KASP marker combination for detecting vibrio harveyi disease resistance of cynoglossus semilaevis and application of KASP marker combination

Abstract

The invention belongs to the technical field of molecular marker-assisted breeding of aquatic animals, and particularly relates to a KASP marker combination for detecting vibrio harveyi resistance of cynoglossus semilaevis and application of the KASP marker combination. The KASP marker combination disclosed by the invention is respectively positioned at the 14: 17 304461 site, the 14: 17 307045 site, the 14: 17 310738 site and the 14: 17 303501 site of the 14 # chromosome of the cynoglossus semilaevis, and the base polymorphism types of the KASP marker combination are C / T, C / G, C / T and A / G respectively. According to the invention, after genotyping is carried out on a to-be-detected individual, the to-be-detected individual is scored according to the vibrio harveyi disease resistance excellent and inferior genotypes, so that the vibrio harveyi disease resistance of the to-be-detected individual is measured; by utilizing the KASP molecular marker combination disclosed by the invention, the vibrio harveyi resistance of the cynoglossus semilaevis can be detected at low cost, high efficiency and high accuracy, and the disease-resistant breeding efficiency of the cynoglossus semilaevis is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted breeding of aquatic animals, and particularly relates to a KASP marker combination for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease and an application thereof. Background Art

[0002] KASP (Kompetitive Allele-Specific PCR) marker is a SNP precise typing method based on PCR technology. Its principle is to design two allele-specific primers and one universal primer, and use fluorescent signals to distinguish different alleles, so as to achieve rapid typing of SNP loci. Due to its high accuracy, low cost and high flexibility, it is widely used in crop and animal breeding, and also plays its unique advantages in human genetic disease detection, pharmacogenomics, and disease susceptibility detection. However, its application in aquatic animal disease resistance detection and disease resistance breeding is relatively lagging, and there is no report on KASP marker in this regard. In the past decade or so, SSR markers have been mainly used for molecular marker-assisted breeding in aquatic animal disease resistance breeding. In recent years, with the rapid development of high-throughput sequencing technology, the use of SNP markers in the whole genome for whole genome selection breeding has become the mainstream. The efficiency of using SSR markers for assisted breeding is very low and the accuracy is not high. Although the efficiency and accuracy of whole genome selection breeding are greatly improved by mining a large number of SNP markers through whole genome sequencing, the cost is too high. Therefore, there is an urgent need to develop low-cost and highly accurate molecular markers and genotyping methods to detect the disease resistance of aquatic animals and conduct disease-resistant breeding.

[0003] The development of the half-smooth tongue sole aquaculture industry is severely restricted by vibriosis, which is mainly caused by Vibrio harveyi. How to detect the half-smooth tongue sole's resistance to Vibrio harveyi disease with low cost, high efficiency and high accuracy has become a major problem in aquaculture and breeding production. In order to improve the disease resistance of half-smooth tongue sole seedlings, the team of Academician Chen Songlin from the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences has established a whole genome selection breeding technology for the resistance to Vibrio harveyi disease. However, this technology relies on sequencing technology for genotyping. The high cost of sequencing limits its large-scale application in half-smooth tongue sole aquaculture and breeding companies. It is urgent to develop a low-cost, high-efficiency and high-accuracy genotyping technology to detect the half-smooth tongue sole's resistance to Vibrio harveyi disease on a large scale, thereby promoting the mass production and promotion of disease-resistant seedlings. Summary of the invention

[0004] The object of the present invention is to provide a KASP marker combination for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease, detect the disease resistance of half-smooth tongue sole by detecting the genotype of specific sites of the individual to be tested, screen out disease-resistant parents or disease-resistant individuals, and use the disease-resistant parents to carry out batch production of disease-resistant seedlings.

[0005] The technical solution of the present invention is as follows: The KASP marker combination for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease of the present invention comprises four KASP markers, namely VH_KASP1, VH_KASP2, VH_KASP3 and VH_KASP4, which are respectively located at positions 14:17304461, 14:17307045, 14:17310738 and 14:17303501 of chromosome 14 of half-smooth tongue sole, and the base polymorphism types thereof are C / T, C / G, C / T and A / G, respectively.

