KASP molecular marker combination for resistance to Vibrio harveyi in Cynoglossus semilaevis and its application in disease-resistant breeding of Cynoglossus semilaevis

Abstract

The present invention belongs to the field of aquatic biotechnology and molecular marker-assisted breeding technology, and specifically relates to a KASP molecular marker combination for resistance to Vibrio harveyi disease in semi-smooth tongue sole and its application in disease-resistant breeding of semi-smooth tongue sole. The KASP molecular marker combination of the present invention comprises three SNP sites, located at sites 17314099, 17325496, and 17355989 of chromosome 14 of semi-smooth tongue sole, respectively. When the genotypes thereof are CC, AA, and GG, respectively, the disease resistance of the semi-smooth tongue sole measured is strong. The present invention also provides primers for detecting the genotype polymorphism of the SNP sites. Utilizing the KASP molecular marker combination of the present invention, it is possible to quickly detect the genotype of Vibrio harveyi disease resistance in a semi-smooth tongue sole breeding population, and then screen for disease-resistant individuals, thereby greatly improving the efficiency of disease-resistant breeding of semi-smooth tongue sole and contributing to the rapid cultivation of new varieties of semi-smooth tongue sole with strong disease resistance.

Description

Technical Field

[0001] The invention belongs to the field of aquatic biotechnology and molecular marker-assisted breeding technology, and particularly relates to a KASP molecular marker combination for resistance to Vibrio harveyi disease in semilaevis tongue sole and its application in disease-resistant breeding of semilaevis tongue sole. Background Art

[0002] Semi-smooth tongue sole (Cynoglossus semi-smooth) is a key marine aquaculture species in my country. In 2017, it was included in the National Marine Industry Technology System. In recent years, the scale of its aquaculture has gradually expanded, primarily through indoor, factory-based flow-through aquaculture and recirculating aquaculture. However, under factory-based aquaculture conditions, due to factors such as high stocking densities, semi-smooth tongue sole is highly susceptible to outbreaks of vibriosis, caused by the bacterium Vibrio harveyi. This disease is extremely difficult to control, with mortality rates exceeding 30% and potentially reaching over 80%. Vibrio harveyi has become a critical limiting factor in the development of the semi-smooth tongue sole aquaculture industry. Therefore, the development of low-cost and efficient molecular markers for disease resistance is urgently needed.

[0003] KASP (Kompetitive Allele Specific PCR) is a molecular biology technique that provides rapid, sensitive, and accurate SNP polymorphism analysis. It is the latest generation of molecular markers with very low detection costs. Currently, KASP molecular markers have been widely used in molecular breeding of crops such as wheat, but their application in molecular breeding of aquatic animals is still in its infancy. In this field, whole-genome selection breeding has replaced microsatellite markers as the mainstream breeding method, offering high selection efficiency and accuracy. However, whole-genome selection breeding requires a large number of SNP markers (at least several thousand) and the genotypes and phenotypes of a large number of individuals (generally over 1,000) to construct a reference population to ensure high selection efficiency. To obtain a sufficient number of SNP genotypes, a large number of individuals from the reference population and candidate populations must undergo whole-genome resequencing or targeted sequencing using breeding arrays to obtain high or very high SNP genotype densities. This results in high sequencing costs per sample and a long genotyping cycle of 1-2 months. The cost of genotyping with KASP markers is significantly lower than the former, and large-scale sample typing can be completed within 3 days.

[0004] Therefore, given the low-cost, high-efficiency, and high-stability characteristics of KASP molecular markers, as well as the urgency of disease-resistant breeding of semi-smooth tongue sole, by developing KASP molecular markers for resistance to Vibrio harveyi disease, accurate genotyping of a large number of samples can be performed in a short period of time, and disease-resistant individuals can be quickly identified, thereby accelerating the disease-resistant breeding process of semi-smooth tongue sole and assisting in the cultivation of new disease-resistant varieties. Summary of the Invention

[0005] The purpose of the present invention is to provide a KASP molecular marker combination for half-smooth tongue sole resistance to Vibrio harveyi disease and its application in half-smooth tongue sole disease resistance breeding, to provide a favorable tool for rapid, efficient and accurate detection of half-smooth tongue sole resistance to Vibrio harveyi disease, and to improve the efficiency of half-smooth tongue sole disease resistance breeding.

[0006] The technical solutions of the present invention are as follows:

[0007] The KASP molecular marker combination for resistance to Vibrio harveyi disease in semilaevis tongue sole described in the present invention comprises three SNP sites, namely Cys_KASP1, Cys_KASP2 and Cys_KASP3, which are located at sites 14:17314099, 14:17325496 and 14:17355989 of chromosome 14 of semilaevis tongue sole, respectively, and the corresponding SNP bases are C / T, C / A and G / T, respectively.

