KASP marker combination for detecting high temperature tolerance of cynoglossus semilaevis and its application

Abstract

The application belongs to the technical field of molecular marker assisted breeding of aquatic animals, and particularly relates to a KASP marker combination for detecting high-temperature tolerance of Cynoglossus semilaevis and application thereof. The KASP marker combination comprises four SNP loci, i.e. 14:20441316, 14:20456272, 14:20820736 and 14:20998894 of chromosome 14 of C. semilaevis, and the base polymorphism types thereof are C / A, C / T, G / A and A / G respectively, and the corresponding high-temperature tolerance advantageous genotypes are CC, CC, GG and AA in turn. The application also provides the matching primers of the KASP marker and a method for measuring the high-temperature tolerance of individuals by using the scoring method. The application can be used for low-cost and high-throughput detection of high-temperature tolerance of C. semilaevis and molecular marker assisted breeding of high-temperature tolerance strains, and has a significant effect.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology for aquatic animals, specifically relating to the KASP marker combination for detecting the high-temperature resistance of the tongue sole and its application. Background Technology

[0002] The tongue sole (Cynoglossus semilaevis) is a marine aquaculture fish. As a cold-water benthic fish, it is quite sensitive to changes in water temperature. Sustained high temperatures in summer, as well as periods of high temperatures during factory farming and pond culture, can easily lead to reduced feeding, stunted growth, weakened immunity, and even large-scale mortality, becoming a significant environmental stressor restricting the stable development of this aquaculture industry.

[0003] Studies have shown significant genetic differences in the tolerance of different individuals and families to high-temperature stress. Therefore, breeding high-temperature tolerant strains is an effective way to improve the survival rate and economic benefits of summer aquaculture. However, phenotypic determination of high-temperature tolerance relies on high-temperature stress tests, which are complex to perform and can cause irreversible damage to the tested individuals, making it difficult to conduct non-destructive screening of breeding populations in the early stages. In addition, traditional phenotypic selection and family selection methods are time-consuming and inefficient. Therefore, how to achieve low-cost, high-efficiency, and high-precision testing of the high-temperature tolerance of *Cocculus semismoothienos* has become a key problem that urgently needs to be solved in current aquaculture and breeding production. Summary of the Invention

[0004] The purpose of this invention is to provide a KASP marker combination for detecting the heat tolerance of tongue sole. This method detects heat tolerance by identifying the genotype at specific loci in individual tongue sole samples. Using the KASP markers and application methods provided by this invention, the heat tolerance of parent tongue sole populations or other cultured populations can be detected at low cost and with high throughput, enabling the development of marker-assisted breeding for heat tolerance.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] This invention provides a KASP marker combination for detecting the heat resistance of tongue sole, comprising four SNP loci, namely HT_KASP1, HT_KASP2, HT_KASP3 and HT_KASP4, located at loci 14:20441316, 14:20456272, 14:20820736 and 14:20998894 on chromosome 14 of tongue sole, respectively, with base polymorphism types of C / A, C / T, G / A and A / G, respectively. The reference genome version of tongue sole is available at: https: / / doi.org / 10.6084 / m9.figshare.32424417.

[0007] The CC genotype at locus 14:20441316 of HT_KASP1 is a heat-resistant genotype, the AC genotype is a normal genotype, and the AA genotype is a susceptible genotype; the CC genotype at locus 14:20456272 of HT_KASP2 is a heat-resistant genotype, the TC genotype is a normal genotype, and the TT genotype is a susceptible genotype; the GG genotype at locus 14:20820736 of HT_KASP3 is a heat-resistant genotype, the AG genotype is a normal genotype, and the AA genotype is a susceptible genotype; the AA genotype at locus 14:20998894 of HT_KASP4 is a heat-resistant genotype, the GA genotype is a normal genotype, and the GG genotype is a susceptible genotype.

[0008] The method for detecting the heat resistance of tongue sole using the KASP marker combination is as follows: The heat-resistant genotype, general genotype and sensitive genotype of each KASP marker are scored as 2 points, 1 point and 0 points respectively. Then, the scores of the four KASP markers of the tested tongue sole individual are added together to obtain the total heat resistance score of the individual (0-8 points). The higher the total score, the stronger the heat resistance of the individual.