[0006] The TT genotype at locus 14:17304461 of chromosome 14 is a disease-resistant genotype, the CT genotype is a general disease-resistant genotype, and the CC genotype is a non-disease-resistant genotype; the CC genotype at locus 14:17307045 of chromosome 14 is a disease-resistant genotype, the CG genotype is a general disease-resistant genotype, and the GG genotype is a non-disease-resistant genotype; the TT genotype at locus 14:17310738 of chromosome 14 is a disease-resistant genotype, the CT genotype is a general disease-resistant genotype, and the CC genotype is a non-disease-resistant genotype; the AA genotype at locus 14:17303501 of chromosome 14 is a disease-resistant genotype, the AG genotype is a general disease-resistant genotype, and the GG genotype is a non-disease-resistant genotype.

[0007] The method for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease by using the KASP marker combination comprises the following steps: (1) The disease-resistant genotype, general disease-resistant genotype, and non-disease-resistant genotype of the four KASP markers were scored as 2 points, 1 point, and 0 points, respectively; (2) The disease resistance of the tested half-smooth tongue sole individuals can be obtained based on the sum of the scores of the four KASP markers. The disease resistance score ranges from 0 to 8. The larger the score, the stronger the disease resistance, and the smaller the score, the weaker the disease resistance. That is, 8 points indicates the strongest disease resistance, and 0 points indicates the weakest disease resistance.

[0008] The present invention also provides detection primers matched with the KASP marker combination for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease, and each KASP marker comprises 2 specific primers and 1 universal primer; The nucleotide sequences of the primers for the VH_KASP1 marker are as follows: Specific primer Fam: 5'-GAAGGTGACCAAGTTCATGCTAAATGTTGAACGTTGAAGAGAAGCC-3', as shown in SEQ ID NO: 1; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTAAATGTTGAACGTTGAAGAGAAGCT-3’, as shown in SEQ ID NO: 2; Common primer: 5’-ACTTAAACCATCCATGGAACAGGAG-3’, as shown in SEQ ID NO: 3; The primer nucleotide sequences of VH_KASP2 marker are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTAAGCAAAACACTTTTCAGCAACAG-3’, as shown in SEQ ID NO: 4; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTAAGCAAAACACTTTTCAGCAACAC-3’, as shown in SEQ ID NO: 5; Common primer: 5’-GCCACTGCTTTAATGTATTCAATT-3’, as shown in SEQ ID NO: 6; The primer nucleotide sequences of VH_KASP3 marker are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTCTGCTTATATGGTAAAACATGAGC-3’, as shown in SEQ ID NO: 7; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTCTGCTTATATGGTAAAACATGAGT-3’, as shown in SEQ ID NO: 8; Common primer: 5’-AGTAAATCCTCCAAACCCGAAGAA-3’, as shown in SEQ ID NO: 9; The primer nucleotide sequences of VH_KASP4 marker are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTTGTATTCTACACAAAAGTACACAA-3’, as shown in SEQ ID NO: 10; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTTGTATTCTACACAAAAGTACACAG-3’, as shown in SEQ ID NO: 11; Universal primer Common: 5’-TACCCAAGACTTTGTTTGCTGGTT-3’, as shown in SEQ ID NO: 12.

[0009] The KASP marker combination and primers for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi can be applied to the detection of the ability of Cynoglossus semilaevis to resist Vibrio harveyi and disease-resistant breeding.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention has developed for the first time a KASP molecular marker combination and primers for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi; and for the first time, it is proposed that after genotyping the individual to be tested, the individual to be tested is scored according to the superior and inferior genotypes of resisting Vibrio harveyi, so as to measure the ability of the individual to be tested to resist Vibrio harveyi; the KASP molecular marker combination of the present invention can realize rapid, accurate and efficient detection of the ability of Cynoglossus semilaevis to resist Vibrio harveyi, can be used for rapid detection of the ability of Cynoglossus semilaevis parent population to resist Vibrio harveyi, and the selected parents resistant to Vibrio harveyi are used for batch production of disease-resistant fry. Description of the Drawings

