KASP marker combination for detecting the resistance of Semilaevis semilaevis to Vibrio harveyi infection and its application

Abstract

The present invention belongs to the technical field of molecular marker-assisted breeding of aquatic animals, and specifically relates to a KASP marker combination for detecting the resistance of half-smooth tongue sole to Vibrio harveyi disease and its application. The KASP marker combination of the present invention is located at positions 14:17304461, 14:17307045, 14:17310738, and 14:17303501 on chromosome 14 of half-smooth tongue sole, and their base polymorphism types are C / T, C / G, C / T, and A / G, respectively. The present invention proposes for the first time that after genotyping the test individuals, the test individuals are scored according to their superior and inferior genotypes for resistance to Vibrio harveyi disease, thereby measuring the test individuals' resistance to Vibrio harveyi disease. Utilizing the KASP molecular marker combination of the present invention, low-cost, high-efficiency, and high-precision testing of half-smooth tongue sole's resistance to Vibrio harveyi disease can be achieved, thereby significantly improving the efficiency of half-smooth tongue sole disease resistance breeding.

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 semilaevis tongue sole to resist Vibrio harveyi disease and an application thereof. Background Art

[0002] KASP (Kompetitive Allele-Specific PCR) markers are a PCR-based method for precise SNP typing. They utilize fluorescent signals to distinguish different alleles using two allele-specific primers and one universal primer, enabling rapid SNP typing. Due to its high accuracy, low cost, and flexibility, it is widely used in crop and animal breeding. It also demonstrates unique advantages in human genetic disease testing, pharmacogenomics, and disease susceptibility testing. However, its application in disease resistance testing and breeding in aquatic animals has lagged behind, and there are currently no reports of KASP markers in this area. For the past decade or so, molecular marker-assisted breeding (SSR) has been the primary method for disease resistance breeding in aquatic animals. In recent years, with the rapid development of high-throughput sequencing technology, genome-wide SNP markers have become the mainstream for whole-genome selection breeding. However, the efficiency and accuracy of SSR marker-assisted breeding remain low. While whole-genome sequencing (WGS) for large-scale SNP marker discovery for genome-wide selection breeding has greatly improved efficiency and accuracy, it is still prohibitively expensive. 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 carry out disease-resistant breeding.

[0003] The development of the half-smooth tongue sole aquaculture industry is severely hampered by vibriosis, primarily caused by Vibrio harveyi. Low-cost, high-efficiency, and high-accuracy testing of half-smooth tongue sole resistance to Vibrio harveyi has become a major challenge in aquaculture and breeding. To improve the disease resistance of half-smooth tongue sole seedlings, a team led by Academician Chen Songlin of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, has established a whole-genome selection breeding technology for resistance to Vibrio harveyi. However, this technology relies on sequencing technology for genotyping, and the high cost of sequencing limits its large-scale application in half-smooth tongue sole aquaculture and breeding companies. There is an urgent need to develop a low-cost, high-efficiency, and high-accuracy genotyping technology for large-scale testing of half-smooth tongue sole resistance to Vibrio harveyi, thereby promoting the mass production and promotion of disease-resistant seedlings. Summary of the Invention

[0004] The purpose of the present invention is to provide a KASP marker combination for detecting the ability of semilaevis tongue sole to resist Vibrio harveyi disease. By detecting the genotype of specific sites of the semilaevis tongue sole to be tested, its disease resistance is tested, and disease-resistant parents or disease-resistant individuals are screened out, and the disease-resistant parents are used for batch production of disease-resistant seedlings.

[0005] The technical solutions of the present invention are as follows:

[0006] The KASP marker combination for detecting the resistance of semilaevis tongue sole to 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 semilaevis tongue sole, and their base polymorphism types are C / T, C / G, C / T and A / G, respectively.

[0007] The TT genotype at locus 14:17304461 on 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 on 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 on 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 on 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.

[0008] The method for detecting the ability of Semilaevis semilaevis sole to resist Vibrio harveyi disease using the KASP marker combination comprises the following steps:

[0009] (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;

[0010] (2) The disease resistance of the tested semilaevis tongue sole 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.

