfish varieties, and has important theoretical significance and application value for the breeding of disease-resistant varieties of cultured shellfish. Brief Description of the Drawings

[0018] Figure 1 It is the polymorphism site map of the promoter region of the RNA helicase gene of the Pacific oyster provided by the embodiment of the present invention.

[0019] Figure 2 It is the sequencing map of different genotypes of the promoter region of the RNA helicase gene of the Pacific oyster provided by the embodiment of the present invention.

[0020] Figure 3 It is the distribution frequency of different genotypes of RNA helicase in the disease-resistant population and sensitive population of the Pacific oyster provided by the embodiment of the present invention, as well as the corresponding chi-square test map. Detailed Embodiments

[0021] Screen out the RNA helicase gene marker related to the disease resistance of the Pacific oyster, establish the corresponding molecular marker-assisted breeding method for the Pacific oyster, and provide gene markers and technical methods for the cultivation of disease-resistant varieties of the Pacific oyster.

[0022] The present invention obtains the disease-resistant related gene marker of the polymorphism site in the promoter region of the RNA helicase gene related to the disease resistance of the Pacific oyster, and thus conducts the disease-resistant gene marker-assisted breeding method. The present invention has the characteristics of strong pertinence, high breeding efficiency, simple and rapid operation, etc., and is applicable to the screening of disease-resistant related markers of shellfish and the breeding of disease-resistant excellent varieties.

[0023] The disease resistance described in the present invention refers to resistance to pathogenic Halomonas salina.

[0024] Example 1

[0025] Obtaining the RNA helicase gene marker related to the disease resistance of the Pacific oyster, which is obtained according to the following steps:

[0026] 1. Polymorphism analysis of the promoter region of the RNA helicase gene in the Pacific oyster [[Crassostrea gigas]]

[0027] Genomic DNA was extracted from the adductor muscle of the Pacific oyster [[Crassostrea gigas]] as a template for gene amplification according to the method described in molecular cloning. The promoter region of the RNA helicase gene in the genomic DNA of 6 individuals from each of the disease-resistant population and the common population was amplified by nested PCR using the primers CgHelicF0, CgHelicR0, CgHelicF, and CgHelicR. The cloned DNA sequences were ligated into T vectors, and 2 clones were selected from each individual for sequencing. The nucleotide sequence obtained by sequencing was 1077 bp (shown as the base sequence of SEQ ID No.1). After alignment and analysis, a total of 12 SNP sites were found. After screening, 8 of these sites showed significant statistical differences between the two populations, namely -224G / T, -371G / A, -383A / C, -448A / T, -487G / T, -716A / T, -737C / T, and -825G / C (see Figure 1 ); the primer sequences were CgHelicF0: 5′-taacgctgagggtaatagaacgg-3′, CgHelicR0: 5′-atggcttacctgaacgctgacc-3′, CgHelicF: 5′-tctgtctcaagttattgcttccatc-3′, and CgHelicR: 5′-catatccctatcgttattcttttgc-3′.

[0028] 2. Screening of RNA helicase gene markers related to disease resistance in the Pacific oyster [[Crassostrea gigas]]

[0029] Genomic DNA was randomly extracted from 40 common individuals and 40 individuals resistant to [[Halomonas pathogenic]] as templates for gene marker screening. A set of nested PCR primers (CgHelicF0 and CgHelicR0, CgHelicF1 and CgHelicR1 primers) were used to amplify the genomic DNA of individuals from the disease-resistant population and the common population by nested PCR. The PCR-amplified RNA helicase promoter region sequence products were purified and then sequenced. Among the 8 polymorphic sites with significant differences between the two populations, 3 genotypes were detected at 5 polymorphic sites, namely -371G / A, -383A / C, -448A / T, -487G / T, and -716A / T, which showed 3 peak patterns on the sequencing maps respectively; for the other 3 sites, 2 genotypes were detected, namely -224G / T, -737C / T, and -825G / C, which showed 2 peak patterns on the sequencing maps respectively (see Figure 2 ).

[0030] The frequencies of different genotypes in the disease-resistant population and the normal population were statistically analyzed (see Table 1). After performing Chi-square Test analysis using SPSS 11.5 software, it was found that the frequencies of individuals with genotypes -224T / T, -371A / A, -383C / C, -448A / A, -487T / T, -716A / A, -737T / T, and -825C / C in the disease-resistant population were significantly higher than those in the normal population. While the frequencies of individuals with genotypes -224G / G, -371G / G, -383A / A, -448T / T, -487G / G, -716T / T, -737C / C, and -825G / G in the normal population were significantly higher than those in the disease-resistant population.

[0031] Therefore, -224T / T, -371A / A, -383C / C, -448A / A, -487T / T, -716A / A, -737T / T, and -825C / C can be used as gene markers related to disease resistance of RNA helicase.