[0011] Figure 1 The genotyping results of 94 Cynoglossus semilaevis samples and 2 blank controls by the VH_KASP1 marker. Red dots represent homozygous TT genotypes, purple dots represent heterozygous TC genotypes, blue dots represent homozygous CC genotypes, and black dots represent blank controls; Figure 2 The genotyping results of 94 Cynoglossus semilaevis samples and 2 blank controls by the VH_KASP2 marker. Red dots represent homozygous CC genotypes, purple dots represent heterozygous CG genotypes, blue dots represent homozygous GG genotypes, and black dots represent blank controls; Figure 3 The genotyping results of 94 Cynoglossus semilaevis samples and 2 blank controls by the VH_KASP3 marker. Red dots represent homozygous TT genotypes, purple dots represent heterozygous TC genotypes, blue dots represent homozygous CC genotypes, and black dots represent blank controls; Figure 4 The genotyping results of 94 Cynoglossus semilaevis samples and 2 blank controls by the VH_KASP4 marker. Red dots represent homozygous AA genotypes, purple dots represent heterozygous AG genotypes, blue dots represent homozygous GG genotypes, and black dots represent blank controls. Detailed Embodiments

[0012] To enable those of ordinary skill 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 embodiments. For those not specifying specific techniques or conditions in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications shall be followed. Unless otherwise specified, the equipment and reagents used in each embodiment are commercially available as normal.

[0013] Example 1 Development of a KASP marker combination for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi (1)Screening of SNP markers related to the ability of Cynoglossus semilaevis to resist Vibrio harveyi In 2022, 240 Cynoglossus semilaevis parental fish collected from 3 main Cynoglossus semilaevis farming areas in China and 600 parental fish of the core family population of Cynoglossus semilaevis preserved by the applicant were used. Male and female individuals with good gonadal maturity were selected from the above parental population to establish families, and a total of 122 families were established. When the fry grew to about 12 cm, 20 fry were randomly selected from each family and mixed in the same cement pond. After stabilizing for 2 weeks, an artificial infection experiment of Vibrio harveyi was carried out by intraperitoneal injection under laboratory conditions. The injection dose was the median lethal dose. Fin samples of all dead individuals (750 samples) and the final surviving individuals (834 samples) were collected to extract DNA, and targeted sequencing and genotyping were carried out using a 20K liquid chip. The genotypes of the above 1584 offspring individuals were imputed using the high-density genotypes of the parents. After filtering, a genome-wide association analysis was carried out in combination with the disease-resistant phenotypes (the phenotype of dead individuals was recorded as 0, and the phenotype of surviving individuals was recorded as 1). Finally, 132 significant SNP markers were screened on chromosome 14, and the 4 most significant SNP markers were selected. The locus information was 14:17304461, 14:17307045, 14:17310738, and 14:17303501 respectively, and their polymorphisms were T / C, C / G, T / C, and A / G respectively. Subsequently, the genotype frequencies of these 4 SNP markers in 750 dead individuals and 834 surviving individuals were respectively counted to determine the dominant genotypes. Finally, the disease-resistant dominant genotype at the 14:17304461 locus was determined to be TT, the disease-resistant dominant genotype at the 14:17307045 locus was determined to be CC, the disease-resistant dominant genotype at the 14:17310738 locus was determined to be TT, and the disease-resistant dominant genotype at the 14:17303501 locus was determined to be AA.

[0014] (2)Design of KASP marker primers and verification of genotyping effect To convert the above 4 SNP markers into KASP markers, first, the sequences of 200 bp upstream and downstream of each of the above 4 SNP markers were extracted, and 2 specific primer sequences (Fam and Hex) and 1 common primer sequence (Common) were designed using software. The final primer sequence information obtained is as follows: The primer sequences for the SNP marker at position 14:17304461 are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTAAATGTTGAACGTTGAAGAGAAGCC-3’, as shown in SEQ ID NO: 1; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTAAATGTTGAACGTTGAAGAGAAGCT-3’, as shown in SEQ ID NO: 2; Common primer: 5’-ACTTAAACCATCCATGGAACAGGAG-3’, as shown in SEQ ID NO: 3. The primer sequences for the SNP marker at position 14:17307045 are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTAAGCAAAACACTTTTCAGCAACAG-3’, as shown in SEQ ID NO: 4; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTAAGCAAAACACTTTTCAGCAACAC-3’, as shown in SEQ ID NO: 5; Common primer: 5’-GCCACTGCTTTAATGTATTCAATT-3’, as shown in SEQ ID NO: 6. The primer sequences for the SNP marker at position 14:17310738 are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTCTGCTTATATGGTAAAACATGAGC-3’, as shown in SEQ ID NO: 7; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTCTGCTTATATGGTAAAACATGAGT-3’, as shown in SEQ ID NO: 8; Common primer: 5’-AGTAAATCCTCCAAACCCGAAGAA-3’, as shown in SEQ ID NO: 9. The primer sequences for the SNP marker at position 14:17303501 are as follows: Specific primer Fam: 5’-GAAGGTGACCAAGTTCATGCTTGTATTCTACACAAAAGTACACAA-3’, as shown in SEQ ID NO: 10; Specific primer Hex: 5’-GAAGGTCGGAGTCAACGGATTTGTATTCTACACAAAAGTACACAG-3’, as shown in SEQ ID NO: 11; Common primer: 5’-TACCCAAGACTTTGTTTGCTGGTT-3’, as shown in SEQ ID NO: 12.