[0009] This invention also provides detection primers for the KASP marker combination used to detect the heat resistance of *Cocculus semismoothienos*. Each KASP marker contains two specific primers (Fam and Hex) and one universal primer. The Fam-specific primer is used to detect sensitive alleles, and the Hex-specific primer is used to detect heat-resistant dominant alleles. The 5' ends of the two specific primers are respectively connected to the FAM fluorescent adapter sequence and the HEX fluorescent adapter sequence. The primer nucleotide sequences are as follows:

[0010] The primer nucleotide sequence (5'-3') labeled with HT_KASP1 (14:20441316):

[0011] Specific primer Fam:

[0012] GAAGGTGACCAAGTTCATGCTACGTGTTCGACGTTATTGTTGACCT, as shown in SEQ ID NO: 1;

[0013] Specific primer Hex:

[0014] GAAGGTCGGAGTCAACGGATTACGTGTTCGACGTTATTGTTGACCG, as shown in SEQ ID NO: 2;

[0015] Common primer: GTTTGTGATGTGGCAGATGTTCACAG, as shown in SEQ ID NO: 3;

[0016] The primer nucleotide sequence (5'-3') labeled with HT_KASP2 (14:20456272):

[0017] Specific primer Fam:

[0018] GAAGGTGACCAAGTTCATGCTGATTTGTTGGCATCATTATGTAACAA, as shown in SEQ ID NO: 4;

[0019] Specific primer Hex:

[0020] GAAGGTCGGAGTCAACGGATTGATTTGTTGGCATCATTATGTAACAG, as shown in SEQ ID NO: 5;

[0021] Common primer: TGTGCAGGCAGATATCCTAAAATGC, as shown in SEQ ID NO: 6;

[0022] The primer nucleotide sequence (5'-3') labeled with HT_KASP3 (14:20820736):

[0023] Specific primer Fam:

[0024] GAAGGTGACCAAGTTCATGCTCAGGTTCACCCTGAAACACAGACA, as shown in SEQ ID NO: 7;

[0025] Specific primer Hex:

[0026] GAAGGTCGGAGTCAACGGATTCAGGTTCACCCTGAAACACAGACG, as shown in SEQ ID NO: 8;

[0027] Common primer: TCCAATCATCGTTCTGATGTCAAAG, as shown in SEQ ID NO: 9;

[0028] The primer nucleotide sequence (5'-3') labeled with HT_KASP4 (14:20998894):

[0029] Specific primer Fam:

[0030] GAAGGTGACCAAGTTCATGCTGATGCACAAACACTGAAAGCTGCTG, as shown in SEQ ID NO: 10;

[0031] Specific primer Hex:

[0032] GAAGGTCGGAGTCAACGGATTGATGCACAAACACTGAAAGCTGCTA, as shown in SEQ ID NO: 11;

[0033] The common primer is: TCTCGGTTTTCAGGAATGGTAGAGG, as shown in SEQ ID NO: 12.

[0034] The KASP marker combination or the detection primers of the KASP marker combination described in this invention are used to prepare products for high-temperature resistance testing of half-smooth tongue sole and for high-temperature molecular marker-assisted breeding.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention is the first to develop a KASP marker combination for detecting the heat tolerance of tongue sole, and also provides corresponding primers. Furthermore, it is the first to propose a method that, after genotyping the tested individuals, scores are assigned based on their heat tolerance dominance and suboptimal genotypes, with the total score used to measure the individual's heat tolerance. The judgment rules are clear and the operation is simple. This method has the advantages of low cost, high throughput, and high accuracy, enabling early, non-destructive, and large-scale testing of the heat tolerance of tongue sole. Individuals with high scores can be selected for parent breeding and seedling production, providing a molecular tool for the breeding of new heat-tolerant tongue sole strains. Attached Figure Description

[0037] Figure 1 The genotyping results of 94 half-smooth tongue sole samples and blank controls using the HT_KASP1 marker can be divided into three genotypes: CC, AC, and AA.

[0038] Figure 2 The genotyping results of 94 half-smooth tongue sole samples and blank controls using the HT_KASP2 marker can be divided into three genotypes: CC, TC, and TT.