[0011] The present invention also provides detection primers matched with the KASP marker combination for detecting the ability of semi-smooth tongue sole to resist Vibrio harveyi disease, wherein each KASP marker comprises two specific primers and one universal primer;

[0012] The nucleotide sequences of the primers for the VH_KASP1 marker are as follows:

[0013] Specific primer Fam:

[0014] 5′-GAAGGTGACCAAGTTCATGCTAAATGTTGAACGTTGAAGAGAAGCC-3′, as shown in SEQ ID NO: 1;

[0015] Specific primer Hex:

[0016] 5′-GAAGGTCGGAGTCAACGGATTAAATGTTGAACGTTGAAGAGAAGCT-3′, as shown in SEQ ID NO: 2;

[0017] Common primer: 5'-ACTTAAACCATCCATGGAACAGGAG-3', as shown in SEQ ID NO: 3;

[0018] The nucleotide sequences of the primers for the VH_KASP2 marker are as follows:

[0019] Specific primer Fam:

[0020] 5′-GAAGGTGACCAAGTTCATGCTAAGCAAAACACTTTTCAGCAACAG-3′, as shown in SEQ ID NO: 4;

[0021] Specific primer Hex:

[0022] 5′-GAAGGTCGGAGTCAACGGATTAAGCAAAACACTTTTCAGCAACAC-3′, as shown in SEQ ID NO: 5;

[0023] Common primer: 5′-GCCACTGCTTTAATGTATTCAATT-3′, as shown in SEQ ID NO: 6;

[0024] The nucleotide sequences of the primers for the VH_KASP3 marker are as follows:

[0025] Specific primer Fam:

[0026] 5′-GAAGGTGACCAAGTTCATGCTCTGCTTATATGGTAAAACATGAGC-3′, as shown in SEQ ID NO: 7;

[0027] Specific primer Hex:

[0028] 5′-GAAGGTCGGAGTCAACGGATTCTGCTTATATGGTAAAACATGAGT-3′, as shown in SEQ ID NO: 8;

[0029] Common primer: 5'-AGTAAATCCTCCAAACCCGAAGAA-3', as shown in SEQ ID NO: 9;

[0030] The nucleotide sequences of the primers for the VH_KASP4 marker are as follows:

[0031] Specific primer Fam:

[0032] 5′-GAAGGTGACCAAGTTCATGCTTGTATTCTACACAAAAGTACACAA-3′, as shown in SEQ ID NO: 10;

[0033] Specific primer Hex:

[0034] 5′-GAAGGTCGGAGTCAACGGATTTGTATTCTACACAAAAGTACACAG-3′, as shown in SEQ ID NO: 11;

[0035] Common primer: 5′-TACCCAAGACTTTGTTTGCTGGTT-3′, as shown in SEQ ID NO: 12.

[0036] The KASP marker combination and primers for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease can be applied to the detection of the ability of half-smooth tongue sole to resist Vibrio harveyi disease and disease-resistant breeding.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention develops, for the first time, a KASP molecular marker combination and primers for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease. It also proposes, for the first time, to measure the ability of the tested individuals to resist Vibrio harveyi disease by genotyping the tested individuals and then scoring them according to their superior and inferior genotypes for resistance to Vibrio harveyi disease. The KASP molecular marker combination of the present invention can achieve rapid, accurate and efficient detection of the ability of half-smooth tongue sole to resist Vibrio harveyi disease, and can be used to quickly detect the ability of half-smooth tongue sole parent populations to resist Vibrio harveyi disease, and the screened parents with resistance to Vibrio harveyi disease can be used for mass production of disease-resistant seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The genotyping results of VH_KASP1 marker for 94 samples of Semilaevis semilaevis and 2 blank controls, red dots represent homozygous TT genotype, purple dots represent heterozygous TC genotype, blue dots represent homozygous CC genotype, and black dots represent blank controls;

[0040] Figure 2The genotyping results of VH_KASP2 marker for 94 semilaevis samples and 2 blank controls, red dots represent homozygous CC genotype, purple dots represent heterozygous CG genotype, blue dots represent homozygous GG genotype, and black dots represent blank controls;

[0041] Figure 3 The genotyping results of 94 semilaevis semilaevis samples and 2 blank controls using the VH_KASP3 marker. Red dots represent the homozygous TT genotype, purple dots represent the heterozygous TC genotype, blue dots represent the homozygous CC genotype, and black dots represent the blank control.

[0042] Figure 4 Figure 4 shows the genotyping results of 94 semilaevis semilaevis samples and 2 blank controls using the VH_KASP4 marker. Red dots represent the homozygous AA genotype, purple dots represent the heterozygous AG genotype, blue dots represent the homozygous GG genotype, and black dots represent the blank control. DETAILED DESCRIPTION

[0043] 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.