[0015] Subsequently, the 5'-ends of the two specific primers of the above 4 SNP markers were ligated with fluorescent linker sequences, and the fluorescent linker sequences were Fam-GAAGGTGACCAAGTTCATGCT and Hex-GAAGGTCGGAGTCAACGGATT respectively, as shown in SEQ ID NO: 13 and SEQ ID NO: 14 respectively. 94 samples were randomly selected from the 1584 DNA samples obtained from the above artificial infection experiment, and another 2 blank controls were set up for PCR amplification.

[0016] The PCR reaction system included 0.001 μL of specific primer Fam (1 μM), 0.001 μL of specific primer Hex (1 μM), 0.003 μL of universal primer Common (1 μM), 0.20 μL of 2×KASP Master Mix, and 20 ng of DNA of the sample to be tested, and ultra-pure water was added to 0.80 μL; the PCR amplification conditions were 94 °C for 15 min, 1 cycle; 95 °C for 20 s, 65 - 56 °C for 60 s, 10 cycles, and the temperature decreased by 0.8 °C in each cycle; 94 °C for 20 s, 57 °C for 60 s, 30 cycles.

[0017] After the reaction, the fluorescence was detected with a microplate reader, and data analysis and genotyping were performed. Finally, it was found that the above 4 SNP loci were all successfully genotyped, indicating that the above 4 SNP markers were successfully converted into KASP markers. The KASP markers corresponding to the above 4 SNP markers were named VH_KASP1, VH_KASP2, VH_KASP3, and VH_KASP4 respectively. The genotyping results of the above 4 KASP markers for 94 samples are shown in Figure 1 、 Figure 2 、 Figure 3 and Figure 4 , in the figure, FAM represents the ratio of the fluorescence of the amplification product of the Fam primer in the sample to the fluorescence of the underlying rox, and VIC represents the ratio of the fluorescence of the amplification product of the Hex primer in the sample to the fluorescence of the underlying rox.

[0018] Example 2 Application of KASP marker combination for resistance to Vibrio harveyi in Cynoglossus semilaevis in disease-resistant breeding To further verify the effect of the present invention in the detection of the ability of Cynoglossus semilaevis to resist Vibrio harveyi and its application potential in the disease-resistant breeding of Cynoglossus semilaevis, this example was specifically carried out, including the following steps: (1) Genotyping of Cynoglossus semilaevis reserve parents and detection of the ability to resist Vibrio harveyi A total of 188 male and female individuals were randomly selected from the semi-smooth tongue sole reserve parent population, and were respectively electronically marked, and the electronic mark numbers were recorded. At the same time, a small amount of fin ray samples were cut from each individual for DNA extraction. Subsequently, all individuals were genotyped according to the method in Example 1, and the anti-Vibrio harveyi disease ability of the above individuals was measured by a scoring method. That is, the disease-resistant genotypes of 4 KASP markers were scored 2 points, the general disease-resistant genotypes were scored 1 point, and the non-disease-resistant genotypes were scored 0 points. Finally, the genotype scores of the 4 markers of each individual were accumulated to obtain the total score of the anti-Vibrio harveyi disease ability of each individual. The specific results are shown in Table 1. Among them, the genotyping of a certain marker of a very small number of individuals failed, the genotype was recorded as N, and the disease-resistant ability score was recorded as 0.