[0008] When the genotype of chromosome 14:17314099 where Cys_KASP1 is located is homozygous CC, the genotype of chromosome 14:17325496 where Cys_KASP2 is located is homozygous AA, and the genotype of chromosome 14:17355989 where Cys_KASP3 is located is homozygous GG, it means that the tested semilaevis tongue sole is resistant to Vibrio harveyi disease.

[0009] The present invention also provides detection primers for the KASP molecular marker combination for resistance to Vibrio harveyi infection in semilaevis sole, wherein each KASP marker comprises two specific primers (Fam and Hex) and one universal primer (Common);

[0010] The nucleotide sequences of the Cys_KASP1-labeled primers are as follows:

[0011] Specific primer Fam:

[0012] 5′-GAAGGTGACCAAGTTCATGCTGTTACTGGGTAGCTTCTCTACGGT-3′, as shown in SEQ ID NO: 1;

[0013] Specific primer Hex:

[0014] 5′-GAAGGTCGGAGTCAACGGATTGTTACTGGGTAGCTTCTCTACGGC-3′, as shown in SEQ ID NO: 2;

[0015] Common primer: 5'-TTCCATGACAATAACACGTTGACC-3', as shown in SEQ ID NO: 3;

[0016] The nucleotide sequences of the Cys_KASP2-labeled primers are as follows:

[0017] Specific primer Fam:

[0018] 5'-GAAGGTGACCAAGTTCATGCTATGCCAGGATTAAGGGGAAATTG-3', as shown in SEQ ID NO: 4;

[0019] Specific primer Hex:

[0020] 5'-GAAGGTCGGAGTCAACGGATTATGCCAGGATTAAGGGGAAATTT-3', as shown in SEQ ID NO: 5;

[0021] Common primer: 5'-GCTTTCACAGAGCAGCAGCTATAA-3'; as shown in SEQ ID NO: 6;

[0022] The nucleotide sequences of the Cys_KASP3-labeled primers are as follows:

[0023] Specific primer Fam:

[0024] 5'-GAAGGTGACCAAGTTCATGCTGACACAATGCACTAGGGTTTGGT-3', as shown in SEQ ID NO: 7;

[0025] Specific primer Hex:

[0026] 5′-GAAGGTCGGAGTCAACGGATTGACACAATGCACTAGGGTTTGGG-3′, as shown in SEQ ID NO: 8;

[0027] Common primer Common: 5′-CAATGCTGAAGAGGATTAGCAGGT-3′, as shown in SEQ ID NO: 9.

[0028] The KASP molecular marker combination and primers for resistance to Vibrio harveyi disease in half-smooth tongue sole can be applied to disease-resistant breeding of half-smooth tongue sole.

[0029] The development of the KASP molecular marker combination for the semilaevis sole against Vibrio harveyi disease comprises the following steps:

[0030] (1) At least 500 semi-laevis tongue soles were artificially infected with Vibrio harveyi to obtain the death time and fin ray samples of all individuals. The artificial infection method of Vibrio harveyi was intraperitoneal injection, and the injection dose was the absolute lethal dose (LD100).

[0031] (2) Fin ray DNA was extracted and genome resequencing and genotyping were performed. Genotype and death time typology were used for whole genome association analysis to screen for significant SNP markers associated with Vibrio resistance. The dominant genotype frequency of the top three most significant SNPs in the last 30% of individuals that died was analyzed to determine the disease-resistant genotype (genotype frequency greater than 0.5).

[0032] (3) Extract 200 bp of flanking sequences upstream and downstream of the three most significant SNP markers. Use software to design two specific primers (Fam and Hex) and one universal primer (Common) for the SNP sites and flanking sequences. Connect fluorescent linker sequences to the 5' ends of the two specific primers. Extract the DNA of the sample to be tested and perform PCR amplification. The PCR reaction system consisted of 0.001 μL of the specific primer Fam (1 μM), 0.001 μL of the specific primer Hex (1 μM), 0.003 μL of the universal primer C (1 μM), 0.20 μL of 2× KASP Master Mix, and 20 ng of the test sample DNA, brought to 0.80 μL with ultrapure water. PCR amplification conditions were 1 cycle at 94°C for 15 min, 10 cycles at 95°C for 20 s, then 65–56°C for 60 s, with the temperature decreasing by 0.8°C each cycle, and 30 cycles at 94°C for 20 s, then 57°C for 60 s. After the reaction, fluorescence was measured using a microplate reader, and data analysis and genotyping were performed. If the Cys_KASP1 marker genotype was CC, the Cys_KASP2 marker genotype was AA, and the Cys_KASP3 marker genotype was GG, the individual was considered resistant; otherwise, it was considered non-resistant.