[0044] Example 1

[0045] Development of a KASP marker panel for detecting the resistance of Semilaevis semilaevis to Vibrio harveyi infection

[0046] (1) Screening of SNP markers associated with Vibrio harveyi resistance in semilaevis tongue sole

[0047] In 2022, 240 half-smooth tongue sole parents collected from three major breeding areas in China and 600 parents from the applicant's core family population were used to establish families. Male and female individuals with good gonadal maturity were selected from these parent populations for family building, resulting in a total of 122 families. When the fry reached approximately 12 cm, 20 fry from each family were randomly collected and mixed in a cement tank. After stabilization for two weeks, they were artificially infected with Vibrio harveyi via intraperitoneal injection under laboratory conditions at a sublethal dose. Fin ray samples were collected from all deceased individuals (750 samples) and the final survivors (834 samples) for DNA extraction. Targeted sequencing and genotyping were performed using a 20K liquid phase array. The genotypes of the 1584 progeny samples were imputed using the high-density genotypes of the parents. After filtering, genome-wide association analysis was performed in conjunction with disease resistance phenotypes (dead individuals were marked as 0 and survivors were marked as 1). Ultimately, 132 significant SNP markers were identified on chromosome 14. The four most significant SNP markers were selected: loci 14:17304461, 14:17307045, 14:17310738, and 14:17303501. Their polymorphisms were T / C, C / G, T / C, and A / G, respectively. The genotype frequencies of these four SNP markers in 750 deceased individuals and 834 surviving individuals were then counted to determine the dominant genotype. The dominant genotype for disease resistance was determined to be TT at locus 14:17304461, CC at locus 14:17307045, TT at locus 14:17310738, and AA at locus 14:17303501.

[0048] (2) KASP marker primer design and genotyping effect verification

[0049] To convert the above four SNP markers into KASP markers, we first extracted 200 bp of sequences upstream and downstream of the four SNP markers. We then used software to design two specific primer sequences (Fam and Hex) and one universal primer sequence (Common). The final primer sequence information was as follows:

[0050] The primer sequences for the SNP marker at site 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, and common primer Common: 5'-ACTTAAACCATCCATGGAACAGGAG-3', as shown in SEQ ID NO: 3;

[0051] The primer sequences for the SNP marker at site 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, and common primer Common: 5'-GCCACTGCTTTAATGTATTCAATT-3', as shown in SEQ ID NO:6;

[0052] The primer sequences for the SNP marker at site 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, and common primer Common: 5'-AGTAAATCCTCCAAACCCGAAGAA-3', as shown in SEQ ID NO:9;

[0053] The primer sequences for the SNP marker at site 14:17303501 are: 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, and common primer Common: 5'-TACCCAAGACTTTGTTTGCTGGTT-3', as shown in SEQ ID NO: 12.

[0054] Subsequently, the 5' ends of the two specific primers for the four SNP markers were ligated with fluorescent linker sequences (Fam-GAAGGTGACCAAGTTCATGCT and Hex-GAAGGTCGGAGTCAACGGATT, respectively, as shown in SEQ ID NO: 13 and SEQ ID NO: 14). PCR amplification was performed on 94 randomly selected DNA samples from the 1584 artificial infection experiments, along with two blank controls.

[0055] The PCR reaction system included 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 Common (1 μM), 0.20 μL of 2×KASP Master Mix, and 20 ng of the test sample DNA, with ultrapure water 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, with the temperature decreasing by 0.8°C each cycle; 94°C for 20 s, 57°C for 60 s, 30 cycles.

[0056] After the reaction, the fluorescence was detected by microplate reader, and data analysis and genotyping were performed. Finally, it was found that the above four SNP sites were successfully typed, indicating that the above four SNP markers were successfully converted into KASP markers. The KASP markers corresponding to the above four SNP markers were named VH_KASP1, VH_KASP2, VH_KASP3 and VH_KASP4 respectively. The genotyping results of the above four KASP markers for 94 samples are shown in Figure 2. Figure 1 、 Figure 2 、 Figure 3 and Figure 4 In the figure, FAM represents the ratio of the fluorescence of the amplified 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 amplified product of the Hex primer in the sample to the fluorescence of the underlying rox.