[0032] Table 1: Chi-square test of the distribution frequencies of different genotypes of RNA helicase in the disease-resistant population and the normal population of Crassostrea gigas

[0033]

[0034] Using the online website SHEsis to construct haplotypes for the loci -224T, -371A, -383C, -448A, -487T, -716A, -737T, and -825C, a haplotype TACATATC significantly related to the disease-resistant population was obtained:

[0035] Table 2. Distribution of each haplotype in the promoter region of the gene in different populations

[0036]

[0037] 3. Rapid screening of individuals carrying gene markers related to disease resistance

[0038] Make holes in the shell of Crassostrea gigas, take 50 mg of the mantle of Crassostrea gigas to extract genomic DNA as a cloning template, and perform nested PCR amplification using CgHelicF0, CgHelicR0, CgHelicF1, and CgHelicR1 as primers. After purifying the PCR products, use a 3730 sequencer for sequencing analysis. Refer to sequencing map 2 to select as many individuals containing genotypes -224T / T, -371A / A, -383C / C, -448A / A, -487T / T, -716A / A, -737T / T, and -825C / C as heat-resistant individuals.

[0039] Example 2

[0040] Marker-assisted breeding method for RNA helicase gene related to disease resistance in Crassostrea gigas

[0041] Using the genotypes -224T / T, -371A / A, -383C / C, -448A / A, -487T / T, -716A / A, -737T / T, and -825C / C in the promoter region of RNA helicase that frequently appear in disease-resistant populations as disease-resistance related RNA helicase gene markers; specifically:

[0042] Propagate as many Crassostrea gigas carrying these disease-resistance related gene markers as possible, breed offspring, clone the RNA helicase gene in the offspring, and study its polymorphism; study the inheritance law of the disease-resistance related RNA helicase gene markers and their relationship with the disease resistance ability of Crassostrea gigas, and select the seedling oysters that contain both the disease-resistance related RNA helicase gene markers and have improved disease resistance ability for multi-generation propagation and cultivation to establish disease-resistant excellent varieties.

[0043] Furthermore, drill holes in the shells of 100 oysters to be selected, take 30 mg of the mantle of Crassostrea gigas to extract DNA as a cloning template, use the primers CgHelicF0, CgHelicR0, CgHelicF, and CgHelicR to amplify the promoter region sequence of the RNA helicase gene by nested PCR, purify the PCR product and perform sequencing analysis. According to the sequencing results, finally select individuals with genotypes -224T / T, -371A / A, -383C / C, -448A / A, -487T / T, -716A / A, -737T / T, and -825C / C (disease-resistant genotypes), and use these individuals as disease-resistant parents to breed and establish families.

[0044]

Claims

1. A gene marker related to disease resistance in Crassostrea gigas, characterized in that: The gene marker related to disease resistance in the Pacific oyster Crassostrea gigas is located in the promoter region of RNA helicase, and its sequence is shown as the nucleotide sequence of SEQ ID No.

1.

2. The gene marker related to disease resistance in Crassostrea gigas according to claim 1, characterized in that: The gene marker locus related to disease resistance in the Pacific oyster Crassostrea gigas is one or several of the sites -224T, -371A, -383C, -448A, -487T, -716A, -737T, and -825C upstream of the start codon of the RNA helicase promoter.

3. The gene marker related to disease resistance in Crassostrea gigas according to claim 2, characterized in that: The gene marker locus related to disease resistance in the Pacific oyster Crassostrea gigas is -224T, -371A, -383C, -448A, -487T, -716A, -737T, and -825C upstream of the start codon of the RNA helicase promoter.

4. A method for obtaining the gene marker related to disease resistance in Crassostrea gigas according to claim 1, characterized in that: Through the polymorphism analysis of the promoter region of the RNA helicase gene in the Pacific oyster Crassostrea gigas; according to the screening of gene phenotypes in the disease-resistant population and the normal population, the gene marker related to disease resistance is obtained.

5. The method for obtaining the gene marker related to disease resistance in Crassostrea gigas according to claim 4, characterized in that: The genomic DNA is extracted from the adductor muscle of the Pacific oyster Crassostrea gigas as the gene amplification template for the promoter region of the RNA helicase, and the primers CgHelicF0, CgHelicR0, CgHelicF1, and CgHelicR1 are used to amplify the nucleotide sequence shown as SEQ ID No.1 by nested PCR technology; The primer sequence CgHelicF0 is 5′-taacgctgagggtaatagaacgg-3′, CgHelicR0 is 5′-atggcttacctgaacgctgacc-3′, CgHelicF1 is 5′-tctgtctcaagttattgcttccatc-3′, and CgHelicR1 is 5′-catatccctatcgttattcttttgc-3′.

6. An application of the gene marker related to disease resistance in Crassostrea gigas according to claim 1, characterized in that: The application of the gene marker related to disease resistance in the Pacific oyster Crassostrea gigas in screening disease-resistant individuals.

7. An application of the gene marker related to disease resistance in Crassostrea gigas according to claim 1, characterized in that: The application of the gene marker related to disease resistance in the Pacific oyster Crassostrea gigas in assisted breeding.

8. A method for breeding disease-resistant varieties, characterized in that: Using the disease-resistant related RNA helicase gene marker described in claim 1, the Pacific oysters carrying this disease-resistant related gene marker are bred to cultivate offspring.

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