[0033] Compared with the existing technology, the present invention has the following beneficial effects: the present invention has developed the KASP molecular marker combination for the first time for the half-smooth tongue sole's resistance to Vibrio harveyi disease. The KASP molecular marker combination can be used to quickly, accurately and efficiently perform genotyping on half-smooth tongue sole, realize batch screening of disease-resistant individuals, and provide strong technical support for disease-resistant breeding of half-smooth tongue sole. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The genotyping results of Cys_KASP1 for 94 samples of Semilaevis semilaevis and 2 blank controls, red dots represent homozygous CC genotype, purple dots represent heterozygous TC genotype, blue dots represent homozygous TT genotype, and black dots represent blank controls;

[0035] Figure 2The genotyping results of Cys_KASP2 for 94 samples of Semilaevis semilaevis and 2 blank controls, red dots represent homozygous AA genotype, purple dots represent heterozygous CA genotype, blue dots represent homozygous CC genotype, and black dots represent blank controls;

[0036] Figure 3 Figure 3 shows the genotyping results of Cys_KASP3 for 94 Semilaevis semilaevis samples and 2 blank controls. Red dots represent homozygous GG genotypes, purple dots represent heterozygous TG genotypes, blue dots represent homozygous TT genotypes, and black dots represent blank controls. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific examples. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Unless otherwise specified, the equipment and reagents used in each example are conventionally commercially available.

[0038] Example 1

[0039] Development of KASP molecular marker combination for resistance to Vibrio harveyi in Cynoglossus semilaevis

[0040] (1) Experiment on artificial infection of semi-smooth tongue sole with Vibrio harveyi

[0041] Methods: A total of 700 half-smooth tongue sole fry (10-15 cm in length) were purchased from a semi-smooth tongue sole breeding company in Tangshan. Artificial infection with Vibrio harveyi was performed using intraperitoneal injection under laboratory conditions. The injection dose was the absolute lethal dose (LD100). The death time of each fish (number of days from infection to death) was recorded, and a small number of fin ray samples were collected and fixed with anhydrous alcohol.

[0042] (2) Resequencing and genotyping

[0043] Fin ray samples of all the deceased individuals were used for DNA extraction and genome resequencing at a depth of 15X. After sequencing, the resequencing data were cleaned and filtered, and SNP genotypes were extracted. Genome-wide association analysis was performed using genotypes and death time phenotypes. Five significant SNP markers were screened on chromosome 14, and the three most significant SNPs (sites 14:17314099, 14:17325496, and 14:17355989) were selected to check the frequencies of dominant genotypes in the last 30% of individuals (210) who died. It was found that the dominant genotype at site 14:17314099 was homozygous CC, the dominant genotype at site 14:17325496 was homozygous AA, and the dominant genotype at site 14:17355989 was homozygous GG. The frequencies of these three dominant genotypes in the last 30% of individuals who died were 62.5%, 61.6%, and 61.5%, respectively.

[0044] (3) Primer design and PCR amplification

[0045] The flanking sequences of 200 bp upstream and downstream of the three most significant SNP marker sites were extracted, and two specific primers (Fam and Hex) and one universal primer (Common) were designed for the SNP sites and flanking sequences using software.

[0046] 14: Two specific primers and one common primer for site 17314099 are: specific primer Fam: 5'-GAAGGTGACCAAGTTCATGCTGTTACTGGGTAGCTTCTCTACGGT-3', as shown in SEQ ID NO: 1, specific primer Hex: 5'-GAAGGTCGGAGTCAACGGATTGTTACTGGGTAGCTTCTCTACGGC-3', as shown in SEQ ID NO: 2, common primer Common: 5'-TTCCATGACAATAACACGTTGACC-3', as shown in SEQ ID NO: 3;

[0047] 14: Two specific primers and one common primer for site 17325496 are: specific primer Fam: 5'-GAAGGTGACCAAGTTCATGCTATGCCAGGATTAAGGGGAAATTG-3', as shown in SEQ ID NO: 4, specific primer Hex: 5'-GAAGGTCGGAGTCAACGGATTATGCCAGGATTAAGGGGAAATTT-3', as shown in SEQ ID NO: 5, common primer Common: 5'-GCTTTCACAGAGCAGCAGCTATAA-3', as shown in SEQ ID NO: 6;

[0048] 14: The two specific primers and one universal primer for site 17355989 are: specific primer Fam: 5'-GAAGGTGACCAAGTTCATGCTGACACAATGCACTAGGGTTTGGT-3', as shown in SEQ ID NO: 7, specific primer Hex: 5'-GAAGGTCGGAGTCAACGGATTGACACAATGCACTAGGGTTTGGG-3', as shown in SEQ ID NO: 8, and universal primer Common: 5'-CAATGCTGAAGAGGATTAGCAGGT-3', as shown in SEQ ID NO: 9.