[0039] Figure 3 The genotyping results of 94 half-smooth tongue sole samples and blank controls using the HT_KASP3 marker can be divided into three genotypes: GG, AG, and AA.

[0040] Figure 4 The genotyping results of 94 half-smooth tongue sole samples and blank controls using the HT_KASP4 marker can be divided into three genotypes: AA, GA, and GG. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical content of this invention, preferred embodiments of the invention are further described below with reference to specific examples. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the equipment and reagents used in each embodiment are all commercially available.

[0042] Example 1: Development of KASP marker assemblages for detecting the high-temperature resistance of tongue sole

[0043] (1) Screening of SNP markers related to high temperature tolerance in tongue sole

[0044] Healthy individuals from the half-smooth tongue sole breeding population underwent an acute heat stress experiment. The fish were subjected to continuous heat stress at a pre-determined temperature (33.5℃). Survival and mortality, as well as survival time, were recorded for each fish. The number of days since death was recorded as the heat tolerance phenotype, for a total of 12 days (1-12, with longer numbers indicating stronger heat tolerance). Fin samples were collected from all individuals for DNA extraction. SNP genotyping was performed on 1260 individuals with recorded heat tolerance phenotypes based on whole-genome resequencing (using the half-smooth tongue sole reference genome version: https: / / doi.org / 10.6084 / m9.figshare.32424417). After filtering, the heat tolerance phenotype and genotype were fitted into a linear mixture model using GEMMA software for genome-wide association analysis (GWAS). Six SNP markers significantly associated with heat resistance were finally screened in the 20.4–21.0 Mb region of chromosome 14 (Table 1), with loci 14:20441316, 14:20447066, 14:20456118, 14:20456272, 14:20820736, and 14:20998894. Based on association analysis and the frequency of associated alleles at each locus, the homozygous genotype of each associated allele was determined to be the dominant heat-resistant genotype, the heterozygous genotype to be the general genotype, and the homozygous genotype of the other allele to be the sensitive genotype.

[0045] Table 1. Information on SNPs significantly associated with the heat tolerance of *Coccus semilaevis* identified by genome-wide association analysis.

[0046]

[0047] (2) KASP marker primer design and genotyping effect verification

[0048] To convert the aforementioned significant SNP markers into KASP markers, 200 bp sequences upstream and downstream of each SNP marker were extracted. Two specific primers (Fam and Hex) and one universal primer (Common) were designed using software, with nucleotide sequences shown in SEQ ID NO: 1-12. The FAM fluorescent adapter sequence GAAGGTGACCAAGTTCATGCT (as shown in SEQ ID NO: 13) and the HEX fluorescent adapter sequence GAAGGTCGGAGTCAACGGATT (as shown in SEQ ID NO: 14) were ligated to the 5' ends of the two specific primers, respectively. Four sites, 14:20441316, 14:20456272, 14:20820736, and 14:20998894, were successfully converted into KASP markers, named HT_KASP1, HT_KASP2, HT_KASP3, and HT_KASP4, respectively. The corresponding thermostable dominant genotypes are CC, CC, GG, and AA, respectively (Table 2).

[0049] Ninety-four DNA samples of *Coccus semilaevis* (covering individuals with different phenotypes, including heat-resistant and sensitive individuals) were selected from the breeding population, with a blank control included. PCR amplification and genotyping were performed using the aforementioned KASP markers. The PCR reaction system included specific primers Fam, Hex, Common, 2×KASP Master Mix, and the DNA samples to be tested, with ultrapure water added to the total volume. PCR amplification conditions were: 94℃ for 15 min, one cycle; 95℃ for 20 s, 65–56℃ for 60 s, 10 cycles, with the annealing extension temperature decreasing by 0.8℃ per cycle; 94℃ for 20 s, 57℃ for 60 s, 30 cycles. After the reaction, fluorescence was detected using a microplate reader, and data analysis and genotyping were performed. The results showed that genotyping was successful at all four loci, and each marker clearly distinguished three genotypes: heat-resistant homozygous, heterozygous, and sensitive homozygous. Figures 1-4 In the figure, FAM represents the ratio of fluorescence of the Fam primer amplification product to the fluorescence of the underlying Rox primer, and VIC represents the ratio of fluorescence of the Hex primer amplification product to the fluorescence of the underlying Rox primer, indicating that the four SNP markers were successfully converted into KASP markers. The distribution of each locus in the three genotypes in the 94 samples is shown in Table 2.