[0019] Table 1 Genotype and scoring table for the detection of the anti-Vibrio harveyi disease ability of 188 semi-smooth tongue sole reserve parents

[0020] (2)Test on the disease-resistant ability of the offspring of the semi-smooth tongue sole resistant to Vibrio harveyi A total of 47 individuals ranked in the top 25% of the total scores among the above 188 candidate parents were selected as the disease-resistant group. At the same time, the parents (47 tails) ranked in the bottom 25% of the total scores were selected as the non-disease-resistant group and were intensively cultivated. After reaching sexual maturity, 5 pairs of male and female parents were respectively selected from the disease-resistant group parents and the non-disease-resistant group parents, and were paired and fry cultured within the group. When the fry grew to 12 cm, 200 fry were randomly fished from the offspring of the disease-resistant group and the non-disease-resistant group respectively, and an artificial infection experiment was carried out by intraperitoneal injection of Vibrio harveyi. The injection dose was the median lethal dose (LD50) of the offspring of the non-disease-resistant group, and the artificial infection survival rates of the two batches of fry were counted. Among them, the infection survival rate of the fry of the disease-resistant group was 95.5%, and the infection survival rate of the fry of the non-disease-resistant group was 56.0%. Thus, it is proved that the KASP marker combination for detecting the anti-Vibrio harveyi disease ability of the semi-smooth tongue sole of the present invention has good application effects in the breeding of the semi-smooth tongue sole resistant to Vibrio harveyi. By using the KASP markers of the present invention, the anti-Vibrio harveyi disease ability of the semi-smooth tongue sole parents was detected and scored, and the semi-smooth tongue sole parents with high scores were selected for the production of disease-resistant fry, and the anti-Vibrio harveyi disease ability of the produced fry was significantly enhanced.

Claims

1. A KASP marker combination for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease, characterized in that, It contains 4 KASP markers, namely VH_KASP1, VH_KASP2, VH_KASP3 and VH_KASP4, which are located at positions 14:17304461, 14:17307045, 14:17310738 and 14:17303501 on chromosome 14 of Cynoglossus semilaevis respectively, and their base polymorphism types are C / T, C / G, C / T and A / G respectively.

2. The KASP marker combination for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease according to claim 1, wherein The TT genotype at position 14:17304461 on chromosome 14 is the disease-resistant genotype, the CT genotype is the moderately disease-resistant genotype, and the CC genotype is the disease-susceptible genotype; the CC genotype at position 14:17307045 on chromosome 14 is the disease-resistant genotype, the CG genotype is the moderately disease-resistant genotype, and the GG genotype is the disease-susceptible genotype; the TT genotype at position 14:17310738 on chromosome 14 is the disease-resistant genotype, the CT genotype is the moderately disease-resistant genotype, and the CC genotype is the disease-susceptible genotype; the AA genotype at position 14:17303501 on chromosome 14 is the disease-resistant genotype, the AG genotype is the moderately disease-resistant genotype, and the GG genotype is the disease-susceptible genotype.

3. The KASP marker combination for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease according to claim 2, wherein A method for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease, comprising the following steps: (1) Score the disease-resistant genotypes, moderately disease-resistant genotypes and disease-susceptible genotypes of the 4 KASP markers, and record them as 2 points, 1 point and 0 point respectively; (2) According to the total score of the 4 KASP markers of the measured Cynoglossus semilaevis individual, the disease resistance of the measured Cynoglossus semilaevis individual can be obtained. The score of the disease resistance is 0-8 points. The higher the score, the stronger the disease resistance, and the lower the score, the weaker the disease resistance.

4. Application of the KASP marker combination for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease according to claim 1 in the detection of the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease and disease-resistant breeding.

5. The detection primer of the KASP marker combination for detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi, characterized in that, Each KASP marker contains 2 specific primers and 1 common primer; The primer nucleotide sequences of the VH_KASP1 marker: the specific primer Fam is as shown in SEQ ID NO: 1, the specific primer Hex is as shown in SEQ ID NO: 2, and the common primer Common is as shown in SEQ ID NO: 3; The primer nucleotide sequences of the VH_KASP2 marker: the specific primer Fam is as shown in SEQ ID NO: 4, the specific primer Hex is as shown in SEQ ID NO: 5, and the common primer Common is as shown in SEQ ID NO: 6; The primer nucleotide sequences of the VH_KASP3 marker: the specific primer Fam is as shown in SEQ ID NO: 7, the specific primer Hex is as shown in SEQ ID NO: 8, and the common primer Common is as shown in SEQ ID NO: 9; The primer nucleotide sequences of the VH_KASP4 marker: the specific primer Fam is as shown in SEQ ID NO: 10, the specific primer Hex is as shown in SEQ ID NO: 11, and the common primer Common is as shown in SEQ ID NO:

12.

6. Use of the primer according to claim 5 in detecting the ability of Cynoglossus semilaevis to resist Vibrio harveyi disease and in disease-resistant breeding.

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