[0049] The 5' ends of two primers specific for the three SNPs were linked to fluorescent linker sequences (Fam-GAAGGTGACCAAGTTCATGCT and Hex-GAAGGTCGGAGTCAACGGATT, respectively, as shown in SEQ ID NO: 10 and SEQ ID NO: 11). PCR amplification was performed on 94 randomly selected DNA samples from the 700 individuals used in the infection experiment, along with two blank controls. The PCR reaction system consisted of 0.001 μL of the specific primer Fam (1 μM), 0.001 μL of the specific primer Hex (1 μM), 0.003 μL of the universal primer C (1 μM), 0.20 μL of the 2× KASP Master Mix, and 20 ng of the test sample DNA, brought to 0.80 μL with ultrapure water. PCR amplification conditions included one cycle of 94°C for 15 min, 10 cycles of 95°C for 20 s, 65–56°C for 60 s, with the temperature decreasing by 0.8°C between cycles, and 30 cycles of 94°C for 20 s, 57°C for 60 s. After the reaction, fluorescence was measured using a microplate reader, and data analysis and genotyping were performed. All three SNPs were successfully typed, indicating that the three SNP markers were successfully converted into KASP markers. The KASP markers corresponding to the three SNP markers 14:17314099, 14:17325496 and 14:17355989 are named Cys_KASP1, Cys_KASP2 and Cys_KASP3 respectively. Figure 1 、 Figure 2 and Figure 3 If the genotypes of the above three loci of a certain semi-smooth tongue sole are CC, AA and GG respectively, then the semi-smooth tongue sole is resistant to Vibrio harveyi disease, otherwise it is considered as a non-disease-resistant individual.

[0050] Example 2

[0051] Application of KASP molecular marker combination for resistance to Vibrio harveyi in disease resistance breeding of Semilaevis semilaevis

[0052] In order to further verify the application effect of the KASP molecular marker combination of the present invention in disease resistance breeding, this example is carried out, which specifically includes the following steps:

[0053] (1) Genotyping of candidate semi-smooth tongue sole

[0054] 384 semilaevis (average weight 45 g) were randomly collected from a colony of semilaevis, all electronically labeled, and the electronic tag number for each individual was recorded. A small number of fin ray samples were also collected, and DNA was extracted. Genotyping was performed according to the method in Example 1. Ultimately, 110 semilaevis showed CC typing using the Cys_KASP1 marker, AA typing using the Cys_KASP2 marker, and GG typing using the Cys_KASP3 marker. These 110 semilaevis were resistant to the disease, while the remaining 274 individuals were not resistant.

[0055] (2) Determination of disease resistance of resistant and non-resistant individuals of Cynoglossus semilaevis

[0056] By scanning the electronic markers of each individual, 110 disease-resistant individuals and 274 non-resistant individuals were selected. Under laboratory conditions, these individuals were artificially infected with Vibrio harveyi via intraperitoneal injection at a median lethal dose (LD50). The mortality rate for the 110 disease-resistant individuals was 8.2%, while the mortality rate for the 274 non-resistant individuals was 69.0%. These results demonstrate that the KASP molecular marker combination of the present invention is effective in breeding disease-resistant semilaevis.

Claims

1. Application of the KASP molecular marker combination for resistance to Vibrio harveyi in the preparation of a product for screening individuals of Semilaevis semilaevis for resistance to Vibrio harveyi; The KASP molecular marker combination comprises three SNP sites, namely Cys_KASP1, Cys_KASP2 and Cys_KASP3, which are located at sites 14:17314099, 14:17325496 and 14:17355989 of chromosome 14 of semi-smooth tongue sole, respectively, and the corresponding SNP bases are C / T, C / A and G / T, respectively; when the genotype of site 14:17314099 on chromosome 14 where Cys_KASP1 is located is homozygous CC, and the genotype of site 14:17325496 on chromosome 14 where Cys_KASP2 is located is homozygous AA, and the genotype of site 14:17355989 on chromosome 14 where Cys_KASP3 is located is homozygous GG, it represents that the semi-smooth tongue sole to be tested is an individual resistant to Vibrio harveyi disease; Each KASP marker contains two specific primers and one universal primer; The nucleotide sequences of the primers for Cys_KASP1 labeling are as follows: the specific primer Fam is shown in SEQ ID NO: 1, the specific primer Hex is shown in SEQ ID NO: 2, and the universal primer Common is shown in SEQ ID NO: 3; The nucleotide sequences of the primers for Cys_KASP2 labeling are as follows: the specific primer Fam is shown in SEQ ID NO: 4, the specific primer Hex is shown in SEQ ID NO: 5, and the universal primer Common is shown in SEQ ID NO: 6; The nucleotide sequences of the primers labeled Cys_KASP3 are as follows: the specific primer Fam is shown in SEQ ID NO: 7, the specific primer Hex is shown in SEQ ID NO: 8, and the universal primer Common is shown in SEQ ID NO: 9.

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