[0050] Table 2. KASP marker locus information, thermotolerant dominant genotypes, and genotype distribution in 94 samples.

[0051]

[0052] Example 2: Application of KASP marker combinations for heat tolerance in half-smooth tongue sole in heat tolerance breeding

[0053] This embodiment further verifies the application effect of the present invention in the detection of the high temperature resistance performance of half-smooth tongue sole and in high temperature resistance breeding, specifically including the following steps:

[0054] (1) Genotyping and heat tolerance scoring of candidate individuals of half-smooth tongue sole

[0055] DNA was extracted from a small number of fin samples of 94 potential parent individuals of *Colophonia semismoothii*. Following the method described in Example 1, all individuals were genotyped according to the HT_KASP1-HT_KASP4 gene sequence. A scoring method was used to assess the heat tolerance of each individual: a heat-tolerant genotype was scored as 2 points, a normal genotype as 1 point, and a sensitive genotype as 0 points. The scores for each individual were summed to obtain a total heat tolerance score (0-8 points). The results are shown in Table 3 (some individuals failed genotyping for a certain marker, and their genotype was recorded as N, with a score of 0). As shown in Table 3, there were significant differences in the total heat tolerance scores among different individuals, which can be used to rank and screen individuals within the population based on their heat tolerance potential.

[0056] Table 3. Genotypes and Scoring Table for High Temperature Tolerance Test of Individual Half-smooth Tongue Sole

[0057]

[0058]

[0059]

[0060] (2) Verification of the high-temperature resistance of offspring of high-temperature resistant parents

[0061] Individuals ranking in the top 25% of heat tolerance scores from the aforementioned parent stock were selected as the heat-tolerant group, and those ranking in the bottom 25% were selected as the sensitive group. After intensive cultivation until sexual maturity, they were paired within their respective groups and raised as fry. When the fry reached 10-12 cm in length, 300 fry were randomly selected from the offspring of both the heat-tolerant and sensitive groups and subjected to a high-temperature stress test at 33.5℃. The survival rates of the two batches of fry under high-temperature stress were recorded. The results are shown in Table 4. The survival rates of the heat-tolerant group's offspring fry after 72 h, 144 h, and 216 h of high-temperature stress were 81%, 45%, and 14%, respectively, while the survival rates of the sensitive group's offspring fry after 72 h, 144 h, and 216 h of high-temperature stress were 54%, 22%, and 0%, respectively. The former was significantly higher than the latter. This demonstrates that the KASP marker combination and scoring method of the present invention has good application effects in the high-temperature resistant breeding of half-smooth tongue sole: by detecting and scoring the high-temperature resistant ability of the parents, the parents with high scores are selected for the production of high-temperature resistant seedlings, which can significantly enhance the high-temperature resistant ability of the offspring seedlings.

[0062] Table 4. Survival rates of high-temperature stress experiments on progeny of heat-tolerant and sensitive populations of *Symplocos spp.*

[0063]

Claims

1. A KASP primer set for detecting the high-temperature resistance of tongue sole, characterized in that, It contains 4 sets of KASP primers, corresponding to 4 SNP sites, namely HT_KASP1, HT_KASP2, HT_KASP3 and HT_KASP4; The primer nucleotide sequences for HT_KASP1 are as follows: specific primer Fam is shown in SEQ ID NO: 1, specific primer Hex is shown in SEQ ID NO: 2, and universal primer Common is shown in SEQ ID NO: 3; The primer nucleotide sequences for HT_KASP2 are as follows: 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; The primer nucleotide sequences for HT_KASP3 are as follows: 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; The primer nucleotide sequences for HT_KASP4 are as follows: 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.

2. The application of the KASP primer set described in claim 1 in the preparation of high-temperature resistance testing and high-temperature resistance molecular marker-assisted breeding products of half-smooth tongue sole.

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