[0057] Example 2

[0058] Application of KASP marker combination for resistance to Vibrio harveyi in disease resistance breeding of Semilaevis semilaevis

[0059] In order to further verify the effect of the present invention in detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease and its application potential in disease-resistant breeding of half-smooth tongue sole, this example was carried out, which specifically includes the following steps:

[0060] (1) Genotyping of Semilaevis semilaevis parental lines and detection of their resistance to Vibrio harveyi

[0061] A total of 188 male and female individuals were randomly selected from a pool of reserve parents of Cynoglossus semilaevis. These individuals were electronically tagged and their electronic tag numbers recorded. A small fin ray sample was collected from each individual for DNA extraction. Genotyping was then performed on all individuals according to the method described in Example 1, and a scoring method was used to assess their resistance to Vibrio harveyi. The genotypes for the four KASP markers were assigned a score of 2, the moderately resistant genotype was assigned a score of 1, and the non-resistant genotype was assigned a score of 0. Finally, the genotypic scores for each individual were summed to obtain a total score for their resistance to Vibrio harveyi. The results are shown in Table 1. A very small number of individuals failed genotyping for a particular marker; these individuals were assigned a genotype score of N and a resistance score of 0.

[0062] Table 1 Genotypes and scores of 188 semilaevis parents for the test of their ability to resist Vibrio harveyi

[0063]

[0064] (2) Disease resistance test of offspring of semi-smooth tongue sole resistant to Vibrio harveyi

[0065] Of the 188 candidate parents, 47 individuals ranked in the top 25% of the total score were selected to form a disease-resistant group. Meanwhile, 47 individuals ranked in the bottom 25% of the total score were selected to form a non-resistant group. After reaching sexual maturity, five male and female pairs were selected from each of the disease-resistant and non-resistant groups. These groups were then paired and reared as fry. When the fry reached 12 cm, 200 fry were randomly selected from the offspring of the disease-resistant and non-resistant groups and artificially infected with Vibrio harveyi via intraperitoneal injection at the half-lethal dose (LD50) of the non-resistant group. The survival rates of the two groups were calculated. The survival rates of the resistant and non-resistant offspring were 95.5%, while those of the non-resistant group were 56.0%. This demonstrates that the KASP marker combination for detecting the ability of half-smooth tongue sole to resist Vibrio harveyi disease of the present invention has a good application effect in breeding half-smooth tongue sole resistant to Vibrio harveyi disease. Using the KASP markers of the present invention, the ability of half-smooth tongue sole parents to resist Vibrio harveyi disease is detected and scored, and half-smooth tongue sole parents with high scores are selected for the production of disease-resistant seedlings. The produced seedlings have significantly enhanced ability to resist Vibrio harveyi disease.

Claims

1. Application of a KASP marker combination for detecting the ability of Semilaevis semilaevis to resist Vibrio harveyi infection in the preparation of a product for screening individuals of Semilaevis semilaevis to resist Vibrio harveyi infection; The KASP marker combination comprises four 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 of chromosome 14 of semilaevis semilaevis, respectively, and their base polymorphism types are C / T, C / G, C / T and A / G, respectively; The TT genotype at the 14:17304461 site on 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 the 14:17307045 site on 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 the 14:17310738 site on 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 the 14:17303501 site on 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; Each KASP marker contains two specific primers and one universal primer; The primer nucleotide sequences for the VH_KASP1 marker 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; Primer nucleotide sequences for VH_KASP2 tagging: specific primer Fam is shown in SEQ ID NO: 4, specific primer Hex is shown in SEQ ID NO: 5, and universal primer Common is shown in SEQ ID NO: 6; VH_KASP3 marker primer nucleotide sequences: specific primer Fam is shown in SEQ ID NO: 7, specific primer Hex is shown in SEQ ID NO: 8, and universal primer Common is shown in SEQ ID NO: 9; Primer nucleotide sequences for VH_KASP4 tagging: specific primer Fam is shown in SEQ ID NO: 10, specific primer Hex is shown in SEQ ID NO: 11, and universal primer Common is shown in SEQ ID NO: 12; 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; the disease resistance of the tested semi-lazy tongue sole individual could be obtained based on the sum of the scores of the four KASP markers of the tested semi-lazy tongue sole individual. The disease resistance score ranged from 0 to 8 points, with the larger the score, the stronger the disease resistance, and the smaller the score, the weaker the disease resistance.

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